Cholinergic Antagonist Homatropine: Validity of Schild Analysis - Lab Report Example

Laboratory Report: Cholinergic Antagonist Homatropine: Validity of Schild Analysis Summary: It is considered necessary in present times to continue with research into how certain chemicals act pharmacologically on parts of the human physiology. This laboratory report describes one such experiment with extracts from guinea pig ileum where the potential drug homatropine, a known antagonist for muscarinic type cholinergic receptors, is tested against the known cholinergic agonist acetylcholine for potency and effectiveness. Acetylcholine is used alone and in combination with homatropine, both being used in varying concentrations, to test how competitive they are in joint action. The results of Schild analysis reveal that the drug homatropine is a competitive antagonist for acetylcholine, both competing exclusively for the muscarinic type cholinergic receptors. The Schild plot slope derived has a perfect unity value leading strongly to this conclusion. This also reveals that both agonist and antagonist compete exclusively for the same subtype of receptors without either getting involved with any other subtypes. The report concludes that this research has been relatively successful and thinks that this is of some value as it is highly essential to use such research as this to disclose the particular nature of the drug so that its pharmacology can be successfully implemented. Contents: 1 Atropine and Homatropine 3-4 2. Research and Aim 4-5 3. Method 5-6 4. Results 6-7 5. Discussion/Conclusion 8-9 Appendix References 1 Atropine and Homatropine: Cholinergic receptor cell types are two – muscarinic and nicotinic. The muscarinic receptors, so called because of their response to muscarine, are to be found mainly in the post-ganglionic parasympathetic effector sites and also in the autonomic ganglion and adrenal medulla. In the latter parts of the nervous system they modulate nicotinic receptor mediated effects (Brown and Taylor, 1996, pp. 142-143). Atropine (dl-Hyoscyamine) and the semi-synthetic homatropine (isopto-atropine) are known muscarinic receptor anatagonists that are usually tertiary amines well able to be absorbed and infiltrate the central nervous system. Nevertheless, they can be converted into the quarternary form by addition of a methyl group to the nitrogen. In this form they are more potent as muscarinic blockers and have increased ganglionic blocking action. Permanently charged quarternary agents do not significantly intrude into the CNS and have limited action there (Brown and Taylor, 1996, pp. 149-150). The atropine/homatropine muscarinic receptor type antagonists have specific depolarization (late EPSP) action in autonomic ganglia. The molecular effects are stimulation of phospholipase C (PLC) with formation of inositol-1, 4, 5 triphosphate () and diacylglycerol (DAG) with increase in systolic (Lefkowitz et al, 1996, p. 119). The atropine/homatropine drug regime is used in ophthalmology for mydriatic and cycloplegic purposes. The drugs inactivate the action of acetylcholine at the choliergically activated muscles of the iris and the accommodative ciliary muscles of the lens. The inactivated muscles of the iris allow dilation of them pupils (mydriasis) and loss of accommodation (cycloplegia). The cycloplegic action is used for occlusion therapy as in the case of treating patients with mild amblyopia (PEDIG, 2002). Inclusion of drug dosage and dosable age and other criteria are eschewed here. 2. Research and Aim: This research experiment uses intact longitudinal muscle-myenteric plexus preparations of guinea pig ileum to determine competitive antagonism of the drug homatropine against the agonist acetlycholine at muscarinic type receptor sites within the cholinergic nerve system in the species ileum. It is expected that electrical stimulation of the contracted ileum will produce relaxation. Application of the agonist alone or a combination of the agonist and antagonist will reduce this relaxation indicating effectiveness of the agonist alone, in cases where it is used alone, or in competitive action against the antagonist (Hebeiss and Kilbinger, 1999). The degree of contraction, in each case, measured in millimeters, will produce indicators of the competitive action of the antagonist. The principal aim of this experimental research is to establish the mode of action and degree of potency of the cholinergic antogonist homatropine, specifically in action against a known agonist acetylcholine. It is necessary to study the selective activeness of particular antagonists as they form a very important part of the present day pharmacologist (Wyllie and Chen, 2007). It becomes necessary to know how such antagonists work on specific receptor sub-types and what role they can possibly play in normal and abnormal body function. It is from the study of their roles that the pharmacologist can devise schema for their potential application in modern drug therapy that often reduces risk to patients inflicted with particular disorders and diseases (Wyllie and Chen, 2007). In this particular research study the potential to study their roles in lieu of receptor sub-types or their action against whole spectrum of agonists is limited. Essentially, this study can produce pointers towards only one potential aim among the many usually associated with such research. This is the potency of the drug (homatropine) that can be safely applied to mitigate agonist (acetylcholine) action, when and if abnormal. 