EXPERIMENT 3: IMPACT ASSESMENT OF DIFFERENT INGREDIENTS ON THE CHARACTERISTICS OF A SUPPOSITORY FORMULATION

Objective

To study the effect of using different base composition on the physical characteristics of a suppository formulation

Introduction

Suppository is a solid formulation with various sizes and shape which is suitable for rectal administration. A good suppository must melt down after administration into the rectal and release the drug for localized or systemic effect.

The drug needs to be dispersed in a suitable suppository base. A good base should not be toxic, does not produce irritation, does not react with the drug, and easy to form into a suppository. Different base composition will affect the rate and release limit of a drug from suppository.

In this experiment, the effect of different base composition on the physical characteristics of the formed suppositories and its effect on the release of the drug from it are studied.

Apparatus and materials :

a)      Apparatus

Weighing boat, spatula, 1 beaker 50ml, 1 beaker 100ml, hot plate, measuring cylinder 5ml,1 set of suppository mould, water bath (37 ̊ C), 1 dialysis beg, threads, glass rod, pipette, cuvette, spectrophotometer UV/Vis

b)      Materials

Polyethylene glycol (PEG) 1000, polyethylene glycol (PEG) 6000, paracetamol

Procedures :

1.      Concentrated paracetamol stock solution was prepared. (10 mg in 5ml distilled water)

2.      Suppository of paracetamol (10 mg) was prepared using formulation :

PEG 1000	PEG 6000	Paracetamol stock solution (1 mg)	amount
6	3	1	10

3.      Suppository was shaped with suppository-mould. The shape, texture and shape of the suppository was described.

4.      One suppository was put in the beaker that filled with 10 ml distilled water (37 ̊ C) and time for the suppository melting was recorded.

5.      One suppository was put in the dialysis beg and the both ends were tightly tied. The dialysis beg was put in the beaker that filled with 50 ml of distilled water that was heated to 37 ̊ C.

6.      For every 5 minutes, the sample (3-4 ml) was pipette d and the diffusion of paracetamol from the suppository was determined by using the spectrometer UV-visible. The distilled water was stirred by the glass rod before the sample was taken.

RESULT AND DISCUSSION

1)      Compare the physical shape suppository and explain.

Group/Characteristic of suppository	3	6	8
Shape	Smooth bullet-shaped	Non-sticky bullet-shaped	Gnitty bullet-shaped
Greasiness	Slightly greasy	Greasy	Least greasy
Hardness	Hard	Hard	Very hard
Colour	Uniform milky	Milky	Transparent

In group 3, they they have 3g of PEG 1000 and 6g of PEG 6000. Next, group 6 have proportion of PEG 1000 (6g) is more than PEG 6000 (3g). Lastly, group 8 absence of PEG 1000 which have 9g of PEG 6000.

Content of PEG 6000 (g)	0	3	6	9
Time required for suppository to melt (min)	35.31	53.20	39.42	49.21
	37.77	48.15	45.31	41.30
	33.48	45.65	54.35	39.45
Average Time (min)	35.52	49.00	46.36	43.32
Standard deviation (SD)	2.1527	3.8461	7.5202	5.1841
+ SD	37.6727	52.8461	53.8802	48.5041
- SD	33.3673	45.1539	38.8398	38.1359

2)      Plot graph time that required for the suppository to melt against amount of PEG 6000 in the formulation. Compare and discuss the result.

            PEG 6000 or also known as Polyethylene Glycol 6000 has high molecular weight of ethylene oxide and it is a water-soluble chemical. In this graph, we can observe that the time taken for the suppository to melt is not directly proportional to the PEG 6000 content in gram. The function of the PEG 6000 content is as a suppository base. As the general knowledge, increasing the mass of the PEG 6000 will make the suppository more solid. Therefore, the time taken for the suppository to melt will be longer as the mass of the PEG 6000 increase. Based on the graph above, the time taken for the suppository to melt with absence of PEG 6000 is 35.52 minutes which the shortest time among the result while the longest time taken for the suppository to melt is the suppository that contain 3g of PEG 6000.

Theoretically, the time taken for the lowest amount of PEG 6000 should be lowest and vice versa but the results we obtained is totally inappropriate. The deviation of the result is majorly effected by the errors occur during conduct the experiment. Defect of suppository made reduction in mass and will reduce the time for suppository to dissolve. Error made during measured, and transferred of the ingredient during making of suppository also may alter the result. There also possibility that suppository does not solid enough when we taken out from the refrigerator. The unsolidified suppository made it easier to be dissolved in water bath. The heating process using water bath also may produce this result. Some of the group might stir the beaker containing suppository which make it faster to dissolve.

3) Plot graph of UV absorption against time. Analyze the result.           

	UV visible absorption
Time (min)	0	5	10	15	20	25	30	35	40	45	50	55	60
Absorption of UV at 520nm	0.025	0.006	0.006	0.008	0.007	0.010	0.010	0.009	0.009	0.032	0.013	0.012	0.015

            The UV spectrometer measures the release of paracetamol from the suppository in the dialysis bag to the distilled water. The release of the drugs from the suppository involved the diffusion mechanism. The water is set to 37 ⁰C to mimic the temperature of human body as suppository will be applied in the human body. Meanwhile, the dialysis bag represents the skin membrane.

