Preservatives in ophthalmic formulations and the urgent need for preservative-free formulation

Topical treatment of ocular diseases is mainly through eye drops. One of the short outcomes of eye drops is using preservatives, particularly for chronic diseases such as glaucoma and dry eye syndrome. Preservatives are chemical compounds that must be incorporated into an eye drop to preserve sterility during storage and use. Their chronic use may cause side effects to the eye, such as irritation, allergy, tear film instability, conjunctival inflammation, sub-conjunctival fibrosis and corneal surface impairment. Ophthalmologists and formulation scientists are moving towards preservative-free formulations. Strategies to manufacture preservative-free formulations are either through changing the container type of formulation or converting it into a solid dosage form. Preservative-free formulations were superior to traditional eye drops by minimizing side effects experienced by the patient.


Introduction
A preservative is a chemical substance added to a product such as food, cosmetics, paints or pharmaceutical preparations to prevent microbial growth during storage and preserve their quality throughout the storage period 1 .By focusing on preservatives in pharmaceutical preparations, it is essential to know that pharmaceuticals are substances manufactured for the benefit of humans to be used in diagnosing, preventing and treating diseases.Pharmaceutical products (in particular preparations with water) are likely to undergo what is called microbial spoilage, and this expression is used when these pharmaceutical products are contaminated with microbes.If a pharmaceutical product is contaminated internally or externally, infections may result and could lead to the loss of an organ or even death.The product must be preserved during storage using a preservative agent to avoid this condition. 2Pharmaceutical products are manufactured in different dosage forms.Each of them has its physical status, whether it is liquid, solid or semisolid.Each of these preparations requires a preservative agent, especially those with higher liquid content and those in which sterility is essential for their use.Preservative antimicrobial efficacy comes from their ability to kill live cells.By that, their toxicity component is a detectable feature not only on the microorganism but on all living cells, including human cells in direct contact with them.It may lead to serious health problems.Preservative-related problems include hypersensitivity, allergic reactions, asthma, and neuron damage, possibly leading to cancer if inappropriately used.According to USP-NF, preservatives are toxic substances and must be low in the final formula to be considered safe.It is essential to know that the quantity of preservative agent relates to the type and nature of active ingredient 3 .

Materials and methods
The ideal properties of preservatives 4 : 1.It does not irritate body tissues when used in the required concentration.2. Must be effective at low concentration 3. Physically and chemically stable to maintain its activity throughout product manufacturing, storage and use.4. Biocompatible with another ingredient in the formula and the product container.5.They have good antimicrobial activity and should exert a broad spectrum of activity against bacteria, whether gram+ve or gram-ve, mold, yeast and other microorganisms.6. Unexpansive.7. Must have the property of high solubility in water and specific buffer solutions.8. Acceptable taste, odor, color and stinging.

Classification of Preservatives:
Preservatives are classified either according to their nature or according to their mechanism of action.There are two main classifications of preservatives according to their nature.
• Natural preservatives are obtained from natural sources such as plants, animals, minerals and microbes, for example, phenolic compounds, essential oils and flavonoids.They are widely used because they do not have broad-spectrum activity and are inactive against the most threatening microorganisms.Also, they must be used in high concentrations to be effective, which may lead to side effects.The solid or foul odor is another obstacle to using natural preservatives, especially in skin products.There is also a possibility of losing their function during manufacturing 5,6 .• Artificial preservatives, which are also called chemical preservatives, are chemically synthesized.They are active in just small concentrations against various microbes.An example of chemical preservatives is sodium benzoate and sorbate (5).Synthetic preservatives are preferred over natural ones due to their higher stability to remain fresh during the long manufacturing, storage and use period.
Although the process of using preservatives extends the shelf life of the products and help keep them free from microorganism, they are considered unhealthy and may lead to minor or severe health problem.There are three main classifications of preservatives according to its mechanism of action: 1-Antimicrobials exert their effect by killing or inhibiting the growth of microorganisms by altering cell membrane permeability and lysing cytoplasmic contents.They are either antibacterial or antifungal.Examples of antibacterial preservatives include quaternary ammonium salts, alcohols, phenols, mercurial and biguanidinesrt.At the same time, benzoic and ascorbic acids and their salts, phenolic compounds such as methyl, ethyl, propyl and butyl p-hydroxybenzoate (parabens) are considered antifungal preservatives.They are both equally important and are frequently used in combination to reach the desirable preservative action for the products 7 .2-Antioxidants: they act by preventing the oxidation of active pharmaceutical products to protect them against degradation due to oxidation; they are classified into three groups: true antioxidants, reducing agents and chelating agents.While both antioxidant and reducing agents interact with free radicals to block the chain reaction and inhibit oxidation, chelating agents form a complex with pharmaceutical ingredients and prevent the degradation of pharmaceutical formulation.In other words, they exhibit a synergistic effect on antioxidants by improving the action of antioxidant preservatives by reacting with heavy metal ions, which catalyze oxidation.Examples of antioxidants are alkygallates, butylated hydroxyanisol, butylated hydroxytoluene, nordihydroguaiaretic acid and tocopherols.While an example of a reducing agent used is ascorbic acid and the potassium and sodium salts of sulfurous acid, an example of a chelating agent used is ethylene diamine tetra acetic acid (EDTA), Polyphosphates and citric acid 8 .3-The ionic buffer system considered a new option for a preservation system, is called SofZia, a combination of multiple components that work together at synergism to give the preservative action.Those include Borate, sorbitol, propylene glycol and zinc 9 .

