INTRODUCTION:

A nanosuspension is a submicron dispersion of nanosized drug particle which are stabilized by surfactants and polymers. A pharmaceutical nanosuspension is defined as very finely dispersed solid drug particles in an aqueous vehicle for oral, topical, parenteral or pulmonary administration. The particle size distribution of the solid particles in nanosuspensions is usually less than one micron with an average particle size ranging between 200 and 600 nm ^1,2.

A nanosuspension not only solves the problems like poor solubility and bioavailability but also improve the safety and efficacy of the drug by altering the pharmacokinetics properties of the drug. Nanosuspensions differ from nanoparticles. Oral route is commonly used and most convenient route for the drug delivery. Oral drug delivery system gained more attention in the pharmaceutical field, due to its more flexibility in designing the dosages from than the other drug delivery systems. In recent years novel drug delivery systems like nanosuspension draws a considerable attention in search for improving bioavailability of poorly soluble drugs ^3,4.

Approximately 40% drug candidate currently available for therapeutic purpose are hydrophobic in nature which affects the bioavailability of such type drug molecules. Many techniques are employed to overcomes is problem, out of which most important are size reduction to nano-size (Formation of Nanocrystals), pH adjustment, use of salt and co-solvents, surfactant, and preparation of lipid formulation ^5,6.

Crystals are defined as presence of high degree of order with a three dimensional periodicity in position of atoms or ions in molecule, with configurationally periodicity of molecules that form the crystal. All chemical entities including drug molecules are able to possess different crystalline forms called as polymorphic form, which have same molecular formula, but arranged in different configuration and showing different physical properties^7,8. Specific polymorphic forms are often considered as important quality-relevant attributes of a drug substance. Polymorphic forms exhibit different dissolution rate, solubility, flow properties, hygroscopicity and behavior under mechanical stress encountered during milling, tableting, agglomeration, etc. Amorphous molecules shows molecular level disorder in configuration, having high internal energy (Chemical potential) i.e. thermodynamically unstable^9,10. These compounds possess higher solubility in aqueous phase as compared to crystalline compounds which possess low internal energy i.e. highly thermodynamically stable with lower solubility. Various techniques are used to enhance the solubility of crystalline drugs which include structural modification by physical and chemical method and other methods like particle size reduction (Micronization/Nanonization), crystal engineering and solid dispersion, use of surfactant, salt formation, and complexation ^11,12.

The drugs classified under BCS class II and IV reported to have problems in their solubility, stability and compatibility which affects their bioavailability and formulation aspects, which can be overcome by making nanocrystals, nanosuspensions during their formulations. This can be used to formulate different dosages forms such as tablets, capsules, parenteral, ophthalmic and nasal preparations ¹³.

Selection Criteria of Drug for Nanosuspension^{14, 15}:

Nanosuspension can be prepared for the API that is having either of following characteristics:

· Water insoluble but which are soluble in oil (high log P) OR API is insoluble in both water and oils.

· Drugs with a reduced tendency of the crystals to dissolve, regardless of the solvent

· API with a very large dose.

ANTIRETROVIRAL THERAPY:

Before people start antiretroviral therapy (ART), health-care provides should initiate a detailed discussion about the redliness of patients to initiate ART, the antiretroviral (ARV) drug regimen, dosages, scheduling, likely benefits, possible adverse effects and the required follow-up and monitoring visits In the case of children with HIV, this conversation should directly involve the caregiver and includes discussion about disclosing their HIV status ^{[16] [17]}. Retesting all people living with HIV before initiating ART is recommended to ensure a correct diagnosis of HIV infection. Initiation of ART should always consider nutritional status, any comorbidity and other medications being taken to assess for possible interactions, contraindications and dose adjustment.

HIV/AIDS has always been one of the most thoroughly global of diseases. The human immunodeficiency virus (HIV) is a lent virus that causes HIV infection and AIDS.

HIV stands for human immunodeficiency virus. AIDS stands for acquired immune deficiency syndrome.

