PRACTICAL WRITE-UP INSTRUCTIONS
In this practical write up, you will be required to produce some graphs and answer some questions on the practical you carried out with the drug “Orlistat”.
A. Calculate Orlistat final concentration and fill in data table
Complete the table below filling in the final concentrations of Orlistat and final absorbance value (A1 – A2). (4 marks)
Tube no. Orlistat final concentration (µM) Absorbance at 410 nm (A1) Absorbance at 500 nm (A2) Absorbance
A1 – A2 = A3
1 0.14 0.04
2 0.19 0.09
3 0.22 0.1
4 0.28 0.1
5 0.4 0.12
6 0.54 0.13
7 0.57 0.15
B.Plot the graphs
Now, you will need to plot two graphs from this data. YOU MUST TITLE AND LABEL THE GRAPHS APPROPRIATELY.
Graph 1. Plot a concentration-response curve of your data (i.e. Orlistat concentration versus Absorbance A3). You may use a graphics package (e.g. Xcel), or, if you prefer, you may draw the graph by hand. (10 marks)
Graph 2. You have been provided with some semi-log graph paper; plot theconcentration-response curve for your dataon this semi log graph paper.Should you have access to a graphical package that will do this for you, you may use this to compile your graph. (10 marks)
C. Estimate Orlistat IC50 value
Fromtheconcentration-response graph on semi-log scale, you will need to estimate the IC50 value for Orlistat. YOU MUST SHOW EVIDENCE OF HOW YOU REACHED THIS VALUE ON YOUR GRAPH (i.e. indicate how you read the IC50 value off the graph)
(5 marks)
ESTIMATED ORLISTAT IC50 VALUE =
D. Refer to the literature for other Orlistat IC50 values
Now you need to compare your Orlistat IC50 value with other, published data on Orlistat. Find references using www.pubmed.comand comment on the estimated IC50 value of Orlistat obtained in your experiment with the literature values (i.e. is it similar or different?).
Note: make sure the article you reference actually used pancreatic lipase in the experiments and make sure you quote the IC50 value of Orlistat from that article and not any other pancreatic lipase inhibitor that may have been investigated.
Ensure that you provide a reference AND MAKE SURE THAT YOU REFERENCE THE ARTICLE CORRECTLY, i.e. in the Harvard style of referencing. If you do not know what this is – look it up!
(6 marks)
CH5011 Pharmacology Practical
Background
The basis of pharmacological intervention is that the drug molecule(s) must be bound to particular constituents of cell/tissues in living systems in order to produce a physiological response/pharmacological effect. This may be in the form of receptor-drug interactions.
The main super families of receptor are:
• Ligand-gated ion channels (LGIC)
• G protein-coupled receptors (GPCR)
• Kinase-linked receptors
• Nuclear/cytoplasmic receptors
However, there are other ways that an action of a drug can elicit a pharmacological response such as by enzyme inhibition, ion channels and carrier molecules. One recently well known drug which inhibits an enzyme is the lipase inhibitor Orlistat (view the YouTube video)1. This is indicated for weight loss in adults who are overweight (body mass index, BMI, ≥28 kg/m2) and should be taken in conjunction with a mildly hypocaloric, lower-fat diet2.
Introduction
Orlistat3 is a potent, specific and long-acting inhibitor of gastrointestinal lipases. It exerts its therapeutic activity in the lumen of the stomach and small intestine by forming a covalent bond with the active serine site of the gastric and pancreatic lipases. The inactivated enzyme is thus unavailable to hydrolyse dietary fat, in the form of triglycerides, into absorbable free fatty acids and monoglycerides. From clinical studies, it has been estimated that 60 mg Orlistat taken three times daily blocks the absorption of approximately 25% of dietary fat.
Typically, in order to evaluate the potency of a drug the EC50 value is used – this represents the concentration of a compound where 50% of its effect is observed. However, for enzyme inhibition the IC50 value is quoted – this represents the concentration of a drug that is required for 50% of response in vitro4.
Aims
The aim of this practical is to estimate5 the IC50 value in vitro for Orlistat using the Pancreatic Lipase Inhibition Assay.
The Pancreatic Lipase Inhibition Assay
The basic principle involves the hydrolysis of the ester (p-nitrophenyllaurate; p-NPL) using the Lipase enzyme and quantification of the yellow coloured p-nitrophenol (p-NP) product at 410 nm. The addition of a lipase inhibitor such as Orlistat will reduce the activity of the Lipase enzyme and hence produce a reduction in the amount of p-nitrophenol formed. In this way it is possible to estimate the concentration of drug (Orlistat) that is required for 50% inhibition i.e., the IC50 value. Figure 1 shows a schematic of the hydrolysis of p-NPL to p-NP by pancreatic lipase and inhibition by Orlistat6.
Figure 1. Schematic of the hydrolysis of p-NPL to p-NP by lipase and inhibition by Orlistat6
Method
YOU WILL BE WORKING IN GROUPS (4 STUDENTS/PER GROUP).
You have been provided the following tubes (labelled as 1 to 7) as listed in Table 1.
Table 1. Details of sample tubes provided Tube 50 µL of Orlistat at a concentration of: Final concentration of Orlistat (in final volume of 1050 µl)
1 2.1 mM
2 630 µM
3 210 µM
4 63 µM
5 21 µM
6 6.3 µM
7 0
FOLLOW THE INSTRUCTIONS BELOW TO CARRY OUT THE EXPERIMENT
1. To each of the tubes labelled 1 -7, add 400 µl of Tris-HCL, being careful not to cross contaminate your tubes.
HINT: hold the eppendorf at a 45° angle and GENTLY pipette your Tris-HCL to the side of the tube (allowing it to trickle to the bottom) – not directly into the fluid at the bottom.
