AU2021105101A4 - Fabrication of lipid based nanocarrier drug delivery for clarithromycin - Google Patents

Abstract

: In the present invention solid lipid nanoparticles were successfully prepared by method namely micro emulsion technique with temperature modulated solidification technique were developed in our laboratory. Clarithromycin was incorporated in the SLNs prepared via micro emulsion with the temperature modulated solidification process. The developed techniques were simple, reproducible, prepared nanoparticles without the need of organic solvents or any sophisticated instruments and have the potential to easily scale up for large scale production. The release pattern of the SLN Dispersion indicates the slow release of the drug, that indicates the it increases the gastric residence time of the drug available to the gastrointestinaltract.

Description

Fabrication of Lipid Based Nanocarrier Drug Delivery for Clarithromycin

Summary: In the present invention solid lipid nanoparticles were successfully prepared by method namely micro emulsion technique with temperature modulated solidification technique were developed in our laboratory. Clarithromycin was incorporated in the SLNs prepared via micro emulsion with the temperature modulated solidification process. The particle sizes of SLN dispersion prepared was below 500 nm. SLN dispersions were stable for 15 days under study. The SLNs appeared to be denser in the core with a well-defined shell and the particle size was in concordance with particle size analysis data obtained from dynamic light scattering. The drug encapsulation efficiency was found to be approximately 95% which may be due to the water insoluble nature of Clarithromycin leading to rapid partitioning into the lipid phase and hence increased encapsulation into the SLNs. The drug loading capacity were up to 8.5%. And release pattern of the SLN Dispersion indicates the slow release of the drug, that indicates the it increases the gastric residence time of the drug available to the gastrointestinal tract.

Background: Drug delivery research is clearly moving from the micro to the nano size scale. Nanotechnology is therefore emerging as a field in the medicine that is expected to elicit significant therapeutic benefits. The development of effective nano delivery systems capable of carrying a drug specifically and safely to a desired site of action is one of the most challenging tasks of pharmaceutical formulation investigators. The nano delivery system mainly includes Nanoemulsions, lipid or polymeric nanoparticles and liposome.

Recent advances in nanoparticulate system, including solid lipid nano particle and polymeric self assemblies are highlighted as potential ideal drug delivery system for poorly soluble, poorly absorbed and labile substances. New function arising from nanosizing such as improved solubility, target ability and adhesion to tissue is described as well as the use of such function in developing a new drug delivery system. Recent advances in drug design and delivery, have also led to the development of an increasing number of

