Formulation And Evaluation of Nasal In Situ Gelling System of Atazanavir

Acquired Immune Deficiency Syndrome¹:

Acquired immunodeficiency syndrome (AIDS), a pandemic and perhaps the deadliest malady has claimed more than 39.9 million people (including 1.4 million children and 38.6 million adults, 53% were women and girls) across the globe living with HIV according to the 2023 World Health Organization (WHO) HIV data and statistics. In 2023, Nearly 0.6 million (6,30,000) people died from HIV-related illness. The global HIV epidemic claimed 69% fewer lives in 2023 since the peak in 2004 (about 2.9 million died). As of the end of 2023, 77% of all people living with HIV were accessing anti-retroviral therapy globally. The causative organism of AIDS is the human immunodeficiency virus (HIV), which debilitates the immune system by triggering the radical loss and dysregulation of the macrophages and CD4+T lymphocytes. Atazanavir (ATV) is an Az peptide HIV-1 protease inhibitor (PI) with activity against Human Immunodeficiency Virus Type 1 (HIV-1). HIV-1 protease is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV-1. Atazanavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature non-infectious viral particles. Protease inhibitors are almost always used in combination with at least two other anti-HIV drugs.

Nasal Cavity: ²

Anatomy Of the Nasal Cavity:

The nasal cavity can be divided into three main regions namely

1. Nasal vestibule
2. Respiratory region
3. Olfactory region

The nasal cavity is the most superior part of the respiratory tract. It extends from the vestibule of the nose to the nasopharynx, and has three divisions:

1. Vestibule–the area surrounding the anterior external opening to the nasal cavity.
2. Respiratory region – lined by a ciliated pseudostratified epithelium, interspersed with mucus-secreting goblet cells.
3. Olfactory region – located at the apex of the nasal cavity. It is lined by olfactory cells with olfactory receptors.

Physiology Of the Nasal Cavity:3

Respiration:

As air is inhaled, the nasal cavity assists in respiration by preparing air for oxygen exchange. Due to the narrow nature of the cavity, inhaled air is rapidly introduced to a large mucosal surface area with a rich supply of blood at body temperature. This process facilitates rapid acclimation of the inhaled air to a temperature better suited for the lungs. Humidification functions to protect the fragile respiratory and olfactory epithelia.

Defense: The nasal cavity also aids in the defense of respiratory tissues. Mucus secretions trap particles and antigens carried into the respiratory system during inhalation. As pathogens become trapped in these secretions, they are bound by secretory IgA dimers (a component of the adaptive immune response), which prevent attachment of pathogens to host epithelium, thus hindering invasion. Mucus can also contain IgE, which is involved in the allergic response and can cause a pathologic type 1 hypersensitivity reaction. Cilia within the nasal cavity also function to propel mucus away from the lungs in an attempt to expel trapped pathogens from the body. The normal bacterial flora in the nasal mucosa also protects from invasion by competing with invading bacteria for space and nutrients.

Olfaction: Additionally, the nasal cavity enables olfaction. Olfaction helps to identify sources of nearby danger or nutrition, as well as influencing mood and sexuality.[11] As air enters the nasal cavity, the turbinate’s function is to direct a portion of the airflow to the higher regions of the cavity. The olfactory cleft is at the roof of the nasal cavity near the cribriform plate. Olfactory receptors located here bind to odorants carried into the nose during inhalation and send signals to the olfactory cortex and other brain regions.

Mechanism Of Nose-To-Brain Drug Delivery: 4

The nasal mucosa has emerged as a useful target tissue for drug delivery when compared to oral drug administration resulting from its accessibility, high blood flow, large surface area, porous endothelial membrane, and its ability to avoid hepatic first-pass metabolism. However, the exact mechanisms of drug delivery for nose-to-brain delivery are not well understood. The pathways involving the combination of the cerebrospinal fluid, vasculature, and lymphatic system have been reported to be responsible for the transport of bioactive agents from the nasal cavity to the brain. However, the properties of the therapeutics and delivery system can result in one predominant pathway. When the drug is deposited on the respiratory epithelium, the drug is absorbed into the systematic circulation and then into the central nervous system. Drug deposited on the olfactory epithelia is transported to the central nervous system through paracellular or transcellular transport via olfactory neurons or olfactory epithelial cells.

