Studying in vitro release and biological assays of ophthalmic emulsions

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Intraocular Drug Delivery Barriers Intraocular drug delivery barriers mainly include tear film, cornea, conjunctiva, sclera, choroid, blood-retinal barrier, and blood-aqueous humor barrier.

Intraocular Drug Delivery Barriers Intraocular drug delivery barriers mainly include tear film, cornea, conjunctiva, sclera, choroid, blood-retinal barrier, and blood-aqueous humor barrier. The fast-flowing tears in the tear film can dilute and wash away the drugs placed on the surface of the eye; the particular sandwich structure of the cornea makes it the function of limiting the penetration of lipophilic and hydrophilic drug molecules at the same time; the fast-flowing blood can quickly clear the conjunctiva, episclera, and Drugs in the choroid tissue; while the blood-aqueous humor barrier and the blood-retinal barrier together constitute the barrier system between blood vessels and the entire ocular tissue, limiting the exchange of substances between blood vessels and ocular tissues. [1]

Solutions, suspensions, emulsions, gels, and ointments are common topical ophthalmic formulations. Traditional ophthalmic formulations often have low bioavailability. Most eye diseases are treated with topical medications. Because eye drops are cheap and easy to prepare, they have been the most commonly used dosage form for ocular administration. Due to the special physiological structure of the eye, after the drug solution is dripped into the conjunctival sac, it is continuously diluted by tears and quickly lost through the nasolacrimal duct, resulting in poor bioavailability. Improving preparations’ bioavailability in the eye is still a challenge for pharmaceutical workers. [2]

In addition to traditional ophthalmic preparations, some new ophthalmic carriers have emerged, including: nano micelles, nanoemulsions, liposomes, nanosuspensions, in situ gels, etc. Next, we will introduce the in vitro release of ophthalmic nanoemulsions, biological assay methods, and preparation processes, as well as the development platform of Medicilon ophthalmic preparations.

Medicilon’s ophthalmic drug preparation services cover four types: eye drops, injections, gels, and eye ointments. It has completed safety research on the preparations of various eye drops and vitreous injection new drugs and helped to obtain clinical approval.

Nanoemulsions are transparent, kinetically stable preparations, and the internal phase droplets are generally 20-200nm (the maximum limit can be up to 500nm).

Ophthalmic o/w nanoemulsions consist of a dispersed phase (oil), a continuous phase (water), and carefully selected surfactants and cosurfactants, which can lower the surface tension of the two immiscible phases of the nanoemulsion.

Microemulsion/nanoemulsion can be used for local drug delivery in the eye. The emulsion is composed of the oil phase, water phase, emulsifier, and co-emulsion agent, which can prolong the contact time between the drug and the corneal epithelial cells and promote the absorption of the drug by the cornea, sclera, or conjunctiva. At the same time, some auxiliary materials can also be added to improve the adhesion of the emulsion. [3]

Microemulsions can solubilize insoluble drugs, protect easily hydrolyzed drugs, and prolong the release time of water-soluble drugs. Ester-containing microemulsions can change the fluidity of biological membranes and have a strong ability to promote penetration. The particle size of the microemulsion is small and uniform, which is good for absorption and greatly improves the bioavailability; the preparation process is simple and can be sterilized by filtration; the viscosity is low. Compared with ordinary eye drops, microemulsion can significantly increase drug concentration, prolong drug ocular retention, increase corneal permeability, improve bioavailability, reduce administration times, and reduce adverse reactions. [4]

In Vitro Release of Ophthalmic Nanoemulsions

Compared with traditional dosage forms (eye drops), ophthalmic nanoemulsions can solubilize slightly soluble drugs, prolong the release of API, use lower drug doses to achieve therapeutic effects, and reduce systemic side effects. The in vitro release study of API from nanoemulsions can determine the release kinetics of drugs and provide preclinical data on the biodistribution and bioavailability of drugs in the eye. Sustained-release formulations can penetrate the drug into the deep layers of the ocular structure after administration. Biorelevant methods for testing in vitro release of ophthalmic products are still under development.