The graph above shows the relationship between the UV absorption at 520nm against time for paracetamol suppository. Based on the graph, it shows decline in the absorption of UV. The flow maintain from 5 until 10mins and increasing at 15mins but decline at 20mins and increasing until 25mins. The flow maintain from 25 until 30mins and also from 35 until 40mins.  Until minutes 40 where there is a sudden increase in the absorption and sudden decrease at 45mins. Theoretically, the UV absorption of the suppository increases when the time increases, which means that the longer the time, the higher the amount of drugs passing through the membrane or the higher the bioavailability is.

As the experiment proceeds, the content in the dialysis bag becomes closer to isotonicity to the distilled water as some of the paracetamol has been moved to the distilled water. This will result in the reduced gradient of the graph when time passes by. If the experiment is continued beyond 60 minutes, it may result in a straight line graph as the concentration of paracetamol in the distilled water equals to the concentration of paracetamol at the dialysis bag.

In this practical, some precaution steps should be considered. The fluid in the beaker must be stirred first before pipetting out to ensure the equal distribution of drug released. Next, the pipette and the plastic cuvette need to be rinsed out using distilled water at each measurement of UV absorption. This is to make sure that there are no any left drug particles remained on the apparatus that may affect the result later. The rope should tie both end of the dialysis bag tightly to prevent leakage of the suppository that has miscible with water. We must also stir the distilled water before sample is taken out for measurements to ensure uniform distribution of the released active ingredient. Besides, the smooth surface of the cuvette should not face the source of UV light and the smooth surface should be cleaned before it is placed into the device.

4)      Plot graph time of UV absorption at 520mm of different formulation of suppositories. Compare and discuss the result.

			UV absorption
T I M E		GROUP	0	5		10			15	20		25			30	35		40	45	50	55	60
I	1 5		0.003 0.012		0.006 0.016		0.053 0.016	0.054 0.026			0.028 0.019		0.029 0.018	0.050 0.021			0.012 0.021	0.008 0.021	0.012 0.097	0.022 0.020	0.005 0.022	0.018 0.024
II	2 6		0.001 0.025		0.018 0.006		0.090 0.006	0.039 0.008			0.105 0.007		0.096 0.010	0.044 0.010			0.088 0.009	0.048 0.009	0.098 0.032	0.052 0.013	0.051 0.012	0.051 0.015
III	3 7		0.026 0.004		0.032 0.006		0.029 0.026	0.024 0.030			0.032 0.042		0.037 0.045	0.067 0.064			0.042 0.063	0.047 0.075	0.052 0.083	0.055 0.099	0.058 0.083	0.060 0.100
IV	4 8		0.060 0.004		0.075 0.005		0.080 0.007	0.084 0.005			0.087 0.006		0.091 0.006	0.093 0.006			0.095 0.009	0.094 0.007	0.102 0.007	0.106 0.009	0.107 0.012	0.109 0.014

Table 1 : Value of UV Absorption with times for all eight groups with different formulation of suppositories, types I, II, III and IV.

Average UV absorption at 520nm
Time(min)	0	5	10	15	20	25	30	35	40	45	50	55	60
I	0.0075	0.0110	0.0345	0.0400	0.0235	0.0235	0.0355	0.0165	0.0145	0.0545	0.0210	0.0135	0.0210
II	0.0130	0.0120	0.0480	0.0235	0.0560	0.0530	0.0270	0.0485	0.0285	0.0650	0.0325	0.0315	0.0330
III	0.0150	0.0190	0.0275	0.0270	0.0370	0.0410	0.0655	0.0525	0.0610	0.0675	0.0770	0.0705	0.0800
IV	0.0320	0.0400	0.0435	0.0445	0.0465	0.0485	0.0495	0.0520	0.0505	0.0545	0.0575	0.0595	0.0615

Table 2 : Value of average UV absorption with times for all four different formulation of suppositories, types I, II, III and IV.

From the graph, Suppository III with 3g PEG1000 and 6g PEG 600 shows the highest increases in UV absorption value compare to other three types of suppositories. Suppository I with 9g of PEG 1000 and Suppository II with 6g PEG 1000 and 3g PEG 6000 show fluctuation trend. At first, Suppository I shows slightly increases in absorption rate and at about 40min, it increase tremendously until 45mins. Suppository III shows obvious fluctuation. Besides, Suppository IV also shows fluctuation trend from the beginning at about 5 minutes and above. In addition, suppository IV has the slowest rate of drug released which accounts for its lowest UV absorption in this experiment. This is because of the high amount of PEG 6000 which contain less amount of hydroxyl group in its structure make it immiscible with water. Therefore the amount of paracetamol to diffuse out of the dialysis bag is smaller and resulted in the less present of paracetamol in the water bath when reading is taken. Besides, PEG 6000 has the characteristic of higher viscosity make it less available to miscible with water. This causes the drug to be difficult to diffuse out into the water. As increased of PEG molecular weight resulted in decreased solubility in water and solvent.