Nasal In-Situ Drug Delivery Systems:5,6

There is a lot of advancements have been seen in oral drug delivery systems in the last few decades, this system has been of limited success in case of drugs undergo extensive first- pass metabolism. Furthermore, it is very difficult for orally administered drugs to transport and enter the CNS because of the existence of the blood-brain barrier (BBB). Retention of drug delivery systems in the nasal cavity prolongs overall nasal transit time, thereby resulting in improved bioavailability for some drugs as well as controlling the drug release to the site. This led to the development of the nasal in-situ gelling system, which can release the drug directly to the CNS through the olfactory nerve and cross the blood-brain barrier.

Aim:

The present work aims to formulate and evaluate a nasal in-situ gelling system for Atazanavir by temperature-triggered system

Objective:

The objectives of the present study are as follows:

• To perform pre-formulation studies such as solubility and melting point determination to characterize Atazanavir
• To carry out drug-excipient compatibility through FT-IR studies to identify any potential interactions that may affect the stability and efficacy of the formulation.
• To formulate an in-situ gel using various drug-polymer ratios based on the temperature- triggered gelation method to optimize gel formation, enhance drug retention, and ensure sustained release for improved therapeutic efficacy.
• To evaluate the formulated in situ gels to ensure stability, efficacy, and patient safety.

MATERIALS AND METHODS

The list of materials and equipment used in the formulation and evaluation studies is illustrated in Table 1&2.

MATERIALS:

Equipment/Instruments:

METHODS

1.1 Preformulation :7,8

Pre-formulation studies were the first step in the rational development of any formulation. It can be designed as an “investigation of physical and chemical properties of the drug substance alone and combined with the excipients”. These studies focus on the physiochemical properties of the new compound that could affect drug performance and the development of an efficacious formulation. The overall objective of pre-formulation testing is generating information useful to the formulator in developing stable and bio-available dosage forms that can be

• To establish physical characteristics.
• To establish its compatibility with the excipient.

1.1.1 Organoleptic properties:

The organoleptic properties of Atazanavir, including colour, odour, and appearance, were evaluated through visual inspection and sensory assessment.

1.1.2 Determination of solubility:9

The pure drug (Atazanavir) was dissolved in different solvents to check their solubility until it formed a precipitate and was recorded. The solvents used are 0.1N HCL, methanol, ethanol, dichloromethane, ethyl acetate, acetonitrile, and water

1.1.3. Melting point determination:¹⁰

A Thiele tube was taken and clamped to a ring stand; the tube was normally filled with clear mineral oil. The oil should be filled to at least 1cm higher than the top triangular arm. Inserted another meter into a one-holed rubber stopper with as lit down one side. Attached the capillary sample to the thermometer with a tiny rubber and or thread. Positioned the capillary tube so that the solid sample is lined up with the middle of the thermos meter bulb. Placed the rubber stopper and thermometer assembly into the Thiele tube, adjusting the height. Heated the apparatus gently on the side arm of the Thiele tube with a Bunsen burner using a back-and-forth motion. However, bubbles should not be seen in the Thiele tube as it warms. Recorded the temperature of the melting range with the appearance of the first visible drop of liquid. At first, it may seem as if the sides of the solid glisten, and the temperature should be recorded when a droplet is seen on the side or bottom of the tube.

1.2 Construction of Standard Curve of Atazanavir

Determination of λmax of atazanavir:11

Stock Solution 1:

100mg (0.1gm) of Atazanavir was weighed & transferred to a 100ml volumetric flask then dissolved in ethanol/SNF & volume was made up to the mark with ethanol/SNF. (1000µg/ml)

Stock Solution 2:

From the above stock solution 1 pipette, out 10ml solution & transfer to 100ml volumetric flask & make up to the volume with ethanol/ SNF. (100µg/ml). From stock solution 2, obtain a concentration of 10µg/ml & scan over the wavelength range of 400nm to 200nm using a UV spectrophotometer against the same solvent as blank. The spectrum of absorbance versus wavelength was recorded & analyzed for the absorbance maximum (λmax) & its Wavelength.

