Studies On Polyquaternium-1and Its Application As Ophthalmic Preservatives

Eyes are the important sense organs for human. However, with the changes of our living habits and environment and with the popularity of paperless office, the number of patients with eye diseases such as asthenopia, keratoconjunctivitis and xerophthalmia increases every year. Then the safety of ophthalmic preparations is a question people always concern about for their direct treatment on the eyes. Most of the ophthalmic preparations in multiple dosage forms are protected against microbial contamination by means of preservatives.In recent years, there have been continuous reports about the ocular irritation and adverse toxic effects induced by preservatives. Current studies have showed that long-term use of ophthalmic preparations containing preservatives for the patients suffering from cataracts, dry eye as well as glaucoma may induce ocular surface changes such as tear film stability and cornea or conjunctiva inflammation, and even cause blindness. Developing gentle preservatives or preservative systems is therefore the key point and mainstream in the development of ophthalmic preparations.Polyquaternium-1(PQ-1) with higher antimicrobial effects and lower ocular irritation has been already used in contact lens care solutions as bactericide. However, there is little application of PQ-1in ophthalmic preparations and few relative literatures and researches in China at present. The research subject aims at studying the application of PQ-1in ophthalmic preparations. Firstly, we did the preliminary study of synthetic route for PQ-1for the purpose of commercial production in China. Secondly, the spectrophotometric method for determination of polyquaternium-1in ophthalmic preparations was established to control its quality. Thereafter, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of PQ-1through double broth dilution method. And the pilot study of the stability of PQ-1and its action mechanism was completed. Then the cytotoxicity of PQ-1to human corneal epithelial cells (HCEpiC) was performed. Finally, we prepared the eye drops preserved by PQ-1and determined its effective concentration range through preservative effectiveness tests (PE test).1Synthesis, structural characterization and molecular weight determination of polyquaternium-1Objective:To establish the synthetic route of PQ-1. Methods:PQ-1was synthesized through copolymerization using N, N, N, N-tetramethyl-2-butene-1,4-diamine (DA),1,4-dichloro-2-butene (DCB) and triethanolamine (TEA) as raw materials. The structure of target compound was characterized by nuclear magnetic resonance spectrometer (NMR) and Fourier transform infrared spectrometer (FT-IR). The molecular weight (MW) and Mv distribution were determined through gel permeation chromatography (GPC). Results:The13C-NMR,^-NMR and IR spectra showed that the structure of the synthetic was in accordance with that of the control sample. The Mw of the products was19432and the Mw/Mn was below3.0. Conclusion:The established synthetic route is simple and easy-operated, and the Mw and Mw distribution of the target compound meet the requirements of ophthalmic PQ-1.2Determination of polyquaternium-1in ophthalmic preparations by differential spectrophotometryObjective:To establish a convenient, fast and accurate method for determination of PQ-1in ophthalmic preparations. Methods:This method is based on a bathochromic shift of the trypan blue absorption which occurs when it is complexed with PQ-1. The contents of PQ-1were determinated by the differential spectrophotometry at wavelength681nm, and the reference solution was trypan blue.Results:The calibration curves showed good linearity in the range between5.00and15.00μg-mL-1(r=0.9996), and the average recovery rate was97.8%(n=9, RSD=0.87%). Conclusion:The established method is easy-operated, fast, low-cost and accurate, which can be applied for the quantitative determination of PQ-1in ophthalmic preparations.3Study on the bacteriostatic activity of polyquaternium-1Objective:To study the bacteriostatic activity and stability of PQ-1and to do preliminary research of the action mechanism. Methods:The MIC and MBC of PQ-1to E. coli, S. aureus, P. aeruginosa, C. albicans and A. niger were determined through double broth dilution method The effects of UV irradiation, temperature, pH and salt concentration on stability of PQ-1were also investigated. The influence of PQ-1on the permeability of bacterial cell membrane was studied through conductimetric method and transmission electron microscope (TEM) was used to observe the ultrastructure of the cells. Moreover, we also studied the effect of PQ-1on the growth curve of the microbes. Results:The MIC and MBC of PQ-1to the test microbes respectively were:E. coli.MLC16μg·mL-1, MBC16μg·mL-1, S. aureus MIC4μg·mL-1, MBC8μg·mL-1, P. aeruginosa MIC16μg·mL-1, MBC32μg·mL-1, C. albicans MIC8μg·mL-1, MBC8μg·mL-1,A. niger MIC64μg·mL-1, MBC128μg·mL-1.The bacteriostatic activity