3. Method: As has already been mentioned in the research section just preceding this section, the guinea pig ileum musculature extract, in contracted form, is electrically stimulated to study potency of agonist (acetlycholine) action either alone or in combination with antagonist (homatropine). Electrical stimulation produces relaxation, measured in millimeters, denoted as response to agonist or agonist/antagonist action. The agonist (acetylcholine) or the agonist/antagonist (acetylcholine/homatropine) is taken in molar concentration. Schild analysis (Wyllie and Chen, 2007) is done on the dose/response parameters to determine competitive degree of antagonism. It is assumed that the same number of receptors are involved each time a different concentration of agonist or combination of agonist/antagonist is applied to the extract. This is to ensure that any difference in response is not due to involvement of variant numbers of receptors (Wyllie and Chen, 2007). In logarithmic form, the Schild equation for agonist (A) and antagonist (B) is: Log(r-1) = log[B] - log (Wyllie and Chen, 2007) Where, r (dose ratio) = ; = Dissociation constant of antagonist; B = Antagonist concentration in . (Wyllie and Chen, 2007) 4. Results: Table 1: Tabulated results of dosage (agonist alone and combination of agonist/antagonist) against response. Acetylcholine Concentration [Molar] ACH Alone ACH + Homatropine 3 x 10-9M ACH + Homatropine 1 x 10-8M ACH + Homatropine 3 x 10-8M 3 x 10-9 3 +/- 1 6 x 10-9 8 +/- 3 No response 3 x 10-8 28 +/- 6 10 +/- 4 No response 6 x 10-8 38 +/- 8 21 +/- 9 5 +/- 2 No response 3 x 10-7 55 +/- 5 43 +/- 7 27 +/- 9 10 +/- 4 6 x 10-7 56 +/- 5 52 +/- 3 38 +/- 7 20 +/- 7 3 x 10-6 ________ 55 +/- 4 53 +/- 6 43 +/- 6 6 x 10-6 ________ ________ 56 +/- 2 54 +/- 5 Note: The median values of all response data are taken. Graph 1: Dose-Response Curves Note: The median values of all response data are taken. Chart 2: Schild Plot Note: 1. All logarithmic functions in this paper are in natural logarithms. 2. Dissociation constant of antagonist is accepted as M as per tabulated data from Table 1. 5. Discussion/Conclusion: It is evident from the results that series of dose-response curves in Graph 1, with the first curve in the series being the control one with no antagonist action, demonstrates perfect right-ward movement. As antagonist is added in increased concentrations from the 2nd curve onwards in the series the peak, though remaining relatively above the 50mm mark, shifts rightwards indicating requirement of higher concentrations of the agonist acetylcholine to produce the same dissociative effect. This can be construed as competitive action between the agonist (acetylcholine) and the antagonist (homatropine) as they seek to engage with the same set of receptors (muscarinic subtype) (Lefkowitz et al, 1996, p. 119). As the antagonist concentration increases it is noticeable that it requires a higher concentration of agonist to achieve the dissociation level where no more responses can be elicited from the receptors (Wyllie and Chen, 2007). From Chart 2, it is evident that the Schild plot has a slope equal to unity and that the dosage ratio is uniformly increasing with the increase in concentration of the antagonist. Thus, in this case the competition between agonist and antagonist is uniform with uniform increase in the dosage ratio as increase in antagonist concentration forces increase in agonist concentration to fuel competition to engage with the same receptor subtypes. It is also indicative from Chart 2 that the value of coincides with the logarithmic value of (10) available as intercept on the X-axis. It is indicative here that, in this experiment, it is proved that acetylcholine and homatropine compete exclusively for the same subtypes of muscarinic cholinergic receptors. Otherwise, if there had been a variety of receptor subtypes that had the ability to engage either the agonist or antagonist the Schild plot slope would have a value other than unity (Wyllie and Chen, 2007). It is also concluded that the dissociation constant for the antagonist for any concentration of agonist is 10and this is borne out by the value of as X-axis intercept in Chart 2. The dissociation constant for acetylcholine working alone is 600 , as per data in Table 1. The report believes that the experiment has been successful in proving that homatropine is an able drug that can exclusively target acetylcholine action in the cholinergic neuronal processes. Appendix: Table 2: Log (ACh Conc. Responses (mm) Standardized. 0.477121 3 0 0 0 0.778151 8 0 0 0 1.477121 28 10 0 0 1.778151 38 21 5 0 2.477121 55 43 27 10 2.778151 56 52 38 20 3.477121 0 55 53 43 3.778151 0 0 56 54 Note: Concentration () values of agonist and antagonist are as per Table 1, Results Section. Table 3: Schild Plot data: Log(r-1) log(B) 1.0986 -1.204 2.3026 0 3.4012 1.0986 References: (PEDIG) The Pediatric Eye Disease Investigator Group, A Randomized Trial of Atropine vs. Patching for Treatment of Moderate Amblyopia in Children, Arch Ophthalmol, 2002, 120; 268-278. Brown, J.H. and Taylor, P., Muscarinic Receptor Agonists and Antagonists, In, Goodman and Gillmans The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) The McGraw-Hill Companies, Inc.,1996, pp. 142-150. Hebeiss, Katalina, and Kilbinger, Heinz, Cholinergic and GABAergic regulation of nitric oxide synthesis in guinea pig ileum, Am J Physiol, Gastrointest Liver Physiol, Vol. 276, Issue 4, G862-866, April, 1999. Lefkowitz, R.J, Hoffman, B.B and Taylor, P. Neurotransmission: The Autonomic and Somatic Motor Nervous Systems, In, Goodman and Gillmans The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, p119. Wyllie, DJA, and Chen, PE, Taking the Time to Study Competitive Antagonism, British Journal of Pharmacology, 2007, 150, 541-555. Read More