1.2.1. Standard calibration curve of Atazanavir in simulated nasal fluid (SNF):

Preparation of SNF 40:

Dissolve 0.74gm of sodium chloride, 0.129gm of potassium chloride, and 0.005gm of calcium chloride in 100ml water.

pH meter Calibration:

The pH of the SNF solution was measured by the pH meter at 25ºC by calibrating the instrument using the three-point calibration method.

Standard calibration curve of Atazanavir in SNF:

Stock Solution 1:

100mg (0.1gm) of Atazanavir was weighed & transferred to a 100ml volumetric flask then dissolved in SNF & volume was made up to the mark with SNF.

Stock Solution 2:

From the above stock solution 1 pipette, out 10ml solution & transfer to 100ml volumetric flask & make up to the volume with SNF.

Serial Solution:

From the above Stock solution 2, pipette out and make suitable dilutions to make 10, 20, 30, 40, and 50 µg/ml concentrations. The absorbance of diluted solution was measured in a UV-visible spectrophotometer at 249nm wavelength.

1.3 Drug Excipient Compatibility Study By FTIR:12

This study is carried out to find out the compatibility between the drug and the various excipients, which will be used in the formulation of a dosage form. The sample disc was prepared by triturating approximately 1 or 2 mg of the sample substance with around 10 - 20 mg of KBr / Potassium bromide it is triturated and the triturates are compressed by a hydraulic press to form a thin disc of around 10-15mm diameter, which will be sufficient to give an IR spectrum of suitable intensity. This disc is then placed in a sample holder and it is scanned in the range of 4000- 400cm^-1in a FTIR spectrophotometer to get a spectrum. The obtained spectra of the drug and the excipients were compared and it was interpreted for the functional group peaks to check for any major interactions.

1.4 Preparation of In-Situ Gel:13

In-situ gel of Atazanavir was formulated by Temperature-triggered method.

Step 1: Drug and isotonicity adjusting agents were solubilized in ethanol and deionized water (30:70) and kept in the refrigerator. After cooling, the required quantity of PF-127 was added and kept at 4°C with periodical stirring to ensure complete dissolution. The required amount of viscosity-enhancing agent HPMC E 50 LV was dissolved with continuous stirring for complete dissolution of HPMC.

Step 2: After cooling, the required amount of PF-127 was added to HPMC solutions and kept in the refrigerator at 4°C for complete dissolution of PF-127. The pH was measured for all the formulations.

Step 3: Then benzalkonium chloride was added and the batches were subjected to a sterilization process and volume was made up to 100ml using deionized water.

Table.5: Formulation Chart of Temperature Triggered Method.

1.5. Evaluation Of Prepared In-Situ Gelling System:14,15,16,17

1.5.1. Physical Appearance & Clarity:

All the prepared batches were checked for their appearance, and clarity (visually).

1.5.2. pH Measurement:

The pH of the prepared solution for all formulations was measured by a digital pH meter at 25 ± 0.5 ?C after it is calibration using standard buffer solutions of pH 4, 7, and 9.2. Then the measurements of pH were recorded as an average of three measurements.

1.5.3. Drug Content Uniformity:

Each formulation 1ml is taken in a 100ml volumetric flask diluted with buffer & shaken to dissolve the drug. The solution is filtered through the Whatmann filter paper. Pipette, out 1ml of the above filtrate & dilute to 10ml with buffer. The content of the drug was estimated spectrometrically by UV at 249 nm.

1.5.4. In-vitro Gelling Capacity:

The cells were cylindrical reservoirs capable of holding 5 ml of the gelation solution pH 6.8 phosphate buffer. Inside the cells, there was a small cavity which was having a capacity of 1 ml to hold the gel sample in place after its formation. 1 ml of the in-situ gelling formulation was carefully placed into the cavity of the cup using a pipette, and 4 ml of the gelation solution was then added slowly. Gelation was observed by visual examination.