Preservatives in ophthalmic formulation
Different pharmaceutical products and formulations use preservatives, including oral, parenteral topical and ophthalmic preparations.According to the USP Pharmaceutical Dosage Form, preservatives are required due to their antimicrobial action in most dosage forms containing water and non-aqueous ophthalmic ointments.Examples of dosage forms that require preservation are solutions, emulsions, gels, drops, sprays, inhalation aerosols and creams.Ocular diseases such as inflammation, infections, glaucoma, dry eye syndrome and cataracts often require topical medications as a treatment, mainly in the form of eye drops.Eye drops are usually prepared in multi-dose containers and require sterility during storage and use due to the wide range of infections that may occur to the eye due to the contaminations of ocular solutions.It is an obligation to use preservatives along with the other formulation ingredients, which was mandated by the US FDA 10 .
In addition to the previously mentioned properties of ideal preservatives, preservatives used in ophthalmic formulations should not affect the tonicity and pH of the eye and cause minimum irritation when used.In addition, they must exert their effect with minimum concentration without affecting the ocular tissues 11 .
For multi-dose ophthalmic preparations, the classes of preservative use are listed in Table 1.Some of the widely used preservatives for ocular delivery include Benzalkonium chloride (BAK), Polyquaterium-1, Cetrimonium chloride, EDTA, Sodium Perborate, SOS, Thimoresal, Chlorobutanol and SofZia.

Benzalkonium Chloride (BAK)
A group of cationic surfactants with high affinity for the microbial membrane as it attaches to the membrane of the microbial agent and disrupts, making them an effective antimicrobial agent Cetrimide Polyquaternium-1 Polyquaternium-42 Benzododecinium bromide(BDB) Cetrimonium chloride Steartrimonium chloride

Mercury derivatives
Behentrimonium chloride Those preservatives contain mercury in their structure, known for their germicidal activity from ancient times and their ability to penetrate the cornea, so it was used as a preservative in ophthalmic preparation in a deficient concentration.It has been 30 years since chlorhexidine started to be used as a preservative agent for ophthalmic drops.In addition, a new study confirms that 0.2% chlorhexidine is an effective sequential combination, especially in fungal infection treatment.However, there are problems with the compatibility problems of chlorhexidine, and it needs to be more satisfactory in a uniform manner with all ophthalmic solutions, making it the less-used preservative day.

Chlorhexidine
Polyhexamethylene biguanide hydrochloride (PHMB) polyaminopropylbiguanides Alcohols polyhexamethylbiguanide The mechanism of action of this class is related to its antioxidant effect gathered with its stabilizing ability on lens membrane integrity and a stimulating effect on Na-K ATPase and membrane sodium pump.These compounds have excellent lipophilic and hydrophilic solubility that give them the privilege to be used in topical eye preparation, as the lipophilic behavior is the factor that controls the trans-corneal penetration of the drug.

