In this article we will discuss about the screening methods for thyroid and anti-thyroid drugs.
The thyroid gland consists of follicular cells which secrete thyroxin (T4) and triidothyronine (T3) and para follicular cells which secrete calcitonin respectively. The main biological effects of T3 and T4 are on growth and development, the calorigenic effect, cardiovascular function and metabolic functions. The primary feedback effect is inhibition of TSH-secretion. These effects can be used for testing thyroid hormone analogues and metabolites. Anti-thyroid drugs can inhibit the iodine uptake and utilisation in the thyroid.
Changes in Metabolic Rate:
Oxygen Consumption Rate:
This method is most commonly used for diagnostic procedures in man as well as for evaluation of thyroid hormones and their derivatives in animals (indirect Calorimetry). It works on the principle that thyroid hormones increase basal metabolic rate, oxygen consumption and carbon dioxide production.
The main requirements include 200 ml wide neck bottles, stop watches and albino mice. Mice are placed individually into 200 ml wide-necked bottles. Groups of 10 mice are used for each dose of test preparation or standard. The bottom of the bottles is covered with filter paper to soak up urine. The bottles are sealed, tilted to a 60° angle and rotated 5 times per min. in a special apparatus. The time when the mice start to undergo an asphyctic seizure is noted.
Immediately after observation of seizures, the mice are released for recovery. Due to the defined muscle work, the test drug increases the time it takes for the mice to start seizure. Average time to seizures is calculated for each dosage and dose-response curves are established. The main disadvantage of this method is that the mice may sometimes die due to convulsions.
Mouse Anoxia Method:
Resistance of rats to oxygen deficiency was decreased when they were treated with thyroid extracts. This increased sensitivity to anoxia forms the basis of an assay method in which mice was dosed with a graded amount of iodinated casein.
The main requirements for the experiment include a closed jar, thermometers, thyroxin and healthy adult albino mice.
20 healthy adult albino mice are used. They are divided into three groups: control, standard and test compound. The standard compound is thyroxine at a dose of 5, 10 and 20 µg per animal given in the form of a subcutaneous injection once daily on 3 alternate days; 2 or 3 days after the last injection they are placed in a closed jar of 1 litre capacity at 23°C and the survival time determined. The end point is taken as the last viable respiration just before convulsions start.
Carbon Dioxide Output:
This assay is based on the principle that thyroid extracts alter the carbon dioxide output of mice. Mice are kept for 24 h in a glass respiratory chamber through which a steady stream of carbon dioxide free air is passed. The carbon dioxide output of the mice is measured. The carbon dioxide in the atmosphere is sucked in KOH solution and the amount absorbed determined by titration. This causes the chamber to cool, so care should be taken that the temperature is maintained at 23°C.
The main requirements for this assay include closed chambers and healthy adult albino mice (16-22 g).
Healthy adult albino mice weighing (16-22 g) are taken and kept at a standard diet. After 8 days, the normal carbon dioxide outputs are determined for 5 consecutive days by placing the mice along with food in closed chambers at 23°C. After 3 weeks, animals are dosed with test substances. Daily doses are adjusted according to body weight. The carbon dioxide output is also determined.
If there is loss of weight of mice, carbon dioxide output is expressed in terms of body weight and if there is gain of weight it is expressed as body surface area.
Anti-Goitrogenic Activity:
Increased secretion of TSH induces thyroid enlargement and weight increase within a few days. In normal animals, the secretion of TSH by the pituitary is regulated by feedback from thyroid hormones.
The administration of goitrogenic compounds which block thyroid hormone synthesis and/or secretion reduces the concentrations of circulating T4/T3 and their pituitary effect, thus releasing TSH from its feedback inhibition. The TSH rise induces hyperplasia of the thyroid follicles as indicated by an increase in thyroid weight. Hyperplasia is prevented by injection of thyroxine or thyroid hormone analogs.
The main requirements include propyl thiouracil (0.1%), thyroxine (10-40 µg/kg) and healthy albino rats (150-180 g).
