The Manufacture And Quality Control Of Pirenzepine Ophthalmic Solution And The Experimental Research Of Its Inhibiting Effect On Form-deprivation Myopia In The Guinea Pigs

Background: Myopia is a very common eye disorder. Nonselective Muscarinicreceptor antagonist, atropine, is believed to inhibit myopic progression and axialelongation. But its clinical application is restricted to a great extent because of sideeffects caused by its similar affinity to all five subtypes of M-receptor. Recent studieshave shown that selective M₁-receptor antagonist pirenzepine, which was injectedintravitreously or subconjunctivally, could inhibit myopic progression on chicken andmammalian model. But the invasive administration protocol does no good to thecompliance of patients. Therefore, a noninvasive form of medication is urgentlyneeded. In the present study, the quality control of pirenzepine ophthalmic solution isevaluated. So was its ocular tolerability, pharmacological effects, as well as itsinfluence on the expression of M₁ and M₄ subtype muscadnic receptors.PartⅠ【Purpose】To evaluate the impurity, stability, irritability and allergic effect of thepirenzepine ophthalmic solution in order to provide evidence for its clinical use.【Methods】High pressure liquid chromatography (HPLC) method was establishedto determining the impurity of three batches of tested samples. Another three batchesof 2ï¼…pirenzepine ophthalmic solution underwent accelerated stability examination at40℃and long-term stability examination at 25℃. In the irritability study, just thesecond part in other word, New Zealand rabbits were used and divided into 4 groups,each of which was administered 1ï¼…, 2ï¼…, 4ï¼…and 8ï¼…pirenzepine ophthalmic solutionin one eye and 0.9ï¼…saline in the other eye. An independent researcher observed thelids, conjunctiva, cornea and iris through the silp lamp and gave grads according toDraized standards. 4 weeks later, all eyes were finally enucleated for histo pathological examination to evaluate the possible toxic effects to ocular structures.As for the last part, the study on the allergic effect, the guinea pigs had their backshaved and pirenzepine ophthalmic solution was applied to the hair-cut area. Thedegree of edema and erythema in the tested area was evaluated. All researches wereperformed in a masked manner.【Results】The impurities of tested pirenzepine ophthalmic solutions were in therequired range. The long-term stability examination at 25℃indicated that The resultsof the other two parts manifested that 1ï¼…and 2ï¼…pirenzepine ophthalmic solution hasless irritancy and better tolerability to the ocular tissue compared with 4ï¼…and 8ï¼…pirenzepine ophthalmic solution. Histological examinations revealed no obviouslytoxic effects in the 1ï¼…and 2ï¼…pirenzepine-treated eyes. The ophthalmic solution isweakly allergenic.【Conclusion】2ï¼…pirenzepine ophthalmic solution is qualified as far as the labeledcontent and impurity are concerned. It is recommended that the storage time of 2ï¼…pirenzepine ophthalmic solution under room temperature was no more than 6 months.1ï¼…and 2ï¼…pirenzepine ophthalmic solution has better tolerability and is suitable forfurther exploration on its pharmacological effects.PartⅡ【Purpose】The aim of part is to study the efficacy of pirenzepine in preventingexperimentally induced form-deprivation myopia in the guinea pigs through topicaladministration, as well as to determine, whether pirenzepine influence the expressionof M₁ and M₄ subtype muscarinic receptor in the retina, choroids and sclera in themyopic model in order to explore the possible action site of pirenzepine.【Methods】Sixty-six guinea pigs underwent monocular deprivation with theireyelids sutured and were divided into 6 groups. 3 groups were treated withpirenzepine ophthalmic solutions whose concentrations were 1ï¼…, 2ï¼…and 4ï¼…respectively; 2 groups, with either atropine or saline; and the last group, left untreated.Data on ocular refraction, in vivo biometric measurements and wet eye weight were collected before and after the experiment. Another thirty-two guinea pigs weredivided into 4 groups and the animals in two groups underwent monocular deprivation(MD). One MD group and one open group were administered retrobulbar injectionwith 0.1ml pirenzepine solution (containing 5 mg pirenzepine hydrochloride) for 14days continuously, and the left two groups were injected 0.9ï¼…saline as control. Theexpression of M₁ and M₄ subtype receptor in the retina, choroids and posterior sclerawere detected with immunohistochemistry and the mRNA level of two receptors inthe three tissues were examined via RT-PCR.【Results】(1) Animals untreated or treated with saline produced-2.31D and-2.25of average axial myopia respectively. Those treated with 1ï¼…pirenzepine ophthalmicsolution produced relative myopia of-1.63D, and those under the treatment of 2ï¼…and 4ï¼…pirenzepine ophthalmic solution only developed a relative myopia of-0.89D and-0.70D (F=9.56, P＜0.05). The significant reduction in myopia in 2ï¼…and4ï¼…pirenzepine-treated animals was caused by significantly less vitreous chamberelongation and axial elongation of the deprived eyes (2ï¼…group: 0.009±0.052mm, 4±group: 0.006±0.078mm) when compared with untreated, saline-treated or 1ï¼…pirenzepine-treated guinea pigs (0.057±0.056mm, 0.064±0.053mm and0.033±0.035mm, respectively). The changes on wet eye weight of untreated and 0.9ï¼…saline treated group also had statistical significance. (2) The retinal pigmentepithelium, choroidal capillary endothelium and sclera of four groups are moderateimmunoreactive to the M₁ receptor antibody. The treatment of MD combined withsaline injection could significantly increase M₁ expression in sclera, choroids andretina as well (ANOVA, F=9.005, 7.26, 10.43). The mRNA level of M₁ receptor in thesclera, choroids and retina of MD-Saline group significantly increase, so did that inthe choroids and retina of MD-pirenzepine group. The choroidal capillaryendothelium and sclera were staining positive to M₄ receptor antibody. Thefluorescence density of retina in MD-pirenzepine group was significantly lower thanthat of the other three groups (ANOVA, F=5.156). The mRNA level of M₄ receptor inthe retina and sclera of MD-P group was significantly lower than that of the otherthree groups (ANOVA, F=12.33, 4.02 respectively).【Conclusion】Topical administration of the M₁-selective muscarinic antagonist, pirenzepine, prevents experimentally induced form-deprivation myopia in guinea pigsby inhibiting axial elongation without obvious damages to ocular tissues. In themammalial myopic model, the expression of Ml receptor rose more greatly than M₄receptor. Pirenzepine might act via M₁ receptor in preventing form-deprived myopia.The changes in M4 receptor expression might be the secondary effect ofpirenzepine.