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SEMINAR ON

INSITU GEL

PRESENTED BY :

DIGANT PATEL,

M.PHARM.

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List of contents

INTRODUCTION DEFINITION ADVANTAGES APPROACHES FORMULATION OF INSITU GEL EVALUATION APPLICATIONS MARKETED FORMULATIONS QUESTIONS REFERENCES

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In situ is a Latin phrase which translated literally as ' In position’ .

In situ gels are drug delivery systems that are in solution form before administration in the body, but once administered, undergo gelation in situ, to form a gel .

Administration routes for in situ gel: - oral, ocular, rectal, vaginal, injectable and intraperitoneal routes.

INTRODUCTION

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ADVANTAGES

Increased contact time.

Improved local bioavailability.

Reduced dose concentration.

Reduced dosing frequency.

Improved patient compliance and comfort.

Its production is less complex and thus lowers the

investment and manufacturing cost.

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Weighed quantities of Timolol maleate, Benzalkonium chloride, EDTA and sodium chloride are dissolved in the pH 4 phosphate buffers under aseptic conditions.

Polyacrylic acid (Carbopol 934p) is slowly added with continuous stirring at a speed of 1,500-2,000 rpm to minimize the formation of the lumps of undispersed mass.

HEC is added with slow stirring to avoid foam formation. Stirring is continued until a clear dispersion is formed.

General method of in situ gel :

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APPROACHES There are six approaches for the in situ gel :

1. Temperature-sensitive hydrogels2. pH-sensitive hydrogels3. Ion-sensitive hydrogels4. Enzyme-sensitive hydrogels 5.Light-sensitive hydrogels 6. Dilution-sensitive hydrogels

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1) Temperature-sensitive hydrogels Temperature-sensitive hydrogels are probably the

most commonly studied class of environment-sensitive polymer systems in drug delivery research.

The use of biomaterial which transits from sol-gel is triggered by increase in temperature, is an attractive way to approach in-situ formation.

The ideal critical temperature range for such system is surrounding physiologic temperature, such that clinical manipulation is facilitated and no external source of heat other than that of body is required for trigger gelation.

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Temperature-sensitive hydrogels are classified into :1) Negatively thermo-sensitive2) Positively thermo-sensitive3) Thermally reversible gels

1)Negatively thermo-sensitive

Negative temperature-sensitive hydrogels have a low critical solution temperature (LCST) and contract upon heating above the LCST. Polymers with low critical temperature (LCST) transition between ambient and physiologic temperature is used for this purpose.

Polymers used in this type of hydrogel are Poly(acrylic acid)(PAA), Poly(acrylamide) (PAAm), Poly(acrylamide co- butyl methacrylate).

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Poly (N,N-diethylacrylamide) (PDEAAm) is also widely used because of its lower critical solution temperature (LCST) in the range of 25°–32°C, close to the body temperature.

Block copolymers made of poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO),Which have LCST at around the body temperature and its sol–gel phase conversion at the body temperature.

A large number of PEO–PPO block copolymers are commercially available under the names of Pluronics (or Poloxamers) and Tetronics

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2) Positively thermo-sensitive

A positive temperature sensitive hydrogel has an upper critical solution temperature (UCST), such hydrogel contracts upon cooling below the upper critical solution temperature.

E.g. poly(acrylic acid) (PAA) polyacrylamide ( PAAm ) poly( acrylamide -co-butyl methacrylate )

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Mechanism

Gelation mechanism.

1. At high temperatures a random coil conformation is assumed.

2. With decreasing temperature, formation of double helices that act as knots is observed.

3. The aggregation of such helices forms the physical junctions of gels.

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(1)

(2)

(3)

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3) Thermally reversible gels

The most commonly used thermoreversible gels are prepared from polymer like….

Poloxamer is triblock copolymer, which is used in ophthalmic drug delivery. It is sol at 20°-25°C And converted in to gel form at body temperature (35°-37°C).