Protease inhibitors used in the treatment of AIDS found to influence the glycoprotein synthesis independently in turn inhibits the growth of HIV. Ritonavir one of the potential protease inhibitor could also act as a substrate for efflux pump that is ultimately preventing the elimination of other co-administered antiretroviral drugs¹⁸.

The purpose of the present study to develop a nanosuspension of ritonavir by the anti-solvent precipitation method with improved the solubility and oral bioavailability. The effect of eudragit RS100, poloxamer 407 (stabilizer) and SLS (surfactant) contents was also studied on the quality attributes of the prepared nanosuspension such as drug content, particle size, entrapment efficiency and stability study ¹⁹.

MATERIALS AND METHODS:

Materials:

Ritonavir was obtained from Mylan Laboratories Limited, Sinnar, Nashik, Poloxamer 407, Eudragit RS100 obtained from Balaji Drug, Gujarat, SLS (Sodium Lauryl Sulphate) and Methanol was obtained from Research-Lab, Mumbai.

Preparation of Nanosuspension method:

The nanoprecipitation method is used for the preparation of ritonavir Nanosuspension. This method is also called as solvent displacement method or anti-solvent precipitation method ²⁰.

The most common method of precipitation used in anti-solvent addition method in which drug is dissolved in suitable organic solvent and this solution mixed with a miscible antisolvent. Rapid addition of drug solution in to the anti-solvent leads to the sudden supersaturation of drug in the mixed solution forms ultrafine drug solids. Precipitation method involves two phases-nuclei formation and crystal growth. When preparing a stable suspension with the minimum particle size, a high nucleation rate and but low growth rate is necessary. Both rates are depending on temperature. In this technique the drug needs to be soluble in at least one solvent which is miscible with non-solvent.

Experimental Work:

1) Preformulation Studies:

1. Solubility studies:

The solubility of the ritonavir was determined by dissolving excess amount of drug in the solvents (water, methanol and ethanol).

2. Physical constant (melting point) determination:

Melting point of ritonavir was measured with the use of Thieles tube apparatus by using paraffin oil, thermometer, thread and burner. The capillary is tight with thermometer and placed in Thieles tube containing paraffin oil, the tube is heated by using burner. The range of temperature when drug just start melting and till it completely melts was noted. Observed values were compared with the reported value.

3. In-vitro drug determination by UV Spectrophotometer:

1. Construction of calibration curve in methanol:

A stock solution of concentration 1000 µg/ml of drug in methanol was prepared separately by dissolving accurately weighed 10 mg of ritonavir 10 ml methanol from this solutions take 1 ml and diluted up to 10 ml with corresponding solvent (100ppm). Appropriate volumes solution were further diluted to obtained final concentrations in range of 10-100 µg/ml. the spectrum of this solution was recorded using UV-visible spectrophotometer ²¹.

4. Drug–excipient compatibility studies:

The drug excipient interaction study was carried out by using physical observation.

Physical observation: the mixture of a drug and each excipients (1:1) ratio placed in separate vials with rubber closure, in order to make hermetically sealed and kept in the environmental test chamber. (Stability chamber, 40℃ at 75 % RH) for 15 days. During the storage, samples were observed for changes if any, such as caking, liquefaction gas formation, and colour change, odour change etc²².

2) Formulation batches of nanosuspensions:

Nine formulation batches were prepared and evaluated for the selection of Best Batch. After the formulation of nanosuspension it was characterized for particle size, Total drug content, entrapment efficiency, and saturation solubility. The low, high and medium concentration of stabilizers selected for the formulation of nanosuspension the final batch subjected to the stability study. The preformulation design of 3² full factorial batches 1 to 9 depicted in table 1.

3. Procedure for Formulation Development of nanosuspensions:

The nanoprecipitation technique is used for the preparation of ritonavir nanosuspension. The pure drug of ritonavir (100mg) was dissolve in (10ml) of methanol to produced organic phase. (Solution 1) The Stabilizers was dissolved in water (40ml) to form aqueous phase. (Solution 2) The solution 1 added into solution 2 drop wised with the help of syringe under the magnetic stirrer for 15 min. After the magnetic stirring the formulation is subjected to agitation with the help of lab stirrer (REMI) at 2000 rpm at 2 hr. ²³.