2. Now, add 450 µl of p-NPL into each tube, following the same pipetting techniques as above
HINT: Be careful tilting your eppendorf as it has more fluid in it!
3. Finally, add 150 µl of the enzyme solution to each tube. You should now have a total volume of 1050 µl. Incubate all samples at 37°C for 30 minutes in the incubator.
4. Whilst you are waiting for incubation to complete, instructions will be provided on the use of the UV/VIS spectrophotometer. After incubation, centrifuge all the tubes at 13,000 rpm for 3 minutes.
5. Using a clean glass/pasteur pipette carefully pipette out the supernatant into a clean plastic micro-cuvette.
HINT: Make sure you have a labelling system for your cuvettes! Do not forget which cuvette corresponds to which concentration of Orlistat!
6. Repeat this procedure for all the tubes ensuring you use a new pipette and cuvette for each tube sample.
7. Set the UV/VIS spectrophotometer to read at a wavelength of 410 nm.
8. Blank the UV/VIS spectrophotometer with deionised water at this wavelength.
9. Analyse the supernatant from all tubes at 410 nm (A1)
10. Record the Absorbance (A1) values in Table 2 (Results section below).
11. Set the UV/VIS spectrophotometer to read at a wavelength of 500 nm.
12. Blank the UV/VIS spectrophotometer with deionised water at this wavelength
13. Analyse the supernatant from all tubes at 500 nm (A2)
14. Record the Absorbance (A2) values in Table 2.
15. Calculate the difference between the Absorbance at 410 nm and 500 nm (A1 – A2 = A3).
16. Record the calculated values (A3) in Table 2.
17. Calculate the final concentration of Orlistat in each tube. Remember the total volume in each tube is 1050 µL7.
18. Record the values in Table 2.
19. Plot a graph of Concentration of Orlistat (x axis) versus Absorbance (A3 values) – y axis. Ensure that all axes are correctly labelled and a title for the plot is provided. You may want to use Microsoft Excel for the plot.
20. Estimate the IC50 value – instructions will be provided via WebLearnafter the practical.
21. Follow instructions below for the practical write-up8.
References / Points
1. http://www.youtube.com/watch?v=vrfO9hS40jA (accessed 5th Nov 2012)
2. http://www.medicines.org.uk (accessed 5th Nov 2012)
3.Orlistat is sold as Xenical or Alli in the UK
4. http://www.fda.gov/ohrms/dockets/ac/00/slides/3621s1d/sld036.htm (accessed 5th November 2012)
5.Due to the nature of the experiment and time frame IC50value is estimated
6. Adapted from: Dolenc et al., (2010). International Journal of Pharmaceutics 396 149–155
7. Example calculation – Tube 1 has 50 µL of an Orlistat solution (concentration 2.10 mg/mL) which is placed in a total volume of 1050 µL and therefore diluted. Therefore, the final concentration of Orlistat in tube 1 is (50/1050 x 2.10) µg/mL = 100 µg/mL
8. Whilst all reasonable effort has been made for the accuracy of the information provided – the author does not accept any liability for any omissions/errors within this document.
Name………………………………………………… Student ID……………………
Results:
Table 2. Absorbance values and final concentration of Orlistat after incubation
Tube no. Absorbance at 410 nm (A1) Absorbance at 500 nm (A2) Absorbance
A1 – A2 = A3
1
2
3
4
5
6
7
WORKING WITH DILUTIONS:
Solutionsused in experiments – particularly drug solutions –often start out at much higher concentrations than actuallyrequired for the experiment (this is mostly to help with storage of the drug and also to save time). It is therefore frequently necessary to dilute these solutions to a desired level before use. This requires a working knowledge of the principles of diluting, dilution factors, concentration factors and the calculations involved (which are very simple).
Once you understand dilution factors, you will be able to make up a solution of drug to any volume with a specific concentration!
Example:
DEFINITIONS:
Aliquot: a measured sub-volume of original sample.
Stock concentration: the concentration of the starting or original aliquot (e.g. the concentrated drug solution)
Diluent: material with which the sample is diluted
Dilution factor (DF): ratio of:
final volume/aliquot volume (final volume = aliquot + diluent)
Concentration factor (CF): ratio of:
aliquot volume divided by the final volume (inverse of the dilution factor)
N.B.
• Dilution/concentrations factors apply regardless of concentration units (i.e. whether the concentration is expressed as mass/volume (e.g. mg/ml) or as molarity (M, mM, µM etc.))
• High dilutions are usually expressed exponentially (i.e.: a solution which has been diluted a million fold is termed a 106 dilution, or is 10-6 concentration).
EXAMPLES
1. If you have a dilution factor of 1:10 that means 1 ml of solution (e.g. drug) is diluted INTO 10 ml of diluent.
In practice, that means: 1ml of solution IN 9 ml diluent.
In other words, a dilution factor of 1:10 is the same as saying “the solution was diluted 10-fold” or the that the solution was diluted by 10 times
Let’s say the starting concentration of our drug solution was 100 µM. What would the concentration be if we diluted the drug solution by a factor of 10 (i.e. a dilution factor of 1:10)?
100 / 10 = 10
New concentration is 10 µM
2. Here is a more complex example involving volumes:
How would you prepare 20 mL of drug solution that needs to be diluted using a dilution factor of 1:50?
1. Work out the volume of drug solution required to make up into 20 ml:
20 (ml) / 50 (dilution factor) = 0.4 ml
i.e. you will need 0.4 ml IN 20 ml to dilute the drug solution
2. How much diluent is required?
20 ml – 0.4 ml = 19.6 ml
3. In other words, to make a drug solution that has been