1. Highly lipophilic drug molecules which may be substrates for intestinal lymphatic transport. 2. Macromolecular biotechnology products which appear to be absorbed into peripheral lymphatic after SC injection and 3. A range of particulate colloidal system (micro particles, liposome) which may facilitate lymphatic transport of drug molecule with little intrinsic lymph directing capacity. (Preface, 2001) Lipids as a carrier, in their various forms, have the potential of providing endless opportunities in the area of drug delivery due to their ability to enhance gastrointestinal solubilization and absorption via selective lymphatic uptake of poor bioavailable drugs. These properties can be harvested to improve the therapeutic efficacy of the drug with low bioavailability, as well as to reduce their effective dose requirement. The physicochemical diversity and biocompatibility of lipids and their ability to enhance oral bioavailability of drugs have made lipid nanoparticles very attractive carriers for oral drug delivery. Lipid nanoparticles based on solid matrix have emerged as potential drug carriers to improve gastrointestinal (GI) absorption and oral bioavailability of several drugs especially lipophilic compounds. The unique properties of lipids viz, their physiochemical diversity biocompatibility and proven ability to enhance oral bioavailability of poorly water soluble, lipophilic drugs through selective lymphatic uptake have made them very attractive candidates as carriers for oral formulations. With the above promises, the emerging field of lipid - based oral drug delivery system (LBODDS) has attracted considerable academic attention. Perhaps, some of the reasons for this include the complexity of their physiochemical properties, challenges is stability and manufacturing at the commercial scale, limited solubility of some poorly water soluble drugs in lipid, their pre-absorptive gastrointestinal processing, a lack of knowledge about the in vivo behavior and influence of co- administered drugs/lipids and finally, the lack of predictive in vitro and in vivo testing methodologies. In spite of these limitations, lipids definitely offer the potential for enhancing drug Bioavailability, though the formulation opportunities are yet to be fully explored. Lipid nanocarriers such as solid lipid nanoparticles (SLN) and nanostructure lipid carriers (NLC) have attracted great attention in oral drug delivery. SLN are composed of biodegradable lipids like highly purified triglycerides, monoglyceride, hard fats, complex glyceride mixture or even waxes which are solid at physiological temperature where as NLC contain a mixture of liquid lipids (oil) and solid lipids in appropriate proportions. Presently, many research groups are trying to explore the possibility of using solid lipid nanoparticles (SLN) as drug carriers. The concept of SLN was first investigated a decade ago to unravel problems associated with other colloidal drug delivery systems, such as instability and non-biodegradability. SLN are widely used to improve bioavailability and to achieve sustained release. Solid lipid nanoparticles are prepared from lipids which are solid at room temperature as well as body temperature. Different solid lipids are exploited to produce solid lipid nanoparticles. A clear advantage of the use of lipid particles as drug carrier system is the fact that the matrix is composed of physiological components that is excipients with generally recognized with safe (GRAS) for oral and topical administration which decreases the cytotoxicity. Solid lipid nanoparticles (SLN) introduced in 1991 represent an alternative carrier system to traditional colloidal carriers, such as emulsion, liposomes and polymeric micro and nanoparticles.SLN are aqueous colloidal dispersions, the matrix of which comprises of solid biodegradable lipids.SLN are sub-micron colloidal carriers ranging from 50-1000 nm, which are composed of physiological lipid, dispersed in water or in aqueous surfactant solution. Each of the currently investigated particulate carriers (polymeric nanoparticles, lipid emulsion, and liposome) possesses specific advantage and disadvantage. The particulate colloidal carriers suffer from certain disadvantages like slow degradation which can cause toxic effects on reticuloendothelial cells. Solid lipid nanoparticles are at the forefront of the rapidly developing field of nanotechnology with several potential applications in drug delivery, clinical medicine and research as well as in other varied sciences. Due to their unique size dependent properties lipid nanoparticles offer the possibility to develop new therapeutics. Solid lipid nanoparticles offer unique properties such as small size, large surface area, and high drug loading capacity and are attractive for their potential to improve performance of pharmaceuticals nutraceuticals and other materials. Oral delivery of drugs with poor aqueous solubility and poor enzymatic and/or metabolic stability is very challenging. However, the advent of nanotechnology has revolutionized the field of oral drug delivery. The review provides an overview of various nano architectures such as nanosuspensions, lipid and polymeric nanocarriers, inorganic nanostructures and describes advantages and challenges associated with their efficient delivery. Among various nano-architectures, only nanosuspensions and spontaneously emulsifying systems have succeeded in reaching pharmaceutical market. Oral route is the most preferred route for drug administration due to greater convenience, less pain, high patient compliance, reduced risk of cross infection, and needle stick injuries. Major portion of the drugs delivery market is occupied by oral drug delivery system. However oral drug delivery is looking for newer avenue due to realization of the factors like low drug solubility, poor GI absorption, rapid metabolism, high fluctuation in the plasma level and variability due to food effects. These factor causes disappointment leading to failure of conventional delivery system. From the last decades, oral drug delivery has taken a new dimension with the increasing application of lipids as carriers for the delivery of poorly water-soluble drugs.