As there are no accepted pharmacopoeial standards in this field, in vitro drug release from these systems is currently being evaluated using various membrane diffusion techniques, including simple dialysis methods, dialysis methods using modified USP Type 4 or USP Type II devices, and Franz diffusion cells. Drug release from ophthalmic nanoemulsions is preferably tested using the USP II method. In this method, the formulation (0.5 mL) is placed in a dialysis bag and in a beaker with a receptor medium. Release tests are usually performed at 34±0.5°C or 37±0.5°C in 900 mL of pH 7.4 phosphate buffer, usually with the addition of 1% sodium lauryl sulfate (SLS) or pH 7.4 buffered saline (PBS ), the paddle speed was set at 50 rpm. The experiment was performed in triplicate (n=3) and lasted 6 hours. Samples were withdrawn at specified intervals and replenished with fresh buffer to maintain a constant media volume. The concentration of the active substance was determined using high-performance liquid chromatography (HPLC) or UV-Vis spectroscopy.

Ince, I.; Karasulu, E.; Ates, H.; Yavasoglu, A.; Kirilmaz, L. A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro and In Vivo Studies. J. Clin. Exp. Ophthalmol. 2015, 06 (02), 1−6.

Ophthalmic Nanoemulsion Biological Test

Research methods of ophthalmic nanoemulsions—the interaction of drugs with other components of nanoemulsions

Understanding the analysis of pharmaceutical nanoemulsions at the molecular level is extremely challenging because of the biphasic nature of the system (oil and water), the relatively low concentration of API in the formulation, and the fact that drug and surfactant molecules can exist in oil and water phases. System dynamics with the continuous exchange between them. Therefore, methods that are sensitive to local and long-range structural changes and interactions within nanoemulsions need to be applied. I will describe thermal and spectroscopic methods that can be used to probe the interactions between the functional components of nanoemulsions.

  1. Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry is a thermal analysis technique used to determine the difference between the heat transferred to a sample and the heat of a reference sample.

  1. Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FTIR) Spectroscopy

NMR spectroscopy is a powerful tool that enables the study of the structure, dynamics, and interactions between components of nanoemulsions at atomic resolution (i.e., APIs, oils, surfactants, and co-surfactants).

The research method of ophthalmic nanoemulsion—biological activity and toxicity test

  1. Evaluation of the antifungal activity of the preparation

The antifungal activity was evaluated using the test drug solution as a reference for nanoemulsions containing antifungal compounds.

  1. Cytotoxicity test

The cytotoxicity determination of nanoemulsion is significant for its safety in eyeball application.

  1. Chicken embryo chorioallantoic membrane test - hen egg test - chorioallantoic membrane (HET-CAM test)

The HET-CAM test is an alternative test to the Draize eye irritation test and can assess the toxicity level of nanoemulsion formulations.

Research methods of ophthalmic nanoemulsions - in vivo studies

Tissue irritation may manifest as excessive lacrimation, conjunctival injection, and corneal swelling or clouding. In vivo histological examination included examination of ocular structures (ie, cornea, conjunctiva, and iris) following administration of the test formulations. Although anatomical and physiological differences exist between species (such as blink frequency or the permeability of the ocular surface), albino rabbits (such as New Zealand rabbits) are most commonly used to evaluate the safety of ocularly administered drugs. The large corneal surface and conjunctival area allow easy observation of any changes occurring after the application of the formulation to the eye. In addition, the rabbit's iris does not contain pigment, allowing observation of capillaries. Post-administration of nanoemulsion formulations can be performed using the Draize test or its modified version, the Low Volume Ocular Test (LVET Test):

  1. Histological examination of eye tissue

Histological examination of rabbit eyes was performed to evaluate possible irritation and pathological changes following ocular application of the formulation. Evaluation was performed on ocular tissue sections fixed in paraffin. Swelling, hemorrhage, and other changes in the retinal epithelium, cornea, and ciliary body are the main indicators of irritation.

  1. Eye irritation test - Draize eye test, low volume eye test (LVET test)

The Draize eye irritation test is traditionally used to evaluate the potential irritation of pharmaceutical and cosmetic preparations to the rabbit eye.

  1. Pharmacodynamic and pharmacokinetic evaluation

To evaluate the pharmacokinetic parameters: Cmax, Tmax and AUC of the API in nanoemulsion form, the test formulation (or reference product) was administered to the conjunctival sac of rabbits, then the aqueous humor was drawn from the anterior chamber and the drug concentration was determined by HPLC . Ligório Fialho and da Silva Cunha applied this method to compare the pharmacokinetic parameters of dexamethasone microemulsions and a reference formulation.

