Available online on 15.06.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2023 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                               Research Article 

Exploring the Potential of Ketoprofen Nanosuspension: In Vitro and In Vivo Insights into Drug Release and Bioavailability

Mahesh N. Sanas¹*, Tejas S. Pachpute ²

Department of Pharmaceutics, Alwar Pharmacy College, Sunrise University, Alwar, Rajasthan, India

^# Both Authors have Equal Contribution

1. INTRODUCTION

The poor water solubility of numerous compounds under development is a recognized factor that often leads to their failure to reach the market. Extensive research and reports have consistently demonstrated that a significant proportion of active compounds in development fall into the category of poor solubility, with a majority belonging to class II drugs according to the Biopharmaceutical Classification System (BCS)¹. This classification system categorizes drugs based on their solubility and permeability characteristics. The prevalence of poorly soluble compounds highlights the need for innovative strategies to improve their solubility and enhance their pharmaceutical properties, ultimately increasing the likelihood of successful development and market introduction^{2, 3}.

The low permeability and low dissolution rate of class-II biopharmaceutical drugs pose challenges to their therapeutic effectiveness. However, by employing innovative nanotechnology-based delivery systems, such as nanosuspension formulations, it becomes possible to enhance the therapeutic activity of these drugs⁴. Nanosuspensions consist of finely dispersed, submicron-sized colloidal particles that encapsulate the drugs. They offer several advantages, including improved drug bioavailability and a wider therapeutic range, which allows for reduced dosing. These characteristics make nanosuspensions a crucial formulation in the quest to enhance the efficacy of class-II drugs⁵.

Numerous strategies have been employed to enhance the solubility of drugs, aiming to overcome the challenges associated with their poor aqueous solubility.   Formulation strategies encompass two methods: the top-down approach, involving the reduction of particle size in larger crystals, and the bottom-up approach, which involves the formation of crystals by precipitating dissolved molecules. High-pressure homogenization and media milling are classified as top-down techniques⁶, while ultrasonication, supercritical anti-solvent precipitation (SAS)⁷, and rapid expansion of a supercritical solution into a liquid solvent (RESOLV) ⁸ are categorized as bottom-up techniques. 

The increase in surface area brought on by the smaller particle size results in increased dissolution. For substances with a high melting point, high dose, and high log p-value, the nanosuspension dosage form is strongly advised.   It is a unique strategy that might improve bioavailability. This technology is noteworthy since it uses fewer excipients than those used in pharmaceuticals, which reduces the risk of toxicity from these components. The bioavailability of BCS class 2 medicines is improved by the nanosuspension method⁹. Enhancing the solubility and dissolution rate of class-II drugs plays a pivotal role in improving their absorption kinetics and extent following oral administration. This factor holds significant importance in the pursuit of optimizing the therapeutic outcomes of these drugs¹⁰.

Anti-solvent precipitation is a viable approach for generating micro- or nano-sized drug particles. In this technique, the drug is initially dissolved in a solvent and swiftly introduced into an anti-solvent, leading to immediate precipitation through rapid desolvation ¹¹. Anti-solvents typically encompass aqueous solutions containing stabilizers like polymers and surfactants. Polymers such as hydroxypropylmethylcellulose (HPMC) and methylcellulose (MC) ¹²and polyvidone (PVP)¹³ are commonly employed due to their capacity to establish robust hydrogen bonds with drugs. These polymers can be adsorbed onto the hydrophobic surface of the drug particles, impeding crystal growth. Nevertheless, conventional anti-solvent precipitation techniques encounter inherent challenges, including difficulty in maintaining particle size post-precipitation. Typically, particles experience rapid growth and exhibit a wide particle size distribution (PSD). Recently, precise control over particle size and PSD has been achieved through the use of a static mixer¹⁴ and a confined impinging jet reactor ¹⁵. Over the past decade, ultrasound has garnered significant attention as an effective means of controlling nucleation and crystallization processes^16.17. Ultrasound irradiation has demonstrated its potential as a viable mixing technique, enhancing mass transfer and expediting molecular diffusion.

Experiments are carried out in a controlled laboratory setting, independent of a live thing, in a process known as in vitro testing. In vitro testing for nanosuspensions may assess many factors such as particle size, drug release kinetics, dissolution profile, and stability. The ultrasonication precipitation technique, which uses ultrasonic waves to break down bigger drug particles into nanoparticles floating in a liquid media, is frequently used to generate nanosuspensions. On the other hand, in vivo, testing entails examining how the nanosuspensions function and what impact they have on a living creature. Pharmacokinetics, tissue distribution, systemic absorption, and therapeutic effectiveness are frequently evaluated using animal models. 