· Evaluation of the Prepared Nanosuspension:

1. Particle size analysis:

The average particle size of all NS formulations was determined by the motic digital microscopy (DMWB1-223). The nanosuspension showing the lowest particle size was selected for further studies. The particle size of the formulated batches was measured in micrometers.

2. Total drug content:

Prepared Nanosuspensions was analyzed for drug content by UV spectroscopic method. Different batches of nanosuspensions equivalent to 10 mg of drug ritonavir weighed accurately and diluted up to 100 ml with methanol. Stock solutions will be diluted with methanol and analyse by UV spectroscopy (LABINDIA 3000⁺) at 234 nm ²⁴.

Table 1: Batches of Design-Experts for Formulation of for Formulation of Ritonavir Nanosuspension (F1-F9).

Ingredients	Formulation Code
Ingredients	F1	F2	F3	F4	F5		F6	F7	F8	F9
Ritonavir (mg)	100	100	100	100	100	100		100	100	100
Eudragit RS100 (%)	200	300	300	400	300	200		400	200	400
Poloxamer 407 (%)	400	400	500	600	600	500		400	600	600
SLS (mg)	10	10	10	10	10	10		10	10	10
Methanol (ml)	10	10	10	10	10	10		10	10	10
Water (ml)	30	30	30	30	30	30		30	30	30
Stirring Speed(RPM)	2000	2000	2000	2000	2000	2000		2000	2000	2000

3. Entrapment efficiency:

The method suitable for determining entrapment efficiency of nanosuspension when fairly high concentration of free drug is present in the supernatant after centrifugation (REMI 12C). 10 ml portion of the freshly prepared nanosuspension was centrifuged at 1000 rpm for 10 min. using centrifuge the supernatant was removed and the amount of incorporated drug was measured by taking the absorbance of supernatant solution at 234 nm by using UV spectrophotometer. (LABINDIA 3000⁺).

Entrapment efficiency was calculated by following formula:

Entrapment Eficiency (%)

= (Winitial drug – W Free drug / × 100)

4. Saturation solubility study:

The saturation solubility of various orally accepted stabilizers was carried out by shake flask method. The solubility of ritonavir in powder form was determined by a shake flask method. Briefly, an excess amount of drug was suspended in 10 ml of water, and the suspensions were shaken and filter through a 0.22µm whatmen filter. The filtered solution was suitably diluted and the ritonavir concentration in the filtrate was analysed by UV analysis method at 234nm.

The solubility of best batch of formulation was measured by centrifugal method. Briefly, 10 ml of nanosuspension was loaded into centrifugal tubes. Samples were centrifuged at 10000 rpm for 10 min. The supernatant solutions were analysed using UV spectrophotometer at 234 nm.

5. Stability Study:

The final formulations were subjected to stability studies as per ICH guidelines. Various parameters such as drug content, entrapment efficiency were measured before and after 30, days of stability ²⁵.

Table 2: Conditions for Stability Study

Duration of study	30 days
Temperature conditions	40º±2ºC
Relative humidity	75± 5 %

Drug Excipients compatibility study:

Drugs are administered and often they are combined with excipients to compound them into a convenient dosage form. Excipients, although pharmacologically inert, result in chemical or physical interaction with drug and interfere with quality and stability of drug products.

The quality of solid dosage form is dependent on physicochemical properties of drug and the excipients. The evaluation of drug–excipients compatibility is based on inherent properties of the excipient. Assessment of possible incompatibilities between the drug and different excipients is an important part of preformulation.

Thermal analysis (DSC) and FTIR has been used extensively used for stability and compatibility studies.

1. FTIR Spectroscopy:

Fourier Transform Infrared (FTIR) (Shimadzu) spectroscopy was performed by placing a Ritonavir with excipient sample in FTIR sample holder. The drug sample was placed and scanned over the range of 4000-400 cm-1. The obtained spectrum was recorded.