Healthy albino rats weighing 150-180 g are used in groups of 8-10 animals. 0.1% propyl thiouracil (PTU) is added to the food or to the drinking water or equivalent doses are given by gavage for 2 weeks. The rats are treated (subcutaneously or by gavage) with various doses of the test compound or the thyroxine standard (10-10 µg/kg). The PTU control group receives only PTU and saline injections.
The rats are sacrificed after 14 days; their thyroid glands are dissected out cleanly and weighed rapidly to avoid evaporation loss. The two- to three-fold increase of thyroid weight by PTU is reversed dose-dependently to normal values by thyroid-active substance. Dose-response curves are plotted for test compounds and the standard and activity ratios calculated.
Inhibition of Iodine Release:
In rats, the release of iodine-131 from the thyroid is inhibited by treatment with thyroxine and the degree of inhibition is related to the dose administered. This phenomenon was used to compare thyroid hormone derivatives with the standard thyroxine.
The main requirements include propylthiouracil, ether, iodine-131 and healthy albino rats.
Healthy albino rats weighing 180-240 g are fed a commercial laboratory chow with or without addition of 0.03% propyl thiouracil (reference compound for thyroid peroxidase inhibition). Food is withheld 8 h before and for 24 h after iodine-131 or iodine-125 is injected intraperitoneally at 25°C. Under light ether anaesthesia, radioactivity over the thyroid region of the neck is determined 40 h later.
This value is taken as zero time and all further counts made at 24 h intervals, are expressed as a percentage of zero time- counts after correction for physical decay of the isotope. After the reading at zero time, the diet is changed to a diet containing 0.03% propyl thiouracil and various doses of the test preparation or the standard are injected subcutaneously at 24 h intervals for a total of four doses. The daily loss of iodine-131 is inversely proportional to the dose of thyroid hormone
The percentage of zero time-counts after 96 h of iodine-131 remaining in the thyroid after the last of 4 doses is plotted against logarithm of the dose. From these dose-response curves, potency ratios are calculated.
Thyroidectomy:
Endocrinological experiments for pharmacological evaluation of thyroid hormones and analogues may be performed in thyroidectomised rats. The thyroid in rats consists of three lobes (left, median and right).
The main requirements include nembutal anaesthesia, surgical equipments and albino rats.
The rat is anaesthetised with nembutal and the legs fixed on a surgical table. The fur of the neck is removed with electric clippers and the area disinfected. A median skin incision of 2.0 cm length is made. On both sides of the neck, large salivary glands and maxillary lymph nodes are found. They are pushed aside, making visible the musculus hyoideus covering the trachea. This muscle is split in the mid-line. The isthmus of the thyroid connecting both lobes is located.
Anti-Thyroid Drugs:
Anti-thyroid drugs are characterised by their ability to interfere with synthesis, release and/or peripheral action of the thyroid hormone, thereby lowering the basal metabolic rate. Decrease of T4/T3 reduces thyroidal inhibition of the pituitary gland; TSH secretion increases and induces a goitrogenic response. This response is used for detecting anti-thyroid drugs.
Inhibition of Iodine Uptake into the Thyroid of Rats:
Propyl thiouracil (PTU) and a wide spectrum of drugs may inhibit thyroid hormone synthesis. Some of these drugs can be used for the treatment of thyrotoxicosis. As a consequence of thyroid peroxidase inhibition, the iodine uptake and content of the thyroid is decreased. This phenomenon is dose-dependent.
The main requirements include potassium iodide, thiouracil and immature albino rats. Groups of immature albino rats of age 26-28 days, weighing 40-45 g, are placed in metabolism cages. They are fed a normal diet, and potassium iodide is added to the drinking water. The test compounds or the reference standard are added in various concentrations to the diet over a period of ten days. The amount of compound that each rat receives is calculated from the total food consumption over 10 days and expressed as milligram daily per kilogram of body weight.
After 10 days of treatment, the rats are sacrificed and the thyroids dissected free from the adjacent tissues. The thyroid is weighed and iodine content determined. In daily doses between 0.1 and 10.0 mg/kg, thiouracil decreases the iodine content of the thyroid depending on the dose. Dose-response curves of test compounds are compared with those of the reference standard for calculation of potency ratios with confidence limits.
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