The drug concentration and its effects on certain tissues or organs are analyzed by administering the nanosuspension formulation to the animals and collecting samples at predetermined intervals. An extensive evaluation of the bioavailability and therapeutic potential of the nanosuspension may be made by integrating the outcomes from in vitro and in vivo testing. Through this procedure, researchers may assess the nanosuspension formulation's capacity to effectively transport the medication, get beyond the drawbacks of the API suspension, and improve drug solubility. The formulation was created using the ultrasonication precipitation process. Finally, these evaluations emphasize the potential of nanosuspensions for enhancing treatment results and accelerating the development of innovative drug delivery methods.

2. METHODS

2.1. Materials

The ketoprofen-loaded nanosuspension was prepared. using precipitation–ultrasonication method. Ketoprofen, obtained with known purity, was formulated into a nanosuspension using appropriate solvents, surfactants such as Tween 80 and PVP-K30, and stabilizers. Optimization of Ketoprofen concentration was performed, and the nanosuspension formulation was characterized for particle size and zeta potential. For the in vivo studies, Wistar rats were used to check the pharmacokinetic parameters, following ethical approval and institutional guidelines. The pharmacokinetic studies utilized a concentration of the ketoprofen-loaded nanosuspension. Sample collection was performed at regular intervals, and subsequent analysis was carried out. Statistical analysis was conducted to interpret the data obtained from all experiments.

2.2 Preparation of Ketoprofen loaded nanosuspension by precipitation–ultrasonication method ^18,19

The precipitation-ultrasonication method was employed to prepare a nanosuspension. The medication was sonicated in methanol for 5 minutes at room temperature to dissolve it. A variety of antisolvent solutions were created by dissolving stabilizers in water, and these solutions were then filtered through a 0.45µm filter. A bath of cold water was used to chill the antisolvent to 3°C. The stabilizer solution containing antisolvent was then rapidly injected with the drug solution using a syringe and needle setup. The mixture was stirred at various speeds under an overhead stirrer, allowing the volatile solvent to evaporate at room temperature for 4-5 hours ²⁰. After precipitation, the sample was immediately transferred to a test tube and subjected to ultrasonication at different time intervals using a 6 mm tip-diameter ultrasonic probe. The nanosuspension was then concentrated by centrifugation at 10,000 RPM for 20 minutes using an AllegraTM 64R Centrifuge (Beckman Coulter, USA). A 40 mL batch size was used for nanosuspension preparation, and an optimized formulation was also prepared through the same method as a physical mixture.

Table 1: The coded and actual values of the variables used in the Factorial design of Ketoprofen nanosuspension

2.2.1 Experimental design using Design Expert Software

To design the formulation of the nanosuspension, it was crucial to identify the variables that could influence its properties. The response surface methodology, specifically the central composite design (CCD), was employed to determine the optimal levels of these variables. Two independent variables, surfactant concentration (%) (X1) and probe sonication time (%) (X2), were investigated at five different concentrations coded as -α, -1, 0, 1, and +α as given in Table 2. 

Table 2: The coded and actual values of the variables used in CCD-RSM of Ketoprofen-nanosuspension

The alpha value was calculated to ensure both rotatability and orthogonality in the design. The dependent variables selected as response parameters were mean particle size (Y1), polydispersity index (Y2), and entrapment efficiency (%) (Y3). A CCD matrix generated by Design-Expert software facilitated the execution of 10 experiments, including 2 factorial points, 3 axial points, and 2 replicates at the center point as given in Table 3. Statistical analysis using ANOVA, based on Fisher's test, was conducted to assess the effects of the independent variables on the responses, with a significance level of p<0.05. The quality of the model was evaluated through the multiple correlation coefficient (R2) and adjusted R2. Contour and three-dimensional surface plots were generated to visualize the relationship and interactions between the coded variables and responses. The optimization goal was to minimize particle size and polydispersity index while maximizing entrapment efficiency²¹.

Table 3: Formulation variables and their responses using CCD

Mean ± SD, (n=3); Coded high values=(-1),  Coded middle/mean values=(0), Coded low values=(+1).

Effect of formulation variables using CCD design The effect of formulation variables on the responses was performed with the help of Design Expert software version 12.  Here, formulation variables are total surfactant concentration and probe sonication time.  The responses are the particle size (Y1), PDI (Y2), and entrapment efficiency (Y3). The CCD has five different levels (-α, −1, 0, +1, +α) to study the effect of the variables. The various formulation run was displayed in Table.