Other polymers are - HPMC, - Xyloglucan, - poly(ethylene oxide)-b-poly(propylene oxide)-b poly(ethylene oxide) (Pluronics®, Tetronics®, poloxamer).

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In this type of formation gel is induced by pH changes.

All the pH-sensitive polymers contain pendant acidic or basic groups that either accept or release protons in response to changes in environmental pH.

The Swelling of hydrogel increases as the external pH increases in the case of weakly acidic (anionic) groups, but decreases if polymer contains weakly basic (cationic) groups.

Polymers used in this type of gel are : Chitosan, polyacrilic acid ( carbopol 940 ), etc.

2) pH-sensitive hydrogels

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PAA is converted sol to gel when PH rise from 4.2 to 7.4 .

At higher pH polymer form bond with mucin which lead to formation of in situ gel.

Chitosan sol form at pH 6.2 ,but when pH rise greater than the 6.2 form gel.

Other polymer are : Hydroxy phthalate latex, Cellulose acetate phthalate.

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3) Ion-sensitive hydrogels:- This system produce gel of the polymer when it get

specific ion in contact. e.g., Alginate (kelton) is used as the gelling agent

in combination with HPMC (Methocel E50Lv) which acted as a viscosity enhancing agent.

Gelrite gellan gum, a novel ophthalmic vehicle that gels in the presence of mono or divalent cations, present in the lachrymal fluid can be used alone and in combinations with sodium alginate as gelling agent.

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In situ formation catalysed by natural enzymes has not been investigated widely but seems to have some advantages over chemical and photochemical approaches.

For example, an enzymatic process operates efficiently under physiologic conditions without need for potentially harmful chemicals such as monomers and initiators.

4) Enzyme sensitive hydrogel

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Cationic pH-sensitive polymers containing immobilized insulin and glucose oxidase can swell in response to blood glucose level releasing the entrapped insulin in a pulsatile fashion.

e.g P(MAA-g-EG) hydrogels are of particular interest in this research.

As described previously, glucose-sensitive hydrogels can be produced by the incorporation of a pH-sensitive hydrogel with immobilized GOD.

A ‘‘squeezing gel’’ is expected when incorporating P(MAA-g-EG) with GOD.

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At high concentrations of glucose, the GOD–glucose reaction produces GlucA, resulting in a decrease in the pH of the environment.

The hydrogels are expected to collapse abruptly with the decrease in pH. It is with this collapse that insulin

could be ‘‘squeezed out’’.

As the glucose concentration decreases by the action of the released insulin, less GlucA is produced, resulting in an increase in pH of the environment.

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Adjusting the amount of enzyme also provides a convenient mechanism for controlling the rate of gel formation, which allows the mixtures to be injected before gel formation.

Polymers used in this type of in situ gel are • 4-hydroxymandelic acid• Poly(acryl amide)• Poly(ethylene glycol acryl amide), etc.

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Photo-polymerisation is commonly used for in situ formation of biomaterials.

A solution of monomers or reactive macromer and initiator can be injected into a tissues site and the application of electromagnetic radiation used to form gel.

Acrylate or similar polymerizable functional groups are typically used as the polymerizable groups on the individual monomers and macromers because they rapidly undergo photo-polymerisation in the presence of suitable photoinitiator. Typically long wavelength ultraviolet and visible wavelengths are used. Short wavelength ultraviolet is not used often because it has limited penetration of tissue and biologically harmful.

5) Light-sensitive hydrogels (Photo-polymerisation)

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e.g A ketone, such as 2,2 dimethoxy-2-phenyl acetophenone, is often used as the initiator for ultraviolet photo polymerization, where as camphorquinone and ethyl eosin initiators are often used in visible light systems.

These systems can be designed readily to be degraded by chemical or enzymatic processes or can be designed for long term persistence in vivo.

Photopolymerizable systems when introduced to the desired site via injection get photocured in situ with the help of fiber optic cables and then release the drug for prolonged period of time.