(+) Gels after a few minutes, dispersed rapidly. (++) Gelation immediately remains for a few hours.

(+++) Gelation immediately remains for an extended period.

1.5.5. Gelation Time:

The time which is taken by the formulation for the transition from liquid phase to gel. The test was carried out by taking 2 ml of liquid in a test tube and keeping it in a water bath. The time taken for transition from solution to gel was recorded.

1.5.6. Gelation Temperature:

Gelling temperature refers to the temperature when the meniscus of the formulation would no longer move upon slanting the test tubes at 90 °C. Miller and Donovan's technique was used to determine the gelation studies. The gelling temperature was determined by placing the test tube, containing a sufficient quantity of the prepared solutions, in a water bath at 4 °C. The temperature of the water bath was increased slowly at a constant rate of 1 °C every 2 min. The temperature at which the gel formation takes place was recorded.

1.5.7. Rheological Studies:

The viscosity was measured using a Brookfield DVT viscometer using an LV-3 spindle. The formulation was taken into a sample holder and angular velocity increased gradually from 0.3, 0.6, 1.5, 3, 6, 12, 30, and 60 rpm with a wait period of 30 sec at each speed. The viscosity measurement was performed at 4⁰C and room temperature i.e. before gelation at 4⁰C and after gelation at room temperature. For all the formulation volume of the sample was adjusted with the mark given in the spindle to measure accurate viscosity. The interpretation of results or viscometer dial reading is converted to a viscosity value in units of centipoise, multiply the reading noted on the dial viscometer by the appropriate factor.

1.5.8. Sterility Studies :18

Tests for sterility were performed for fungi, aerobic and anaerobic bacteria by using fluid thioglycollate media and soya casein digest media

1.5.8.1. Sterility (negative control) test:

Alternate thioglycolate media was incubated at 30-35 ºC and soya bean casein digest medium at 20-25 ºC for 7 days. (No growth of organisms occurs)

1.5.8.2. Growth promotion (positive control) test:

The sterile media is inoculated with about 100 viable micro-organisms and incubated as per the specified conditions. The test was found satisfactory based on the evidence of growth within the stipulated days (7 days). Nasal preparations should be sterile and must be checked for the presence of any bacteria or fungi before it is used. Since the formulation is a nasal preparation, so according to the I.P. procedure two containers were selected for sterility test. The test samples were labeled as ‘negative control’, ‘test’, and ‘positive control’.

1.5.8.3. Test for aerobic bacteria:

Twenty ml each of sterile alternative thioglycolate was transferred to 3 tubes aseptically. The tube labeled as positive control was inoculated with viable aerobic microorganism Bacillus subtilis aseptically. 2.5 ml of the nasal preparation was added to the tube labelled as a test. Then incubate all three tubes at 30-35 ºC for 7 days.

1.5.8.4. Test for anaerobic bacteria:

Twenty ml each of sterile alternative thioglycolate was transferred to 3 tubes aseptically. The tube labeled as positive control was inoculated with viable anaerobic microorganism Bacteroides vulgates aseptically. 2.5 ml of the nasal preparation was added to the tube labeled as a test. Then incubate all three tubes at 30-35 ºC for 7 days.

1.5.8.5. Test for fungi:

Twenty ml each of sterile soya bean-casein digest medium was transferred to 3 tubes aseptically. The tube labeled as positive control was inoculated with candida albicans aseptically. To the test tube labeled as ‘test’, 2.5 ml of the sample was transferred. The third test tube was labeled as a negative control. All three tubes were incubated at 20- 25 ºC for 7 days.

1.5.9. Isotonicity Studies:19

Isotonicity is an important characteristic of the nasal drug delivery system. Isotonicity has to be maintained to prevent tissue damage. Formulations are mixed with a few drops of blood and observed under a microscope at 45x magnification and compared with standard marketed nasal formulations. The shape of the blood cell is compared with standard marketed nasal formulation as per IP.