  1. API biodistribution to the eye compartment

To determine the biodistribution of the drug in different regions of the eye and in the blood, Akhter et al. instilled a drop of cyclosporin A nanoemulsion into the eyes of albino rabbits at specified time intervals. Then, in the collected biological samples (i.e., aqueous humor, conjunctiva, eyeball cornea, and blood from the marginal ear vein), API concentrations were determined using high-performance liquid chromatography (UPLC) to assess the presence of cyclosporine A in aqueous humor. Distribution and structure of the eye.

Preparation technology of ophthalmic nanoemulsion

The self-emulsification method is most commonly used to prepare ophthalmic nanoemulsions. In this method, the aqueous phase is added portion-wise to a mixture of oil and preselected surfactants and co-surfactants with gentle agitation at room temperature. The preparation of nanoemulsions using the self-emulsification method proceeds in three stages: in the first stage a two-phase, homogeneous lipid phase consisting of oil and lipophilic surfactant in a water-miscible solvent, and a homogeneous lipid phase composed of water and hydrophilic surfactant The second phase composed of the agent. In the second step, an o/w emulsion is formed instantaneously during the addition of the lipid phase to the aqueous phase under continuous magnetic stirring, and finally, in the third step, the water-miscible solvent is removed by evaporation under reduced pressure.

Quality control of ophthalmic nanoemulsion

The quality control of ophthalmic preparations is based on pharmacopoeia standards and extended by internal product quality standards, which are essential to ensure product quality and are defined by the manufacturer. Including evaluation of pH value, osmolarity, viscosity, clarity, compatibility with eyes, sterility, etc. These indicators should be regularly tested not only as part of batch release, but also during the process (as intermediate stability data) and at the end of the stability study. Therefore, it is crucial to identify critical quality attributes (CQAs) that are directly related to product quality, efficacy and toxicity, as they should form the basis of product quality control. CQAs for ophthalmic nanoemulsions include not only process-related aspects such as particle size distribution, but also the purity and stability of the drug substance and key excipients after processing and during storage, as well as the sterility and safety of multi-dose products. Bacteriostatic efficiency. Additionally, industry quality control includes final product testing of nanoemulsions in containers. This means additional testing to assess packaging integrity, draw volume, interactions between nanoemulsion components (drug substance and excipients) and packaging materials, and weight change determination during storage.

Medicilon Ophthalmic Preparation Development Platform

The Medicilon Preparation Department can undertake the development of dosage forms, including ophthalmic liquid preparations and ophthalmic semi-solid preparations. Medicilon has solutions, suspensions, emulsions, gels, ointments, creams, and other technologies. platform. The completed project categories include eye drops 1, 2, and 4, all of which have been successfully declared and are currently undergoing clinical trials. At the same time, Medicilon can undertake preclinical research in ophthalmology. The ophthalmology platform has a special intraocular drug delivery technology and is equipped with an advanced ophthalmic surgery microscope. Animal species such as dogs, miniature pigs, and non-human primates achieve unique fine-grained dosing.

Medicilon Heidelberg laser ophthalmology diagnostic instrument SPECTRALIS®HRA + OCT

[1] Zhang Shanshan, Zhu Jing, Zhao Yongyue, Zhang Wei, Miao Zhenyu, Guo Jianjun, Bu Haizhi. Ophthalmic drug delivery technology and its research and development overview [J]. Chinese Journal of Clinical Pharmacology, 2015, 31(07): 580-583.

[2] Yang Long, Chen Lingyun, Wei Gang. Research progress of ophthalmic lipid nano-preparation [J]. Chinese Journal of Pharmaceutical Industry, 2016, 47(12): 1592-1599. DOI: 10.16522/j.cnki.cjph .2016.12.022.

[3]. Chen Zhi, Feng Yang, Zhu Ronggang. Research progress of ophthalmic pharmaceutical preparations [J]. Food and Drugs, 2012,14(05):213-216.

[4]. Jin Yiguang et al. Application of nanotechnology in drug delivery.