The photo-reactions provide rapid polymerization rates at physiological temperature.

Further more, the systems are easily placed in complex shaped volumes leading to an implant formation.

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UV-sensitive hydrogels :- leuco derivative molecule bis(4-di-methylamino) phenyl methyl leucocyanide. Visible light-sensitive hydrogels :- poly(N-iso-propyl acrylamide)

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6. Dilution-Sensitive Hydrogels

This type of hydrogel contains polymer that undergoes

phase transition in presence of higher amount of water.

By having a system undergoing phase transition as a

consequence of dilution with water, a higher amount of

polymer can be used.

e.g lutrol

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FORMULATION OF INSITU GEL

For ophthalmic :- Drugs -For ophthalmics in-situ gel suitable candidates are

moxifloxacin hydrochloride Linezolid Gatifloxacin

Polymers- They are responsible for providing gelling properties. -Examples:-

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HPMC E 50 LVHPMC K 4MXanthan gum , Hydroxy ethyl cellulose , Carbopol 934P

Preservatives:- -These include Benzalkonium chloride chlorhexidine acetate

Solvents:--Distilled water

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For Nasal :-

Drugs -For Nasal in-situ gel suitable candidates are Vitamin B12 Chlorpheniramine maleate,Ondansentron Flunarizine hydrochloride, Zolmitriptan Salbutamol sulphate

Polymers-They are responsible for providing gelling properties. -Examples:-sterculia foitida, Pluronic F68 , Carbopol 934P Pluronic F127 , Chitosan Pluronic 407 , Gellan gum

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Preservatives:- -These include :- methyl paraben propyl paraben benzalkonium chloride

Solvent :- Distilled water

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For Parenteral insitu gel :-

Drugs :- Following drugs use in perentral in situ gel :- e.g. Gatifloxacin Doxycylin Leuprolide etc.

Polymers :- They are responsible for providing gelling properties. -Examples:- poly(n-isopropylacrylamide) sodium alginate poloxymer , Alginic acid Gellan gum , Pluronic F127 Carbomer etc.

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Buffer solution :- Following buffers are used : examles :- acetate buffer citrophosphate buffer etc.

Solvent :- Distilled water

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For oral insitu gel

Drugs :-example Clotrimazole, Diltiazem Ofloxacin, Clarithromycin Nifedipine , Omeprazole Polymers They are responsible for providing gelling properties. Examples:-Gellan and sodium alginate

-Chitosan -Carbopol

-Gallan gum -Xanthan gum, -Xyloglucan

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Preservatives:- These include

-Methyl paraben -Propyl paraben -Benzalkonium chloride

Flavoring agent:- -Aspartem -saccharine sodium etc.

Solvents:- -Purified water

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EVALUATION

pH Clarity Texture analysis Gelling capacity Gel strength Rheological studies Sol-Gel transition temperature Fourier Transforms Infrared Spectroscopy Drug content estimation In vitro drug release studies Accelerated stability studies

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pH :

pH of prepared in situ gel is measured with pH meter.

Clarity :

Clarity test is done by visual inspection of each container

under a good light, To check against reflection into the

eyes and viewed against a black and white background,

with the contents set in motion with a swirling action.

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The firmness , consistency and cohesiveness of formulation are assessed using texture analyzer, which mainly indicates the syringeability of sol , so the formulation can be easily administered

in-vivo.

Higher values of adhesiveness of gels are needed to maintain an intimate contact with surface like tissues.

Texture analysis :

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Gelling capacity :

The gelling capacity of the prepared formulation is

determined by placing a drop of the formulation in a

vial containing 2 ml of freshly prepared simulated tear

fluid and visually observe. The time taken for its

gelling is noted down.

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This parameter is evaluated using a Rheometer.

Depending on the mechanism of the gelling of gelling agent used, a specified amount of gel is prepared in a beaker, from the sol form .

This gel containing beaker is raised at a certain rate, so pushing a probe slowly through the gel.