1.5.10. In-vitro Drug Diffusion Studies:20

In-vitro drug diffusion studies were carried out using a modified Franz diffusion cell. The apparatus consists of a cylindrical glass tube which was opened at both ends. The gel sample (1 ml) was spread uniformly on the surface of the eggshell membrane (previously soaked in water overnight) and was fixed to one end of the tube such that the preparation occupied the inner circumference of the tube. The whole assembly was fixed in such a way that the lower end of the tube containing gel just touched (1-2mm deep) the surface of the diffusion medium. i.e., 200 ml of pH 6.8 phosphate buffer contained in a 200 ml beaker which was placed in a water bath and maintained at 37±2°C. The eggshell membrane acts as a barrier between the gel phase and the pH 6.8 phosphate buffer. A quantity of 1 ml samples was withdrawn from receptor fluid at the interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 hrs. The released drug was estimated by spectrophotometer at 249 nm and 1 ml phosphate buffer pH 6.8 was replaced each time.

RESULT AND DISCUSSION

1. Preformulation Studies:

1.1. Organoleptic Properties:

Drug description: This study is important as organoleptic property determination is a preliminary test for every drug. The nature, color & odour of Atazanavir comply with the specifications that are mentioned in various literature sources. Descriptions of drugs are shown in Table 5.

Table.5: Description of drug.

1.2. Determination of Solubility:

Solubility: Solubility studies were carried out in different solvents as per I.P. and observations were shown in Table 6. Solubility analysis is important because the drug has to dissolve in the solvents and also in the dissolution medium used. Atazanavir was found to be freely soluble in methanol, ethanol, and dimethylformamide, slightly soluble in water, and soluble in SNF. Solubility studies were carried out in different solvents as per I.P. and observations were showed in Table:6.

1.3.     Melting Point Determination:

Melting points were carried out and observations were shown in Table.7. The Melting point of Atazanavir was found to be 208ºC, which is within the near standard range of 209 ºC, which showed that the procured pure drug (Atazanavir) which is free from impurities. All readings are an average of three determinations (n=3).

2. Construction of Standard Curve of Atazanavir:

Estimation of Atazanavir was carried out at wavelength 249 nm in simulated nasal fluid. The value of the regression coefficient was found to be 0.9980 which showed a linear relationship between concentration and absorbance. The regression equation generated was y=0.1588x. By using the value of the regression coefficient % CDR was calculated. It was observed that the concentration ranges of 2-10µg/ml obeyed Beer’s Lambert’s law. Atazanavir was estimated using UV spectrophotometer measured at 249 nm using simulated nasal fluid.

Table.8: Estimation of Atazanavir measured at 249 nm using UV-Spectrophotometry.

Figure.10: Atazanavir measured at 249 nm using UV-Spectrophotometry.

1. 1. Drug Excipients Compatibility Study By FT-IR

The physical mixture of Atazanavir and excipients was subjected to FT-IR to identify any interaction between them. The IR spectra of Atazanavir 1651.12cm^-1, 1246.06cm^-1, 2958.90 cm^-1, 1548.90 cm^-1, 3358.18cm^-1, wavenumber as major peaks were found when it is mixed with excipients. There was no appearance or disappearance of any characteristic peak of the drug, which confirms the absence of chemical interaction. Hence the excipients Pluronic F 127, HPMC E50LV which were observed to be compatible with Atazanavir were selected for further development of the formulation.

Figure.11: FT-IR Spectrum of Atazanavir.

Table.9: Interpretation of FT-IR spectral data of Atazanavir.

Figure.12: FT-IR Spectrum of Pluronic F-127.

Table.10: Interpretation of FT-IR spectral data of Pluronic F-127.

Figure13: FT-IR Spectrum of HPMC E 50 LV

Table.11: Interpretation of FT-IR spectral data of HPMC E 50 LV.

figure.14: FT-IR Spectrum of Atazanavir+HPMC E 50 LV and Pluronic F-127.