2. Differential scanning calorimetry (DSC):

DSC (METTLER) can be used to detect the physical compatibility among the drug and polymer, by determining the thermal behaviour of pure ritonavir, eudragit RS100, poloxamer 407, SLS and physical mixture of ritonavir + eudragit RS100 + poloxamer 407 + SLS and selected formulas ^{[26] [27]}.

RESULTS AND DICUSSIONS:

1. Melting point:

The melting point was determined by capillary method and it was found to be and complies with I.P.2014.

Table 3: Melting Point of Ritonavir.

Parameter	Observed	Reported standard	Inference
Melting point	120-125ºC	119-123ºC	Complies with I.P.

2. UV- Visible spectrophotometric characterization:

Determine of Calibration Curve ritonavir in methanol:

The Ritonavir with scanning range of 200-400 nm. The maximum wavelength of the drug was found to be 234 nm in methanol.

Figure 1: Calibration Curve of Ritonavir in Methanol

Total drug content:

The drug content of all NS formulations was found to be greater than 80%. Indicating suitability of these methods for particle size reduction.

Table 5: Total Drug Content of Formulations.

Formulation Code	Total Drug Content (%)
F1	82.64
F2	84.25
F3	80.79
F4	85.27
F5	81.69
F6	87.99
F7	96.90
F8	92.57
F9	94.89

Figure 7: Total Drug Content of Formulated Nanosuspension.

Entrapment efficiency:

The entrapment efficiency of all formulations was found to be in the range of (80.11-94.29%) the entrapment efficiency of F7 was high when compared to other formulations.

Table 6: Entrapment Efficiency Data of Formulations.

Formulation Code	Entrapment Efficiency (%)
F1	82.70
F2	81.56
F3	85.21
F4	81.39
F5	85.57
F6	80.11
F7	94.29
F8	87.49
F9	91.89

Figure 8: Entrapment Efficiency of Formulated Nanosuspension.

Saturation solubility studies:

Formulations	Saturation Solubility (µm/ml)
Pure drug	13.65
Nanosuspension	66.75

Figure 9: Comparison of Solubility of Ritonavir and its Nanosuspension

Stability Study:

Table 7: Stability Data of F7 Formulation of Nanosuspension.

Time Period	Entrapment Efficiency	Total Drug Content
Initial (0days)	94.29%	96.19%
Average Storage
1 month	93.65%	96.03%

CONCLUSION:

The obtained results of the present study demonstrate that nanoprecipitation technique was employed to produce nanoparticles of ritonavir, a poorly water-soluble drug, for the improvement of solubility and bioavilability. In this process, the particle size of ritonavir can be obtained in the nano-size ranges, by adjusting the operation parameters. In conclusion, the appropriate selection of process parameter and formulation parameters we can conclude that nanoprecipitation method is a simple and effective approach to produce nanosized particles of poorly water soluble drugs with enhance solubility.

LIST OF ABBREVATIONS:

1. SLS – Sodium Lauryl Sulphate

2. ART – Antiretroviral Therapy

3. HIV – Human immunodeficiency virus

4. AIDS – Acquired immune deficiency syndrome

5. UV – Ultra violet

6. NS – Nanosuspension

7. ICH – International Conference on Harmonization

8. DSC – Differential Scanning Calorimeter

9. FTIR – Fourier Transform Infrared

10. IP – Indian Pharmacopoeia

11. RTV – Ritonavir

12. RPM – Revolution Per Minute

13. TDC – Total Drug Content

14. EE – Entrapment Efficiency

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

We are grateful to the teacher’s and Principal of Loknete Dr. J. D. Pawar College of Pharmacy, Manur, Tal. Kalwan for their helpful guidance.

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Received on 30.05.2021 Modified on 12.06.2021

Res. J. Pharma. Dosage Forms and Tech.2021; 13(4):297-304.

DOI: 10.52711/0975-4377.2021.00049

Formulation Code	Average Particle Size (µm)
F1	0.012
F2	0.015
F3	0.017
F4	0.014
F5	0.013
F6	0.016
F7	0.006
F8	0.007
F9	0.009