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3: Drug Plasma-Protein Binding

Equipment and Materials

1 M Tris buffer pH 8

7 % bovine serum albumin solution (in Tris buffer at pH 8)

2.5 mM sulphadimidine

2.5 mM sulphanilamide

2.5 mM Phenylbutazone

Ammonium Sulphate

1.5M HCl

0.5% sodium nitrite

0.5% ammonium Sulphamate

0.05% N-(Naphthyl)ethylenediamine

15 x 2ml Eppendorf tubes (per pair)

16 test tubes + rack (per pair)

Automated pipettes capable of delivering 200 – 2000 μL

Colorimeter and cuvettes capable of reading at 545nm

Introduction

Many substances including drugs combine with plasma proteins especially albumin. The amount of drug protein binding has profound effects on the metabolism, distribution and excretion of the drug in the body. It is therefore important to discover early on in the drug development process the plasma protein binding properties of a given drug. While much has been made of the importance of binding interactions as a source of unwanted drug interactions, present evidence suggests that this is not as important as was once thought.

In this practical the relative binding properties of Sulphadimidine and Sulphanilamide will be investigated. For reference, the structures of these molecules are given on the cover of this booklet.

 

Protocol

This Practical should be conducted in Pairs

Label 6 Eppindorf tubes 1-6 and prepare according to the following table:

 

Tube no	Sulphadimidine / μL	Sulphanilamide / μL	Phenylbutazone / μL	Albumin in buffer / μL	Tris buffer / μL	Distilled water
1	300				600	300
2	300			600		300
3	300		300	600
4		300			600	300
5		300		600		300
6		300	300	600

 

• Mix well and leave at room temperature for at least 5 minutes.
• While waiting you can weigh out six 0.7g (+ or – 0.01g) ammonium sulphate portions for the next step. Use the smallest plastic weighing boats provided.
• Add 0.7g of ammonium sulphate to each tube then shake thoroughly.
• Centrifuge at 12000 rpm for 10 minutes.
• There should be a lump of milky white solid in the bottom of the tube and a clear supernatant, if this is not the case spin for a further 5 minutes.
• If the solid collects at the top of the tube you may have to sample past it. Take care not to get any solid into the pipette.

Serial Dilutions (for calibration plots).

You have been provided with 2.5 mM solution of sulphadimidine and sulphanilamide. From these solutions you will perform a serial dilution to obtain the following concentrations in solution. Use a further 9 Eppindorf tubes labeled 7-15 to make up the dilutions according to the table below. This time there is no need to centrifuge.

Tube no	[Sulphadimidine] / mM	[Sulphanilamide] / mM
7	1.25
8	0.63
9	0.31
10	0.15
11		1.25
12		0.63
13		0.31
14		0.15
15 (Blank)	0	0

Tip: Notice that each solution is half the concentration of the previous one, e.g. solution 8 could be made by made up by adding 1 cm³ (1000 μL ) of solution in tube 7 and diluting with 1cm³ of water and so on.

Bratton-Marshall Assay:

You will now estimate the concentrations of each drug in all 15 solutions contained in the Eppendorf tubes. This will be done using the Bratton-Marshall assay.

• Label a set of short test tubes 1 – 15 and add 100 μL of supernatant from the Eppendorf tubes (1 – 6) and the serially diluted solutions (7 – 15) in number order. It is essential not to confuse tube labels during this stage.
• Now check that you have 15 tubes each containing 100 μL

 

The Concentrations of Sulphanilamide and sulphadimidine in each tube can be determined using a modified Bratton-Marshall method. Iit is very important that you follow this procedure exactly.

 

1. To each of the tubes add 2cm³ of 1.5M hydrochloric acid followed by 0.1cm³ of 0.5% NaNO₂ shake and leave for 2 minutes only.

 

1. Add 1 cm³ of 0.5% ammonium sulphamate to each tube, shake and leave for 3 minutes

 

1. Add 1 cm³ of 0.05% N-(naphthyl)ethylenediamine to each tube, shake and allow to stand for 15 minutes for the colour to develop

 

1. Measure the absorbance at 545 nm, remembering to zero the spectrometer using your blank solution (tube 15).

It is good practice to read solutions from low to high absorbance using the same cuvette. This minimizes interference between one solution and one cuvette and another. We therefore suggest the following reading order, using tube 15 as the blank.

 

Reading order	Tube number	Abs (545nm)
1	14	0.045
2	13	0.089
3	12	0.134
4	11	0.209
RINSE WITH DISTILLED WATER
5	6	0.308
6	5	0.429
7	4 (control)	0.531
RINSE WITH DISTILLED WATER
8	10	0.054
9	9	0.121
10	8	0.198
11	7	0.345
RINSE WITH DISTILLED WATER
12	3	0.323
13	2	0.456
14	1 (control)	0

Write up

• Use your absorbance data from tubes 7 – 15 to draw two calibration plots; one for sulphadimidine (tubes 7-10) and one for sulphanilamide (tubes 11-14). Remember to include the 0, 0 point as the origin of each plot.
• Taking the control tubes (1 and 4, no plasma) as 0% binding, calculate the percentage binding of each drug in the presence and absence of phenylbutazone (structure is given on the cover).
• What effect does adding phenylbutazone have? Why is this the case?
• Calculate the mass of each drug used in each Eppendorf tube – why is it an advantage to the drug development process to do this sort of analysis on such a small scale?
• Suggest other analytical techniques that could be used to establish the amount of unbound drug in the supernatant layer.

References and further reading

 

1. Rang, H.P. Dale M. M. Ritter, J.M. Flower, R. (2007) Pharmacology; Churchill Livingstone.
2. Rang, H.P; (2006) Drug Discovery and Development; Churchill Livingstone.
3. Bratton A. C. Marshall E. K. A new coupling Component for sulphanilamide Determination. J. Bio. Chem. 128 (2),537
4. Wheipton R. Watkins G. Curry S. H. Bratton-Marshall and Liquid-Chromatographic Methods Compared for Determination of Sulfamethazine Acetylator Status. Clin. Chem. 27/11, 1911-1914 (1981)

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