Benzyl alcohol
Those came in a homogeneous series of paraben with antimicrobial effects, convenient against molds and yeasts.However, they may be considered irritable to the eye and cause epithelium cell damage even in a minimum concentration that is effective against microorganisms and suitable for preservatives.This group has an antimicrobial effect.A type of combination preserving system, it has a synergistic antimicrobial effect compared to each preservative alone.

Sofzia, Multiple component
Borate, sorbitol, propylene glycol, zinc SofZia is a component preservative system using the ionic buffer process to give its preservative action; it is one of a kind type.
Benzalkonium chloride BAK is a quaternary ammonium compound and one of the most used preservatives in ocular preparations, especially drops.It contributes to the preservation of approximately 70% of all the ocular formulations in the market, and its action is related to its surface active structure.Currently, the use of BAK is declining due to several disadvantages noticed over the years.BAK has deposition properties in ocular tissue, such as trabecular meshwork, corneal endothelium, lens, and retina after topical drop installation.This may lead to corneal damage, especially with chronic use or when used in high doses.According to a previous study comparing the toxicity of BKA to other preservatives, BAK with 0.01 % (w/v) had higher toxicity than most of the other preservatives 19 .The classification of toxicity was thiomersal (0.01%)>BAK (0.01%)>chlorobutanol (0.5%)>methyl paraben (0.01%)>sodium perborate (0.02%)>ethylene diamine tetra acetic acid (EDTA).Some of the problems associated with the use of BAK includes: • Inflammation and allergic reaction of the surface of the eye • Fibrosis of the sub conjunctiva • Causes dry eye and exacerbation of dry eye condition due to tear film instability • Necrosis, cellular apoptosis, and endothelial cell destruction may even reach the need for corneal transplantation in patients who already suffer from kerastoconjectivites sicca.• Impairment of the corneal surface • Failure of filtering surgery for a patient with glaucoma after chronic use of topical formulation with BAK as a preservative.
A study that compared the use of BAK eye drops with the preservative-free formulation in vitro using human conjunctival goblet cells concluded that the formulation with BAK caused a decrease in the goblet cells' density compared with the preservative-free formulation.Also, the study found that the effect of BAK on the goblet cells is time and concentration-dependent.The use of BAK in higher concentrations or extended periods is linked to ocular surface damage 20 .Another study was focused on the safety and efficacy of antiglaucoma medication, 0.5% timolol, and eye drops, as the study showed the benefit of switching patients with glaucoma from BAK-preserved Timolol drops to the preservative-free formulation.The theory was that BAK helps penetration of Timolol into the cornea and contributes to the efficacy enhancement of Timol eye drops.This study proved that the preservative-free formulation is equally effective in lowering intraocular pressure compared to the formulation with BAK 21 .

Polyquaternium-1(polyquad):
It is one of the quaternary ammonium preservatives in artificial tears.When applied, it has a superficial effect on epithelial cells, so it is considered less toxic than BAK.However, it reduces the density of goblet cells in the conjunctiva, reducing the production of aqueous tear film 22 .

Cetrimonium chloride:
It is one of the most widely used detergent-type preservative agents.It has tremendous antiseptic properties but has the disadvantage of causing inflammation and keratinization of the eye's limbus, conjunctival stroma and epithelium 10 .

EDTA (Mercury derivative compound)
Disodium-ethylene diamine tetra-acetate is a type of mercury derivative, and its preservative activity is due to its ability to chelate and deactivate heavy metals.It is also used in ophthalmic preparations as a buffering agent to manage the sensitization of the eye to external chemicals or changes in the pH.Long-term use and repeated administration may damage the ocular tissue cells.Another example of a mercury derivative preservative is thimerosal.It was previously used due to its good antimicrobial properties, but currently, it was cast away from approximately all ocular preparations due to its toxicity.One is the drastic toxic effect and damage to the nervous system, and it causes cell retraction and cessation of mitotic activity. 19.

Phenyl mercuric nitrate or acetate:
Polymercuric nitrate, followed by polymercuric acetate to a lesser extent, is used as a preservative in eye drops.It is characterized by its stability, no irritation and wide range of activity against bacteria and fungi.However, it has a problem of being slow in its action, especially against severe inoculation with Pseudomonas aeruginosa, incompatibility with halides, robust absorption by rubber, and its ability to cause mercurialities 23 .