BRIEF DESCRIPTION OF THE DRAWINGS Other objects, features, and advantages of the embodiment will be apparent from the following description when read with reference to the accompanying drawings. In the drawings, wherein like reference numerals denote corresponding parts throughout the several views. Preferred embodiments of the present invention are herein further described, by way of non limiting example only, with reference to the accompanying tables, in which:

Table 1: Formulations prepared with different types of lipids to optimize the particle size Table 2: Formulation prepared by stearic acid and Glyceryl Mono Stearate Table 3: Optimized formulations prepared to form finished product Table 4. Analytical method for characterization of the drug Table 5: In vitro release Profile of the Drug Fig: A stable micro emulsion view

Fig2. IR spectrum of sample Fig 3. Area vs concentration curve Fig 4. SEM solid lipid nanoparticles Fig 5. Cumulative drug release vs time

MODES FOR CARRYING OUT THE PREFERRED EMBODIMENTS The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, the singular forms "a", "an", "the" include plural referents unless the context clearly dictates otherwise. Further, the terms "like", "as such", "for example", "including" are meant to introduce examples which further clarify more general subject matter, and should be contemplated for the persons skilled in the art to understand the subject matter. Although this invention has been described in conjunction with the exemplary embodiments' below, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the exemplary embodiments of the invention as set forth above are intended to be illustrative and not limiting. Various changes may be made without departing from the spirit and scope of the invention.

Infra-Red Spectroscopy

The infrared spectrum of clarithromycin, were obtained in KBr pellet. The 20mg sample was sent for the IR analysis. The spectral peaks have been assigned to various molecular vibration; these are contained in following table:

HPLC Method A simple, sensitive and rapid reversed phased high performance liquid chromatographic method developed for the quantitation of the Clarithromycin. Like erythromycin, it has no conjugated double bond in the lactone ring hence significant UV absorbance is only obtained at wavelength below 210nm. Hence HPLC method was chosen as analytical method for the drug Clarithromycin. Preparation of stock solution The mother stock for the preparation of standard calibration curve for HPLC was prepared by dissolving 100mg of the drug in to the 100ml of methanol in a volumetric flask of 100ml.The solution was kept in the Sonicator for 2min.The solution prepared was of concentration 1000pg/ml of strength. Preparation of standard drug solution and validation samples The mother stock was treated as working stock, from which dilutions of 100,150,200,250,300pg/ml solution for the standard calibration curve were prepared. All the dilutions were prepared in the mobile phase. The blank sample was containing HPLC grade methanol only. Preparation of Buffer solution 0.067M Monobasic potassium phosphate buffer of pH 4.00 was prepared by weighing accurate 9.11g of KH 2PO4 in 1000ml of volumetric flask. Deionized water was added to dissolve it, volume was made up by 1000ml. The pH of the buffer solution was maintained by dilute solution of the o-phosphoric acid, by the use of pH meter. The buffer then was filtered by Vacuum filtration assembly using filter size of .2pm. Preparation of Mobile Phase Mobile phase was prepared by mixing 70 volumes of HPLC grade methanol and 30 volumes of 0.067M Monobasic potassium phosphate buffer of pH 4.00. The mobile phase was sonicated for min before use. Chromatographic Condition

Column: Column C1 8 , 250mmx4.6 mm i.d, packed with octadecylsilane bonded to porous silica pm particles. Mobile Phase: A mixture of 70 volumes of Methanol and 30 ml of 0.067M of monobasic potassium phosphate buffer of pH 4.00. Flow Rate: 1ml/min Spectrophotometer UV Detector: Set at 210nm. Injection Volume: 20pl fixed volume injection HPLC Method Validation A method validation provides documented evidence and a high degree of assurance that an analytical method employed for a specific test is suitable for its intended use. Linearity The linear range of detectability that obeys Beer's Law is dependent on the compound analyzed and the detector used. The working sample concentration and sample tested for accuracy should be in the linear range. Acceptability of the linearity data is often judged by examining the correlation coefficient and y-intercept of the linear regression line for the response (area) vs. concentration. The linearity was studied in the concentration range of 100-300pg/ml of concentration with n=3. The linearity data demonstrates acceptable linearity for Clarithromycin over the range of 80 to 120% of the target concentration. The result for assessment of linearity of the HPLC method was demonstrated by the correlation coefficient (r2). The data obtained from the linearity experiments are presented in figure: Accuracy Accuracy is the measure of the closeness of the test result obtained by that method to the true value. The accuracy is usually determined by one of the four approaches: > First accuracy can be assessed by analyzing a sample of known concentration (reference material) and comparing the measured value to the true value.