2.2.2 Statistical analysis and optimization

Using Design Expert, version 11, the analysis of variance (ANOVA) helped to demonstrate the process' of statistical optimization. The level of significance for all fitted models is (p<0.05). Using CCD design, the fitted models were further assessed to produce data showing how factors affect responses. For the various replies, the response surface plot and 3-D plot were created.

2.2.3 Selection of optimized batch: 

Selection of optimized batch based on the parameters such as Particle size, Entrapment efficacy, and Polydispersity index using the statistical software Design-Expert (version 7.0.0; Stat-Ease, Inc., Minneapolis, Minnesota, USA) to create the design matrix for the study of optimization data.

By using the optimized batch, further In vivo, in vitro, and stability studies were carried out.

2.3 In vitro release study of an optimized batch of Ketoprofen-nanosuspension

It is good knowledge that a number of variables, including the composition and structure of the nanosuspension, affect how quickly a medication is releases from a nanocarrier. Ketoprofen nanosuspension development was examined for its in vitro releases ²².

Using a dialysis bag approach, in vitro release of ketoprofen was carried out in pH 1.2 HCL and pH 6.8 phosphate buffer saline solution (PBS). Before the experiment, the dialysis bags were hydrated in phosphate-buffered saline, pH 6.8. Ketoprofen-containing nanosuspension has been placed in dialysis bags. After adding pH 1.2 HCL to a beaker containing 100 mL of phosphate buffer saline pH 6.8, the dialysis bags were knotted at both ends and inserted inside. The beaker was kept at 37°C while being stirred continuously at 100 rpm.

At regular intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 24 hours, the sample was taken out. The trial was carried out at pH 1.2 HCL for the first two hours and pH 6.8 for the next twenty-four hours. To keep the sink condition, the 3mL sample of dissolving media was withdrawn and replaced with a new buffer at the same temperature. By using a UV-visible spectrophotometer (UV 1700, Shimadzu, Japan) set to a maximum wavelength of 260 nm, the quantity of ketoprofen in the aliquots was evaluated ²³.

2.4 In vivo study of Ketoprofen-nanosuspension

The current investigation adhered to the ARRIVE guidelines as well as the UK Animals (Scientific Procedures) Act, 1986 and its related guidelines. Male Wistar rats were given the protocol and approved it in accordance with standards. They were maintained at typical, controlled environmental temperatures of 30 to 32^oC ²². 

The Institutional Animal Ethics Committee (IAEC) and the Committee for Control and Supervision of Experiments on Animals (CPCSEA) committee both accepted the animal study protocol. 

In this investigation, male Wistar rats weighing 190–200 g was used in the nanosuspension containing KTP release trials. Before the trial, they had completed dietary supplementation of pellets and water. Rats were starved overnight before to the experimentation²³. Rats were administered anesthesia on the day of the experiment using a combination of 10% ketamine (0.35 mL/kg) and 2% Xylazine (0.25 mL/kg) solution ²⁴. 

To conduct the investigation, two groups of six rats each were used. The groups were labeled, and an oral gavage was used to deliver a 40mg/kg oral dosage of API suspension and nanosuspension (made with 1% Na CMC).

Table 4: Grouping of animals for the administration of dose of the drug

Using a 22-gauge catheter, a roughly 1.0 mL blood sample was taken from the rat's orbital plexus at predetermined intervals of 0, 1, 2, 3, 4, and 6 hours. Centrifugation was used to separate the plasma, which was then kept at - 40^oC for analysis for 20 min at 2000 rpm.  

Pharmacokinetic Analysis

The non-compartmental parameter was used to calculate the drug's bioavailability, T_max (time of occurrence), and C_max (maximum plasma concentration). For the estimate of pharmacokinetics parameters, Microsoft Excel for Windows 10 was utilized in conjunction with the PK solver 2.0 software.

2.5 Accelerated stability studies of Ketoprofen-nanosuspension ²⁵

The shelf life (storage or transit) of the nanosuspension goods that are intended for the market should be steady. In general, a commercial product must have a shelf life of at least one year in order to be pharmaceutically acceptable, have a good drug retention capacity, and retain particle size throughout storage. Therefore, the factors that need to be evaluated include drug leakage, particle size increase, and Zeta potential ^26,27

Studies on produced nanosuspension's stability were conducted for up to a month, and stability was determined by the nanosuspension's EE percentage, polydispersity index, and particle size ²⁸.

3. RESULTS      

3.1 In vitro release study of an optimized batch of Ketoprofen-nanosuspension

Description of the in vitro dissolution profile is provided in Fig.1. In contrast, lyophilized KTP-nanosuspension released 30% of the medication in less than 1 hour while API-suspension released just 10% of the drug in that time. The presence of the surfactant Tween 80 in the suspension causes the medication to be released from the nanosuspension. Poor water solubility, poor wettability, and maybe particle aggregation was blamed for the inadequate drug release from the API suspension. The combination of the surfactant in the nanosuspension and the increased drug release was the cause. Additionally, the size decrease of the nanosuspension was responsible for the increase in the drug dissolution rate.