Stabilized Oxychloro Complex SOS (Purite):
It is the type of oxidative complex or what is called soft preservative.It consists of 99.5% chlorite (ClO2), 0.5% chlorate (ClO3), and trace amounts of chlorine dioxide (ClO2) as a mixture at equilibrium.It has a good effect as a bactericidal, fungicidal, and even virucidal, with a good safety profile compared to other preservatives.It has the property of converting to the standard component of the tears, such as sodium ions, chloride ions, oxygen, and water, so it has a perfect histopathological effect on the cornea's epithelium.It may be considered one of the best preservatives used in ophthalmic preparations 23 .

Sodium Perborate:
It is considered the first oxidative preservative used in ocular formulations, especially eye lubrication drops.Once applied to the eye, it interacts with water and is converted to oxygen and catalase, producing hydrogen peroxide as a byproduct.
The antimicrobial effect is related to the effect of hydrogen peroxide.It is considered safe and gentle compared to other well-known preservatives such as BAK.
The disadvantages encountered with its use are the destruction of the precorneal tear film and the destabilization of the membrane and cell wall.They may also be considered a direct cause of mutagenesis when used in a large concentration or chronically used 23 .

Chlorhexidine acetate (CHA):
It is a biguanide preservative and has been used for ophthalmic preparations at a concentration of 0.005 % (w/v).It has a wide range of activity against gram-positive and harmful bacteria but low activity against spores and some types of bacteria, especially Proteus and Pseudomonas species.It is known to be less irritant with low toxicity.It is highly active in solutions with neutral or slightly alkaline pH, and its activity is reduced when combined with organic matter such as serum and phospholipid.There is an incompatibility between the CHA and anionic compounds, and when used in high concentrations, it is irritant to the mucous membranes 19,23 .

Chlorobutanol:
It is one of the detergent types of preservatives with broad-spectrum antimicrobial activity that acts by denaturing the microbial cell membrane.The denaturing effect is not limited to the microbes, but it also affects the cells of the ocular tissue, which is responsible for the toxic effect of chlorobutanol.The side effects include irritation to the eye, retraction of the cells of the ocular tissue, impairment of normal cytokinesis and the movement of the cell and impaired mitotic activity.In addition, it obstructs the properties of the corneal epithelial, such as transport and barrier activity, and inhibits the imposition of oxygen by the cornea.In addition to its effect on ocular tissues, chlorobutanol is not considered stable when stored at room temperature 19 .

Methyl paraben and propyl paraben:
They are a parabens type of preservatives that are recommended to be combined for synergist effect.Parabens have a distinct wide range of antimicrobial activity with low toxicity.When used separately, a higher concentration is required, which may cause irritation and toxicity to the eye.Although the use of parabens may cause epithelial cell destruction, it is considered safer compared to other preservatives such as Thi, BAK and CBL 24 .

Sorbate (Sorbic acid):
It is one of the most widely used carboxylic acid types of preservative.It is used in a concentration between (0.1-0.2) % (w/v).It has the advantage of being safe with lower effects on epithelium than other preservatives when used in low concentrations.However, an investigation demonstrated that at high concentrations, it affects the epithelial cells of the eye, reducing movement and preventing mitotic activity, but no cell death was observed.The main problem with sorbate is the restricted effect against microbes and the difficulty of removing microbial agents alone 25 .

N-hydroxy-methyl-glycinate / disodium-ethylene diamine tetra-acetate (NIG/EDTA):
It is a new type of preservative that came from combining two preservatives NIG 0.002% with EDTA 0.1%.The combination is characterized by safety and low toxicity on the corneal cells of the eye compared to each type alone.Also, it has a synergistic antimicrobial effect compared to each preservative alone.The side effects encountered with the combination depend on the concentration or the recurrence of use 26 .

Sofia:
It is a new ionic buffer system for preserving ophthalmic preparations composed of Borate, sorbitol, propylene glycol and zinc.It is safe when used because it is converted to elements that are present in the tear film, so it is considered gentle and non-irritating with a low cytotoxic effect on ocular surfaces.Although it is considered safer than conventional preservatives, SofZia is a less effective antimicrobial agent.However, it has some effects due to the summative effect of its components, like zinc 27 .A study comparing BAK with SofZia concluded that the protection from microbial contamination provided by BAK is more effective than that offered by SofZia 28 .