The changes in the load on the probe can be measured as a function of depth of immersion of the probe below the gel surface.

Gel-Strength :

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Rheological studies :

The viscosity measurements are carried out using

Brookfield viscometer. The in situ gel formulations are

placed in the sampler tube.

The samples are analyzed at 37°C ± 0.5°C by a

circulating bath connected to the viscometer adaptor

prior to each measurement. The angular velocity of the

spindle is increased 1 to 4 and the viscosity of the

formulation are measured.

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Sol-Gel transition temperature :

The sol-gel transition temperature may be defined as that temperature at which the liquid phase makes a transition to gel.

Gelation point is considered as the temperature where formulations would not flow when test tubes are tilted to 90° angle as the temperature is gradually increased.

While in case of pH and ion dependant polymer there is change in pH or contact with nasal fluid they get change from sol to gel.

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Fourier Transforms Infrared Spectroscopy:

Fourier-transform infrared (FT-IR) spectra are obtained using an FT-IR spectrometer . The pure drug mixed thoroughly with potassium bromide, an infrared transparent matrix, at specified ratio. (usually 1:5 ratio).

The KBr discs are prepared by compressing the powders at a pressure of 5 tons for 5 min in a hydraulic press.

Scans are obtained at a resolution of 4cm-1 ,from 4000 to 400 cm-1 .

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Drug content estimation :

The drug content estimation is carried out by diluting 1

ml of prepared formulation in 100 ml of distill water and

analyzed using UV-visible spectrophotometer at

appropriate wavelength.

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In vitro drug release studies :

In vitro release study of in situ gel solution is carried out by using Franz diffusion cell.

The formulation is placed in donor compartment and freshly prepared simulated tear fluid in receptor compartment.

Between donor and receptor compartment dialysis membrane is placed (0.22μm pore size).

The whole assembly is placed on the thermostatically controlled magnetic stirrer.

Formulation is accurately pipetted into donor chamber.

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1ml of sample is withdrawn at predetermined time interval of 1hr for 6 hr and same volume of fresh medium is replaced.

The withdrawn samples are diluted to 10ml in a volumetric flask with respective solvent and analyze by UV spectrophotometer at respective nm using reagent as blank.

The drug content is calculated using the equation generated from standard calibration curve.

The % cumulative drug release is (%CDR) calculated.

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Accelerated stability studies :

Optimized sterile formulation is subjected to stability testing.

Sterile optimized formulation is filled in glass vials, closed with gray butyl rubber closures and sealed with aluminium caps.

The vials contain optimized formulation are kept in stability chamber, maintained at 40°C ± 2°C and

75 % ± 5 % RH for one month. Samples are withdrawn weekly and estimated for

drug content, pH, visual appearance, gelling capacity and in vitro drug release.

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For Ophthalmic Formulation following tests are carried out :-

Antimicrobial Activity Ocular irritation studies Isotonicity Evaluation

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Antimicrobial Activity

Antimicrobial activity is determined by agar diffusion test employing cup plate technique. The drug is allowed to diffuse through a solid agar medium.

The standard minimum inhibitory concentration of controlled and developed formulations containing drug are prepared.

A total of 60 ml of nutrient agar media is prepared and sterilized at 15 lb/sq-inch pressure for 18 minutes in an autoclave; 0.5 ml of microorganism suspension is poured into the above medium which is maintained at temperature of 52°C to 58°C.

This will be done in an aseptic condition. Immediately 20 ml of the microbial agar suspension is poured into each petriplate.

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After solidification of the media, sterile standard solutions and the developed formulations are diluted suitably with sterile distilled water (test solutions) and poured in to the cup of sterile nutrient agar Petri plates.

After allowing diffusion of the solutions for 2 hours, the agar plates are incubated at 37°C for 24 hours.

The Zone of inhibition (ZOI) is measured around each cup and compare with that of control.

The entire operation is carried out in a laminar airflow unit. Each formulation solution is tested in triplicate.

Both positive and negative controls are maintained throughout the study.