of PQ-1was not affected by UV irradiation and pH, but was decreased with the rise of temperature and salt concentration. The value of electric conductivity increased with the extension of the action time of PQ-1. By using the TEM, we found that after exposed to the PQ-1, the bacterial surface became rough and had some pellet substance coming out and clear folds and rupture were found on the cell walls. From the growth curve, PQ-1with MIC significantly inhibited the multiplication of the test microbes. Conclusion:PQ-1with low concentration showed strong bacteriostatic activity. It was relatively stable to UV irradiation and pH, but was heat-sensitive and easily deactivated in hyperosmotic salt solution. The action mechanism of PQ-1may be through increasing the permeability of bacterial cell which results in the leakage of electrolytes including K+in the cell.4Study on the cytotoxicity of polyquaternium-1to human corneal epithelial cells in vitroObjective:To investigate the cytotoxicity of PQ-1to cultured HCEpiC in vitro. Methods:The model of HCEpiC in vitro was established. HCEpiC were incubated with PQ-1solutions at various concentrations (0.01%,0.005%,0.001%,0.0005%) for a period of10min,25min,1h and2h respectively. Observation of cell morphology was made through inverted phase contrast microscope and the cell viability was determined by MTT assay and live/dead cell staining. The benzalkonium bromide and ethylparaben with commonly used concentration were chosen for comparison in the study. Results:(1) Morphologic changes:With the extension of exposure time, HCEpiC incubated with0.01%PQ-1,0.005%PQ-1and0.01%benzalkonium bromide became loose and round and cell breakage occurred gradually.0.03%ethylparaben didn’t affect the cell morphology within1h, but a few cells became loose and deciduous after being treated for2h. The HCEpiC cultured in0.001%PQ-1and0.0005%PQ-1were normal in morphosis.(2) MTT assay:The MTT assay showed that0.01%PQ-1,0.005%PQ-1,0.01%benzalkonium bromide and0.03%ethylparaben consistently and dramatically caused decreases in the viability of HCEpiC (P<0.05). However, there’s no statistical difference of cell viability between treated group of0.001%PQ-1or0.0005%PQ-1and the control group (P>0.05).(3) Live/Dead cell staining:Only a few live cells existed in the treated groups of0.01%PQ-1,0.005%PQ-1and0.01%benzalkonium bromide, while the number of live cells remained at a high level in the groups of0.001%PQ-1and0.0005%PQ-1.0.03%ethylparaben had no obvious effect on the cell activity within1h, but dead cells increased after being treated for2h. Conclusion:PQ-1with high concentration have some cytotoxic effect on HCEpiC, however, PQ-1with commonly used concentration is relatively less toxic than the other two preservatives, which demonstrates that PQ-1is quite an ideal preservative for ophthalmic preparations.5Feasibility research on the eye drops preserved by polyquaternium-1Objective:To investigate the compatibility between PQ-1and common ophthalmic excipients and to study the preservative application of PQ-1in eye drops. Methods:(1) The compatibility between PQ-1and common ophthalmic excipients such as phosphate buffer, borate buffer, sodium hyaluronate (SH), edetate disodium (EDTA-2Na), hypromellose (HPMC), polyvinylpyrrolidone-K30(PVP-K30), ethylparaben and Tween80, was studied through determining the clarity and light transmittance of the mixed solutions respectively.(2) The influence of the Mw and concentration of SH, the concentration of PQ-1and the pH value on the clarity of the SH-PQ-1solutions was evaluated.(3) The taurine eye drops preserved by0.0001%PQ-1and0.0001%PHMB was prepared, in which SH with relatively low Mw but up-to-standard and0.05%Tween80were added. The preservative effectiveness of the eye drops was tested.(4) The preliminary study of the applicative concentration range of PQ-1in compound sodium chloride eye drops was completed. Results:(1) PQ-1was compatible with the common ophthalmic exipients except SH. The solution became opalescent when PQ-1and SH were mixed together.(2) The clarity of the mixed solution of PQ-1and SH tended to be negtively proportional to the Mw and concentration of SH and to the concentration of PQ-1, but pH value (5-8) was demonstrated to have little influence on the clarity.(3) The preservative effectiveness of the taurine eye drops preserved by0.0001%PQ-1and0.0001%PHMB was proved to meet the evaluation standard and the character of the eye drops was qualified.(4) The preservative effectiveness of the compound sodium chloride eye drops would be up-to-standard when the concentration of PQ-1was not less than0.0006%. Then the applicable concentration range of PQ-1in the eye drops was preliminarily intended to be0.0006-0.0009%. Conclusion:PQ-1has the potential to be applied in the ophthalmic preparations as preservatives. However, the long-term stability of the prepared two kinds of eye drops and how to make PQ-1and SH compatible with each other needs further work in the future.