0. 2. Evaluation Of Prepared In-Situ Gelling System
1. Physical Appearance & Clarity:

Physical Appearance & Clarity: The in-situ gels formulations (F1- F9) are evaluated for their appearance & clarity. All formulations (F1-F9) were transparent in their appearance and their clarity is clear as shown in Table 13.

Table.13: Physical appearance & clarity (F1-F9)

2. pH Measurement

The pH Measurement is very important for nasal preparation otherwise it leads to irritation to the nose. Formulations F1 to F9 were checked for their pH using a calibrated pH meter. The pH of the formulations was found to be in the range of 6.33–6.47. The measurement of the pH of each formulation was in triplicate and the average values along with standard deviation are noted in Table 14.

Table.14: pH measurement of F1-F9 formulation

Drug Content:

Drug Content: The drug content of the developed formulations F1 to F9 was found to be in the range of 94.97 ± 0.07% to 92.55 ± 0.40%. which were within the limit (not < 94% and not > 106%) as specified in U.S.P. Estimation were performed in triplicate. This study concludes that the drug is uniformly distributed in the formulation. The result of drug content estimation of all formulations is shown in Table 18 and Figure 17.

Table.18: Drug content of formulation F1-F9

Determination Gelation Time, Gelation Temperature:

Gelling Time: Gelling time of the formulations was assessed by visual examination. Gelling time was found to vary according to the concentration of the polymer used proportionally. The gelling time of formulations F1-F9 was found to be 60-50 seconds. Observations of the study were tabulated in Table 17.

Gel Temperature Determination: Gel temperature determination of formulations F1-F9 was carried out using a test tube. The gelling temperature was found to vary according to the concentration of the polymer used. The gelling time of formulations F1-F9 was found to be 28-35⁰c. Observations of the study were tabulated in Table 17.

Table.17: Gelation time, Gelation temperature of in-situ gelling formulation (F1-F9)

Rheological Studies:

Viscosity: The rheological properties of the solutions are important in viewing their proposed oral administration. The formulation should have an optimum viscosity, which then undergoes a rapid sol-gel transition due to the triggering of the polymer due to temperature. The prepared formulation was evaluated for their rheological property using Brookfield viscometer at various shear rates before gel formation i.e. at 40 ?C and after gel formation at 300 ?C. The order of viscosity of all formulations was found in the following order F9 > F8 > F7 > F6 > F5 > F4 > F3 > F2 > F1 as shown in Table 15-16 and Figure 16. The formulations showed a marked increase in viscosity with increasing concentration of pluronic F127 and HPMC E50LV. This showed that the viscosity of formulation decreases with an increase in shear rate. The formulations obey non-newtonian flow (pseudoplastic flow).

Table.15: Viscosity of formulation F1-F9 Before gelation

Table.16: Viscosity of formulation F1-F9 After gelation

Figure.16: Viscosity of temperature-triggered formulation F1-F9 (after gelation)

Sterility Studies:

Sterility Studies: The selected formulation was subjected to sterility study by using various media i.e. fluid thioglycolate media and soybean casein digest media the tests are carried out for aerobic, anaerobic bacteria and fungi. The prepared media was used as nutrient media for the growth of microorganisms. The selected best formulation F5 was subjected to a sterility test which showed a negative sign i.e no formation of colonies. The formulation F5 was found to be sterile and is shown in Table 19.

*(-) sign suggests negative results (No growth of microorganisms)

**(+) sign suggests positive results (Formation of colonies of microorganisms)

Isotonicity Studies:

Isotonicity Study: The selected formulation F5 was subjected to isotonicity which is compared with the marketed nasal formulation. The blood cell with formulation F5 is compared with the blood cell of marketed nasal formulation which didn’t show any difference in cell shape. The isotonicity study of formulation F5 is shown in Figure no 18, 19.

Figure.18: Blood cells with marketed.

Figure.19: Blood cells with formulation F5 nasal formulation as standard.

IN-Vitro Drug Release Studies:

The in-vitro release studies of formulations F1-F9 was carried out for 12 hrs. Among all formulations F5 released 92.3% of drug. So, Formulation F5 was selected as a best formulation. (shown in Table: 20).

Table.20: In-vitro drug release studies.