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Ocular irritation studies :

The Draize-irritancy test is designed for the ocular irritation potential of the ophthalmic product prior to marketing.

According to the Draize test, the amount of substance

applied to the eye is normally 100μl is placed into the lower cul-de-sac with observation of the various criteria made at a designed required time interval of 1hr, 24hr, 48hr, 72hr, and 1 week after administration and rabbits are observed periodically for redness, swelling, watering of the eye.

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Isotonicity Evaluation

Isotonicity is important characteristic of the ophthalmic preparations. Isotonicity has to be maintained to prevent tissue damage or irritation of eye.

Formulations are mixed with few drops of blood and observed under microscope at 45X magnification and compared with standard marketed ophthalmic formulation

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For Nasal formulation following test is carried out :-

Determination of Mucoadhesive Strength

The mucoadhesive strength is determined by using the modified method.

The mucoadhesive potential of formulation is determined by measuring a force require to detach the formulation from nasal mucosal tissue.

A section of sheep nasal mucosa is fix on each of two glass slides using thread .

Gel is placed on first slide and this slide place below the height adjustable pan. While another slide with mucosal section is fix in inverted position to the underside of the same pan.

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Both the slides with gel formulation between them held in contact with each other, for 2 min to ensure intimate contact between them.

Then weight is keep rising in second pan until slides get detached from each other. The mucoadhesive force express as the detachment stress in dyne/cm2

is determine from the minimal weight that detach the mucosal tissue from surface of each formulation.

Mucoadhesive Strength (dyne/cm2) = mg/A,

Where, m = weight required for detachment in gram, g = Acceleration due to gravity (980cm/s2), A = Area of mucosa exposed

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For Oral formulation (floating ) following test is carried out :-

In-vitro floating study :

Floating study is carried out in 500 ml of 0.1 N HCl (pH 1.2) in a beaker.

Accurately measured 10 ml of solution is added to HCl. Time required for immersing of gel on surface after adding solution (floating lag time) and total floating time are measured.

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APPLICATIONS

A.Oral Delivery.B.Parenteral DeliveryC.Ocular DeliveryD. Vaginal DeliveryE. Dermal and Transdermal DeliveryF. Nasal Delivery

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Pectin, xyloglucan and gellan gum are the natural polymers used for in situ forming oral drug delivery systems.

The potential of an orally administered in situ gelling pectin formulation for the sustained delivery of paracetamol has been reported.

The main advantage of using pectin for these formulations is that it is water soluble,

so organic solvents are not necessary in the formulation.

In situ gelling gellan formulation as vehicle for oral delivery of theophylline is reported.

A. Oral-delivery

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The formulation consisted of gellan solution with calcium chloride and sodium citrate complex.

When administered orally, the calcium ions are released in acidic environment of stomach leading to gelation of gellan thus forming a gel in situ.

An increased bioavailability with sustained drug release profile of theophylline

Its study in rats and rabbits was observed from gellan formulations as compared to the commercial sustained release liquid dosage form.

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B. Parenteral Delivery One of the most obvious ways to provide sustained

release medication is to place the drug in a delivery system and inject or implant the system into the body tissue. Thermoreversible gels mainly prepared from poloxamers are predominantly used.

The suitability of poloxamer gel alone or with the addition of hydroxy propyl methyl cellulose (HPMC), sodium carboxy- methylcellulose or dextran was studied for epidural administration of drugs in vitro.

The compact gel depot acted as the rate-limiting step and significantly prolonged the dural permeation of drugs in comparison with control solutions.

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Pluronic F127 gels which contained either insulin or insulin-PLGA nanoparticles can be useful for the preparation of a controlled delivery system.

Like, poloxamer gels were tested for intramuscular and subcutaneous administration of human growth hormone or with the aim to develop a long acting single dose injection of lidocaine.

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C. Ocular Delivery

The efficacy of ophthalmic hydrogels is mostly based on an increase of ocular residence time via enhanced viscosity and mucoadhesive properties.