Result
Preservative-free formulation necessity: All the currently used preservatives in the clinic have a negative impact on ocular tissues, such as irritation, allergy, tear film instability, conjunctival inflammation, subconjunctival fibrosis and corneal surface impairment.This is particularly evident when eye drops treat chronic conditions such as glaucoma 23,29 .

Containers
Pharmaceutical companies are developing special containers with specific requirements to avoid contamination of the formulation during storage and use.In standard multidose containers, the formulation is squeezed to deliver the drops, and when squeezing, removed air is drawn back inside the bottle to replace the dispensed liquid.The air drawn back is usually contaminated.Contamination through the drawback air must be prevented for multidose containers to be used without preservatives.To overcome the problem, several strategies are used, such as backflow prevention, an airless system that prevents air from entering the container during use, a filter for bacterial prevention and dose control dispensers.Some of the currently used techniques will be discussed.
Ophthalmic Squeeze Dispenser (OSD) 37 The ophthalmic squeeze dispenser (OSD) patented Tip-Seal mechanism by Aptar Pharma.The OSD Tip-Seal technology includes a sealing membrane, spring mechanism, and microbiological filter to protect the formulation from bacterial ingress through flowing air Figure 1.Upon application, the eye drop bottle will be squeezed, and the pressure inside the system will be increased, allowing the formulation to enter the delivery channels.The pressure will build up until the Tipsealing Seal's membrane opens, allowing the drop to escape.Once the drop is released, the Tip-Seal closes, and any incoming air is sterilely filtered through the 0.2 µm filter membrane.An example of this system in the market is Restasis® MultiDose, which contains cyclosporine eye emulsion.

Continuous Mono Dose system (COMOD system) 38
The Continuous mono dose system COMOD, patented by URSAPHARM, is a new system developed to eliminate the use of preservatives through an entirely airless application.The liquid is placed within a flexible, airtight pouch in the container without contact with the surrounding air.During use, when the particular pump system is activated, the air drifts into the space between the pouch and the container to balance the pressure.A negative pressure is created, which causes the inner pouch to contract and remain closed to avoid contamination.The pumping process will compress the liquid in the solution container.A ball valve, spring and piston seal the outlet valve tightly.Rapid opening delivers a fixed amount of the product.
The valve closes immediately afterward to prevent the backflow effect, as shown in Figure 2.An example of this system in the market is HYLO-COMOD, which contains sodium hyaluronate.

ABAK Bottle
The ABAK® is a dispenser system that delivers eye drops without contamination and can be used for 3 months after opening without preservatives.This innovative dispenser is composed of complex and precise parts.The container's flexible walls are made of low-density additives-free polyethylene.The dispenser comprises a locking system with a temper-evident ring and a neutral microporous pad regulating air and liquid flow.A patented surface treatment renders the neutral microporous hydrophilic polymer membrane partially hydrophobic.The ABAK system is completed with rounded protective tips.Once the drop is expelled, the membrane filters the residual drop and the error to compensate for the depression 39 .An example of this container in the market is the Hyabak ® , a uniquely formulated hypotonic drop that contains Hyaluronic acid and Actinoquinol.

Aero Pump 3K system
It consists of three parts: a filter matrix, valve, and coil of oligodynamic effect, as in Figure 4.The container preservative-free formulation is separated from the orifice using the valve.During use, the pump within the system is activated, creating a negative pressure on the product inside the dosing chamber.The created pressure will open the valve, and a drop will be released.Once the dosing process is complete, the system will be sealed by a microbiological valve that prevents leaking and backflow of the product.Then, the pump will return to its starting position, and the aliquot will be withdrawn to the dosing chamber.The subsequent air inflow will pass through a special filter to compensate for the vacuum within the container.
Furthermore, an antimicrobial silver coil at the orifice prevents microbial growth in that semi-open area through an oligodynamic effect.The oligodynamic effect means that heavy metals in small amounts will have an antimicrobial effect on the formulation 40 .Examples of this system in the market include is • Duokopt ®, which contains Dorzolamide and Timolol.