> The second approach is to compare test results from the new method which results from an existing alternate, well-characterized procedure that is known too accurate. > The third approach is based on the recovery of known amounts of the analyte. This is performed by spiking analyte in blank matrices. For assay method spiked samples are prepared in triplicate at three levels over a range of 50-150% of the target concentration. The percent recovery should be then calculated.

> Fourth approach is the technique of standard additions, which can also be used to determine recovery of spiked analyte. This approach is used if it is not possible to prepare a blank sample matrix without the presence of analyte. In the present study, a number of different dilutions were prepared in the triplicate; percent recoveries of the response (area/concentration) were calculated. Precision The precision is the measure of degree of repeatability of the analytical method under normal operation, and normally expressed as the percent relative standard deviation for a statistically significant number of samples. The precision data was assessed by the three replicate samples analyzed at each level in the accuracy studies. Method of preparation of solid lipid nanoparticles Preparation of solid lipid nanoparticles via micro emulsion method was performed at a temperature above the melting point of the lipid. Formulations were prepared using Glyceryl Monostearate as the solid lipid, Tween 80 as the surfactant, and deionized water as the dispersion medium. Appropriate quantity of lipid was melted at 80°C in a beaker, under continuous stirring. 2% aqueous solution of surfactant was also kept at same temperature as lipid melt. Weighed the quantity of the drug was added in the lipid melt. Keeping the temperature constant, the aqueous surfactant solution was added slowly in to the lipid phase with stirring, and mixed at the same temperature. This micro emulsion was transferred to the probe Sonicator chamber keeping the constant temperature for required time. Maintained the temperature of the Chiller at -20°C. A transparent, thermodynamically stable system was formed when the compounds are mixed in the correct ratio for the micro emulsion formation. This micro emulsion was rapidly cool down to temperature up to -15° C with the help of the Chiller containing Propylene glycol and deionized water in the ratio of 50:50 ratio to maintain the temperature at -20° C. Finally, the lipid particles solidified to form the SLN Dispersion of optimum size. This method was based on the temperature modulated micro emulsion method.

Preparation of the solid lipid nanoparticles was performed in the three steps:

• Selection of type of lipid

• Selection of concentration of lipid

• Final formulation by Optimizing the time of processing

SELECTION OF TYPE OF LIPIDS SLN 2 and SLN 4 were selected for the further optimization on the basis of the analysis of the reports of the size distribution and the poly dispersity index. And also the Sonication time were also increased to get a stable SLN dispersion.

EVALUATION PARAMETERS OF SOLID LIPID NANOPARTICLES Determination of particle size of the SLN Dispersion The mean diameter and zeta potential of the SLN formulations were measured by the use of instrument Malvern Zeta Sizer Nano ZS. The Malvem Zeta sizer instrument is used for the enhanced detection of small or dilute samples at very low or high concentration using dynamic light scattering with NIBS (Non-Invasive Backscatter Optics). Dynamic Light Scattering is used to measure particles and molecules size. This technique measures the diffusion of particles moving under Brownian motion and converts this to size and size distribution using the Stokes Einstein relationship. NIBS are incorporated to give high sensitivity simultaneously with the highest size and concentration range. All the particle size analysis was performed by the analysis of intensity of the size of the particles as well as the volume distributed data of the formulation. The 100pl of SLN dispersions was diluted with 1ml of deionized water for the analysis of the particle size and zeta potential of the SLN Dispersions. Drug entrapment efficiency of the SLN

Drug encapsulation efficiency of the SLN dispersions were determined by the indirect method,

2ml of the dispersion was filled in the dialysis bag keep in the 100ml of the media and kept for 24hrs.1ml of the media was sample out and analyzed in the HPLC. The concentration of un entrapped drug was calculated.