Since resulted swollen hydrogel is aqueous based, it is very comfortable in the human eye.

Among these polymers, in situ gels are preferred since they are conveniently dropped in the eye as a solution, where it undergoes transition into a gel. Thermosensitive, specific ion sensitive or pH-sensitive hydrogels have been examined for their potential as vehicles for ocular drugs.

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Formulations based on a thermoplastic graft copolymer that undergo in situ gelation have been developed to provide prolonged release of active ingredients such as non oxynol-9, progestins, estrogens, peptides and proteins.

A mucoadhesive thermosensitive gel containing combination of poloxamers and polycarbophil exhibited increased and prolonged antifungal activity of clotrimazole in comparison with conventional PEG-based formulation.

D. Vaginal Delivery

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Thermally reversible gel of Pluronic F127 was evaluated as vehicle for the percutaneous administration of indomethacin. In-vivo studies suggest that 20% w/w aqueous gel may be of practical use as a base for topical administration of the drug.

Poloxamer 407 gel was found suitable for transdermal delivery of insulin.

The combination of chemical enhancers and iontophoresis resulted in synergistic enhancement of insulin permeation.

E. DERMAL AND TRANSDERMAL DELIVERY

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Nasal formulations of chlorpheniramine maleate and tetrahydrozoline hydrochloride were investigated.

The findings suggest that liquid formulations of chlorpheniramine maleate facilitate the instillation into the nose and the hydrogel formed on the mucous membrane provide controlled drug release.

F. NASAL DELIVERY

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MARKETED FORMULATIONS

It is a timolol maleate ophthalmic gel formulation of Merck and Co.

Which is supplied as a sterile, isotonic, buffered, aqueous gel forming solution of timolol maleate.

Dosage strengths 0.25% and 0.5% in market . Each ml of Timoptic 0.25% contains 2.5 mg of timolol (3.4 mg of timolol maleate ).

pH of solution is approximately 7.0 Inactive ingredients include gellan gum (Temperature and Ion sensitive polymer ) , tromethamine , mannitol and water for injection and the preservative used is benzododecinium bromide 0.012%.

(1) Timoptic :-

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Oncogel is a frozen formulation of paclitaxel in Regel.

It is a free flowing liquid below room temperature which upon injection forms a gel in-situ in response to body temperature.

hGHD-1 is a novel injectable depot formulation of human growth hormone (hGH) utilizing Macromed's Regel drug delivery system for treatment of patients with hGH deficiency.

2) Oncogel

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AzaSite is a marketed product of InSite Vision.

AzaSite is a topical ophthalmic solution of azithromycin formulated in DuraSite.

AzaSite is supplied as a sterile aqueous ophthalmic

formulation designed for topical administration.

The recommended initial dose of the drug is instill 1 drop in the affected eye(s) twice daily, eight to twelve hours apart for the first two days and then in still 1 drop in the affected eye (s) once daily for the next five days.

(3) AzaSite:

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(4) Pilopine HS:

Pilopine HS is a marketed product of Alcon Laboratories. Pilopine HS (pilocarpine hydrochloride ophthalmic gel) 4% is a sterile topical ophthalmic aqueous gel which contains more than 90% water and employs Carbopol-940 (pH Sensitive Polymer), a synthetic high molecular weight cross linked polymer of acrylic acid, to impart a high viscosity.

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(5) Akten™:

Akten™ is an HPMC-based gel of lidocaine hydrochloride for ocular surface anesthesia.

Akten™ contains 35 mg of lidocaine hydrochloride per mL as the active ingredient.

Akten™ also contains Hypromellose, Sodium Chloride, and Purified Water as inactive ingredients.

The pH may be adjusted to 5.5 to 7.5 with Hydrochloric Acid and/or Sodium Hydroxide.

The recommended dose of Akten™ is 2 drops applied to the ocular surface in the area of the planned procedure. Akten™ may be reapplied to maintain anesthetic effect.

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