Novelia -Multi-Dose Closing Tip System
It uses a similar technology as the Ophthalmic Squeeze Dispenser but with some crucial differences.It features a silicone tube-based valve mechanism named Pure-Flow™ Technology, and the container is vented via air diffusion through a silicone membrane.Pure Flow technology means that the container has a one-way valve to prevent the solution from being reintroduced into the container, thus preventing contamination of the liquid in the container 42 .To ensure microbial integrity, silver is added to the plastic material of the actuator, protection cap, and silicone valve for the design currently available, as shown in Figure 6.An example of this container in the market is: • LIKETERES® contain Carboxymethyl cellulose, Lecithin and amino acid.

Airless Antibacterial Dispensing System(AADS)
To dispense a drop from the new container, the patient clicks the bottle's base rather than squeezing the sides, causing the liquid to compress.The AADS systems have multiple valves and airless pumps that work together to dispense a single drop.An example of this dispenser in the market is Visine®Pure Tears.

Bottle squeezing Valve opening
Valve reclosing

Single dose packaging of eye drops:
Standard eye drops are usually in multidose containers.When the container opens for use, the possibility of contamination increases, requiring preservatives.To avoid the problem, single-dose containers were developed.Each dose is packaged in a single plastic container to be used and discarded, as in Figure 7.  Preservative-free solid ocular drug delivery system: They are solid dosage forms to be used in the eye without preservatives.They are manufactured in different shapes and sizes.Because solid dosage forms are stable, no preservative is needed.Different dosage forms were investigated, such as solid eye drops (Minitablets), minidiscs, inserts and collagen inserts 46 .

Solid eye drop (Minitablets):
It is a biodegradable solid dosage form in the shape of a minitablet with a diameter of less than 3 mm.They can be manufactured for a single dose (fast disintegrated) or sustained release.Once placed in the eye, it can be dissolved immediately to form a solution or converted to a gel form and can be used for both hydrophilic and hydrophobic drugs.Table 3 represents some of the investigated minitablets for ocular delivery.

Ocular solid inserts 69 :
Ocusert® is an example of a solid insert that releases a drug from a reservoir through the microporous membrane at a constant rate.Ocusert® pilo-20 and Ocus-ert® pilo-40 were the first FDA-approved ocusert for pilocarpine delivery to treat glaucoma.They release the drug at a constant rate and provide a near-constant concentration in ocular tissues for 7 days, and they are preservative-free.The reservoir is fabricated from alginic acid pilocarpine, surrounded by an ethylene vinyl acetate rate-controlling membrane, and impregnated with titanium dioxide.An annular ring for visibility in the insertion and removal of the insert surrounds the reservoir.
Another example of FDA approved preservative-free insert is Artificial Tear Insert/ LACRISERT®.The insert is 3.5 mm long with a 1.27 mm diameter and contains 5 mg of hydroxy propyl cellulose.It is a preservative, and once placed in the cul de sac, it swells in ocular fluids, forming a hydrophilic layer that stabilizes the tear film and keeps the cornea moist until it completely dissolves within 14-18 hours after application.Another preservative-free solid insert that FDA approves is Dextenza®.It is a 0.4 mg dexamethasone insert intended to be placed in the punctum of the eye to release dexamethasone for one month to treat pain and inflammation after ocular surgery.Another widely investigated type is collagen corneal inserts (shields).Because of collagen biodegradability, biocompatibility, low antigenicity, and well-known safety profile, collagen has become a handy carrier for delivering various drugs and agents.They are shaped in the form of contact lenses and stored dry and hydrated before use, so no preservative is needed for their manufacture.
Another system under development is the New Ophthalmic Delivery System (NODS).It consists of a solidified paper handle and a flag made of polyvinyl alcohol that contains the active ingredient and is attached to the handle with a soluble membrane.Compared to conventional eye drops, this system ensures the delivery of a specific drug dose to the eyeball and increases the bioavailability of the active ingredient without preservatives.