Entrapment efficiency =weight of initial drug-weight of unbound drug

weight of total drug in formulation x 100

DRUG LOADING CAPACITY Drug loading capacity of SLN dispersion was calculated by the given formula

Drug loading capacity (%)=weight of initial drug-weight of unbound drug total weight of the formulation x 100

In vitro release of the drug was studied by the dialysis bag diffusion technique method. The dialysis bag of (12000-140000 Da) could retain the particles of solid lipid nanoparticles and allow the drug transfer in to the phosphate buffer of pH 7.4 10mg/ml equivalent amount of drug from the SLN dispersion was filled in the dialysis membrane which is presoaked for about 4 hrs in deionized water. 2ml of SLN Dispersion was transferred into the dialysis bag with the two ends sealed by the thread. Then bag was placed in the beaker containing 100ml of phosphate buffer. This beaker was placed in the stirrer keeping the temperature at 37°C and for stirring at 100 rpm. The phosphate buffer in the beaker was removed at each sampling point and fresh dissolution media of same amount was added in the beaker. The concentration of the Clarithromycin was determined by the HPLC. Physical stability of the SLN Dispersion Physical stability of the SLN Dispersion was studied by the observation of size and zeta potential of the formulation. Samples were stored for 15 days for the physical stability studies. The physical stability of this SLN determine by Zeta potential.

RESULTS AND DISCUSSION IR Spectroscopy

The given spectra show the various peaks which confirm the presence of the pure clarithromycin drug. The Spectral assignments of the drug Clarithromycin were similar to the Standard group assignment values. This confirms the presence of the pure chemical entity as Clarithromycin.

Analytical Method Analytical method for characterization of the drug by HPLC Standard calibration curve for the drug Clarithromycin was prepared in the range of the concentration 100-300pg/ml of the solution. 100mg of Clarithromycin was dissolved in 100ml of HPLC grade methanol results in 1000pg/ml concentration. All the dilutions were prepared from this solution using mobile phase as diluting media. The dilutions were prepared in triplicate in ml of volumetric flask. Size of the solid lipid nanoparticles

The size of the SLN Dispersion was found to in the range of 200-400nm.Here are the reports of the intensity of the with volume distribution. The zeta potential of the SLN Dispersion was found to be in the range of -40-5Mv. Here's attach all the respective zeta potential reports Entrapment efficiency of solid lipid nanoparticles

Entrapment efficiency of the SLN Dispersion were found in the range of 85-90%. Drug loading capacity of solid lipid nanoparticles

The drug loading capacity of the final finished product were found in the range of 7.5- 8.5 %. In vitro release of the drug was studied and found that the formulation exhibits the property of the oral sustained release drug delivery, means that the SLN dispersion prolongs the residence time of the drug in the body. The release study was performed for 48hrs. The 30% of the drug was released in first 5 hrs. and the remaining 70% of the drug was released up to 48hrs. Physical Stability of solid lipid nanoparticles The physical stability study results that the particle size of the SLN Dispersion were tend to increase up to 50-100nm on storing the product under refrigerated conditions. The Zeta potential of the formulation decreases up 5Mv.

The foregoing descriptions of exemplary embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to best explain the principles of the disclosure and its practical application, to thereby enable others skilled in the art to best utilize the disclosure and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions, substitutions of equivalents are contemplated as circumstance may suggest or render expedient but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present disclosure.

Claims (6)

1. Claims: 1. Herein we claim a Formulation which exhibits lipid based nanocarrier drug delivery for clarithromycin and shows sustained drug release of drug on the site.
2. 2. The formulation claimed in 1 exhibits the property of the oral sustained release drug delivery, means that the SLN dispersion prolongs the residence time of the drug in the body
3. 3. The formulation claimed in 1 has localized drug delivery which decrease incidence of getting adverse effects.
4. 4. The formulation claimed in 1 has capacity to deliver both lipophilic and hydrophilic drug candidate.
5. 5. The formulation claimed in 1 has better control over release kinetics of encapsulated compounds.
6. 6. We also claim the method for Preparation of solid lipid nanoparticles via micro emulsion method.