          In suppository formulation, PEG 1000 and PEG 6000 are polyethylene glycol polymer which are also mixture of two or more grades of macrogols and acts as water soluble base. PEG 1000 is fairly firm solid whereas PEG 6000 is wax-like polymer. They are chemically stable, nonirritating, miscible with water as well as mucous secretions, and can be formulated, either by molding or compression, in a wide range of hardness and melting point. They do not melt at body temperature, but dissolve to provide a more prolonged release.

          Presence of PEG will slow down the UV absorption rate. Based on the graph, it is shown that when the amount of PEG 6000 increases, the UV absorption value of the suppositories will decreases. But, increasing with time, the UV absorbance value will increase. Increase in molecular weight of PEG from 1000 to 6000 will cause an increase in viscosity. Thus less penetration of UV will be obtained for suppository with higher PEG molecular weight which is PEG 6000 compared to PEG 1000 as described in the absorbance value in suppository IV. However, in this case based on the graph, the best formulation of suppository with higher value of UV absorbance is suppository III with 3g of PEG 1000 and 6g of PEG 6000.

The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light passing through a sample, and compares it to the intensity of light before it passes through the sample. The ratio is called the transmittance, and is usually expressed as a percentage. The spectrophotometer measures the intensity of light reflected from a sample, and compares it to the intensity of light reflected from a reference material.There are several precaution steps that must be taken when using the UV spectrophotometer. Firstly, don't touch the optical surface of cuvettes with fingers to avoid erratic reading. Then, do not overfill or under fill the cuvette, the cuvette should be three-fourth full. Besides, preserve the instrument from dust particles. Moreover, never wash the cuvettes with chromic acid. Wash the cuvette with the distilled water and then with methanol before storage.

There are several errors occur during this experiment which make the result inaccurate. For example, stirring the distilled water in the beaker is not done in every step before taking the sample to be measured in UV-visible spectrometer. Moreover, environmental factors such as saliva may accidentally spill into the beaker affecting the solution leading to inaccurate measurement. Besides, it may be paracetamol powder is not homogenously dispersed in the suppository base and this will affect the absorption of UV light. Hence, all these errors should be avoided during experiment to get an accurate result.

 5) What is the function of each ingredients used in preparation of suppositories? How the different amount of PEG 1000 and PEG 6000 affect the physical characteristics of suppositories formulation and the released of drug from the formulation?

         In suppositories formulation, the ingredients been used are polyethylene glycol 1000, polyethylene glycol 6000 and paracetamol. Each ingredient has different function. Polyethylene Glycol is one of example of base used in suppositories formulation. Function of polyethylene glycol is water soluble/ water miscible bases that contained polyethylene glycol (PEG) polymers. PEG offers rapid delivery of the active pharmaceutical ingredient with minimal manufacturing issues. Paracetamol, is the active ingredient used in this formulations. Paracetamol used to relieve mild moderate pain such as headaches, migraine, toothache colds and flu. .PEG is available in wide range of molecular weight. Different ratios of low and high molecular weight of PEG can be altered to alter the hardness of the suppositories. Therefore, in this experiment, higher molecular weight of PEG, PEG 6000 will form more harder suppositories compared to lower molecular weight PEG, PEG 1000. Polyethylene glycol suppositories do not melt at body temperature but dissolve slowly in the body’s fluids. The base need not be formulated to melt at body temperature. Thus, we need to prepare suppositories from polyethylene glycol mixtures having melting points considerably higher than body temperature. This property permits a slower release of the medication from the base once the suppository has been inserted, and permits convenient storage of these suppositories without need for refrigeration and without danger of their softening excessively in warm weather. The higher the amount of PEG 1000 used , the suppositories formed are smooth, hard semi-solid, oily and even white in colour while the more the PEG 6000 is used will give smooth, wax-like, uneven white in colour andrelatively harder than PEG 1000 because PEG 6000 will give more solid products. Polyethylene glycol does not melt at body temperature, but dissolve to provide a more prolonged released drug. Drug release depends on the base dissolving rather than melting. The melting point is often around 50°C.  Higher proportions of high molecular weight PEG produce preparations which release the drug slowly and are also brittle.  Less brittle products which release the drug more readily can be prepared by mixing high PEG with medium and low polymers of PEG.

Conclusion
From the suppositories that we compound, there are torpedo in shape, smooth on the surface, hard to be pressed, and white in colour. This is because our group use higher amount of PEG1000 compared to PEG6000. So, the suppositories formed are smooth, hard and even white.
The suppository that contained 3g of PEG 6000 took about 49 minutes to melt at 37^oC. While, the absorption of UV at 520nm shown a slowly increase proportional to the time.

Reference

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iii.The Pharmaceutics and Compounding Laboratory. UNC. http://pharmlabs.unc.edu/labs/suppository/intro.htm

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