Point of view in preservative-free formulations
Despite their disadvantages, eye drops are the primary dosage for treating eye diseases.However, their chronic use is problematic and needs to improve treatment adherence, mainly due to preservatives.Preservative-free formulations can solve the problem, with many pharmaceutical companies moving in that direction.However, their use still needs to be improved; part of the problem is cost and difficulty.Containers as a single unit for preservative-free formulations are complex in design and use expensive mechanisms to ensure sterility during use.However, using single-unit dose containers is expensive and unfriendly to the environment because of the use of the plastic container for each dose.A unique applicator or packaging will be required for a solid dosage form, which may increase the cost of the drop.Since it is a chronic condition, many patients still prefer traditional eye drops due to ease of application and cost.The next step will be innovating methods for lowering the cost of preservative-free formulations.

Discussion
A survey done by 249 ophthalmologists in 1999 that included 4107 patients with glaucoma concluded that the ocular symptoms that appeared in patients on medication with preservatives were more predominant than in patients on medication without preservatives 30 .A study reported by Jaenen et al. in 2007 demonstrated that the incidence of ocular signs and symptoms was significantly (p < 0.0001) higher in patients receiving preserved eye drops and that the incidence of these signs and symptoms significantly (p < 0.0001) by switching to a preservative-free formulation or by reducing the amount of preservative containing treatment 31 .
A study done in 2002 based on a survey done by ophthalmologists in their private clinics among 4107 glaucoma patients who were following topical medication for glaucoma, the majority of which was preserved, and then those patients were assisted for the clinical signs and symptoms in the ocular tissue and if there is any sign of ocular side effect or toxicity and the result of the survey verify that the detected ratio of patient with ocular toxicity was high in those patients how were on preserved glaucoma medications 32 .
A survey was done in Italy, Belgium, and Portugal on 9,658 patients who also use glaucoma topical eye treatment, and 30 and 50% of Patients were detected with dry eye ocular symptom s33 .In the US, a survey of patients who have one, two or three eye drops and the relation of the number of treatments with the side effects or prevalence of dry eye syndrome in patients on glaucoma medication confirmed that the percentage of patients suffering from dry eye increase by increasing the number of eye drops 34 .
A study included two groups of patients.Each group was on either preserved or preservative-free latanoprost topical medication.Then the following points were assisted, which include the corneal/conjunctival staining score, Ocular Surface Disease Index (OSDI, Allergan, Inc., Irvine, CA, USA) score, hyperemia score, tear break-up time (TBUT), adherence, and drug tolerance and according to the result that was gathered from this study it proved that patient on preservative-free latanoprost where The stinging/burning sensation was less severe and lasted less time in the preservative-free group than in the preserved group.Overall, preservative-free latanoprost demonstrated better ocular tolerance as measured by hyperemia and stinging/burning symptoms after higher adherence than preserved latanoprost 35 .
During the last 20 years, the term preservative-free formulation has progressed among ophthalmologists and formulation scientists.Eliminating preservatives in eye formulations, particularly eye drops, can enhance the efficacy of treatment in particular chronic conditions.The need for preservative-free formulations led to the development of new dosage forms and delivery systems, some of which have reached the clinic, and some are still under development 36 .Strategies used to achieve that goal are related to the container or dosage form used.Moreover, the drop passes over the silver coil in the tip as it is released.The valves close after the drop is expelled, preventing air from entering the bottle.This Pfizer's new dispenser includes multiple features that prevent bacteria from entering the container and contaminating the contents 43 .
The advantages of single-dose packaging are eliminating the need for preservatives and accurate dosing since each dose is packaged separately.Table 2 represents some available single-dose eye drops in the market 23,36 .They are sterile, thin, drug-impregnated, solid devices in the form of discs, films or rods.They are inserted into the conjunctival sac to allow medication to reach the ocular surface.Like minitablets, they are either dissolved for single use or converted to a gel for sustained release formulation 70 .Their advantages are increasing the contact between the drug and the conjunctival tissue, sustaining the release of the drug, reducing systemic absorption, increasing patient compliance and eliminating the use of preservatives compared with an aqueous solution.Different methods are used for their preparation depending on their shape and intended use 71 .

Conclusions
In conclusion, there is a shift towards using preservative-free formulation among patients with chronic conditions.

Figure 5 .
Figure 5. Functioning of the Novelia system with example 42 .