The Korsmeyer-Peppas release kinetics was shown to be the best match model for the comparison of release kinetics (correlation coefficient (r2= 0.8956)).

Figure 1: The lyophilized KTP nanosuspension shows the best performance as compared to the API pure suspension

3.2 In vivo pharmacokinetic study

Male Wistar rats were used to determine an in vivo pharmacokinetic investigation. Fig. 2 and Table. 5 both showed the pharmacokinetic profile. With the use of the Windows 10 MS Excel-based PK Solver software, the pharmacokinetic profile was calculated. The comparison between the API-suspension and KTF-nanosuspension is shown in Fig.

Figure 2: The pharmacokinetic profile

Table 5: API-suspension and KTP-nanosuspension

 T_max; time to attain maximum concentration, C_max: maximum concentration, AUC: Area under Curve.  Values are expressed as mean±SD, (n=3). 

The pharmacokinetic profile of the API suspension and KTP-nanosuspension showed a significant difference.  The absorption rate is confirmed by Tmax 2 h, Cmax 0.9320.54, and AUC0-t 0.8422.01. In comparison to API suspension, the nanosuspension had a greater AUC and Cmax. This was brought on by the KTP's amorphous form in the nanosuspension. The addition of a mixture of surfactants, such as Tween 80 and PVP K-30, was the cause of the higher Cmax rate. As a result, nanosuspension increased the drug's bioavailability and simultaneous solubility. Additionally, the nanosuspension's larger particle size and higher surface area boost the solubility and bioavailability while facilitating absorption.

3.3 Stability of Optimized Ketoprofen–nanosuspension

As per ICH Q1A(R2) stability guidelines, the temperature and RH conditions for accelerated stability studies are maintained as follows: 252^oC /60%5%RH

Table 6: Stability studies of optimized batch

It was noted that the formulation remained stable at room temperature for 3 months as shown in Table 6. Ketoprofen nanosuspension is hence stable at room temperature. The findings of the stability analysis showed that the optimized batch proved highly stable at room temperature.

4. DISCUSSION

The study successfully demonstrated the potential of nanosuspensions as a drug delivery method for improving the solubility, bioavailability, and therapeutic effectiveness of ketoprofen. The formulation and design were based on lipid-based drug delivery systems and nanotechnology principles, which took into account factors such as particle size, stability, and drug release kinetics. The desired characteristics of the nanosuspension were achieved by optimisation utilizing statistical tools and experimental design techniques. By carefully adjusting formulation components including lipid type, surfactant concentration, and homogenization method, the study was successful in developing a nanosuspension with a suitable particle size, high drug loading, and good physical stability. 

Characterization results revealed that the lipid-based ketoprofen nanosuspension had much smaller particles than conventional formulations. The reduced particle size allowed for an increase in the absorption and solubility of the medicine. The increased surface area and quicker drug release kinetics of the nanosuspension further improved its high bioavailability and therapeutic efficacy. The findings demonstrate that by increasing the poor water solubility of ketoprofen, this nanosuspension has the potential to enhance its therapeutic effects. Additionally, the developed nanosuspension offers a structure for further investigation, including in vitro and in vivo experiments.

In conclusion, our thorough analysis combined in vitro and in vivo research to examine the bioavailability of ketoprofen. The findings showed that the nanosuspension formulation has a great deal of potential for successfully delivering ketoprofen into the body while addressing the drawbacks of the conventional API suspension of ketoprofen. Additionally, because of the nanosuspension's reduced particle size and increased solubility characteristics, its therapeutic effect was enhanced. These findings emphasise the potential therapeutic uses of ketoprofen and add to the increasing amount of information on cutting-edge drug delivery technologies, particularly nanosuspensions. Ketoprofen's treatment limits may be addressed, and its solubility can be improved, due to the detailed analysis of the ketoprofen loaded nanosuspension. 

Acknowledgement

We would like to express our sincere gratitude to Sunrise University, Alwar Pharmacy College, Alwar, Rajasthan, India for providing us with the research facility required to conduct our study. The support extended by the institution was instrumental in successfully completing the research project. We also thank the faculty members and staff for their cooperation and assistance throughout the project. Their guidance and expertise were invaluable in shaping the research direction and methodology. We are grateful for the opportunity to have access to the facilities and resources provided by Sunrise University, Alwar Pharmacy College.

Conflict of Interest: No Conflict of interest

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