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Herein, we have investigated the in vitro drug release from an in situ forming gel formulation including Triamcinolone Acetonide (TR) and PLGA (poly-d,l-lactide-co-glycolide) in the presence of Glycofurol (GL) and Hydroxyapatite nanoparticles (HA) as additives. We found that in the presence of these additives the drug release rate is improved. In this study we used the Taguchi method to optimize the experiment conditions and to reduce the initial burst release so 9 different formulations were compared over a period of time. In the absence of any additive, the initial burst release after 1.5 days was 8% TR. At the optimized conditions obtained by the Taguchi method, burst release decreased to 1.3%. Triamcinolone Acetonide strongly affected the release and the burst of the drug. PLGA-HA combination demonstrated a significant pH buffering capability that prevents tissue inflammation at the injection site which is the result of acidic degradation products of PLGA. Hydroxyapatite is the hydroxyl endmember of the complex apatite group. Therefore, the hydrophobic agent can be an attractive additive for modified release rate of drug from PLGA-based drug delivery systems.
Keywords: Triamcinolone Acetonide, Glycofurol, Hydroxyapatite, PLGA, In Situ, Taguchi Method.
In the recent years, research and development of new injectable drug delivery systems have improved noticeably (1-3). This consideration powered by the advantages of these drug delivery systems, which includes easy to use, target delivery for a site-specific organ/cell (4-6), enhanced delivery periods, decreased undesirable side effects common to most forms of drugs by reduction of body drug dosage, and much more comfort and patient compliance. (7).
The in-situ forming implants are one of common techniques in field of injectable drug delivery systems which has grown in the past few years. In this method, drug and biodegradable polymer are dissolved in an organic solvent. This liquid formulation administered by subcutaneous injection which generates a (semi-) solid depot. This drug delivery system is a good alternative for implant devices for a number of reasons; first, the process causes much less pain in comparison to implants, which requires local surgery. Secondly, it is possible for localized or systemic drug delivery to maintain for a long period of time, ranging from one to several months. By getting constant, 'infusion-like' plasma level time profiles, parental depot systems could minimize side effects. Some of other advantages of this technique are dose reduction from the avoidance of peaks and valleys, and enhancement of patient compliance by reducing frequency of the application. In situ forming depot systems are relatively simple to produce from polymers gained from this approach; this makes a manufacturing advantage. The costs of the production process is less for this method since microspheres have to be washed and isolated (8).
One of the alternatives of conventional biodegradable systems over the last decade has been in situ forming biodegradable drug delivery system due to its patent-friendly and cost-effective characteristics. There are in situ forming implants and in situ forming microparticles (ISM) depending on the produced depots in body. To form an in situ forming implant, the basis is a drug-containing biodegradable polymer solution in a biocompatible organic solvent. When the solution enters the body, the solvent diffuses in the aqueous environment. This leads to polymer precipitation and formation of an implant at the injection site (9-13). The most popular solvent uses in this method is N-methyl-2-pyrrolidone (NMP).NMP is classified as a Class II solvent (ICH Guidelines) and has good biocompatibility (13). The most successful biodegradable and biocompatible polymer used in developments of medicine is Polylactide-polyglycolide (PLGA). The reason for this success is that PLGA undergoes hydrolysis in the body to produce the biodegradable metabolite monomers (14). Lactic acid and glycolic acid are the biodegradable and bio compatible metabolites of PLGA (Fig. 1).
Numerous properties of PLGA on drug release such as molecular weight, lactide/glycolide ratio, and thermal functional groups have been thoroughly investigated in popular PLGA systems. To make polymer degradation and drug release more rapid, it is appropriate to use a lower molecular weight PLGA. If the lactide content decreases, it will result in a slower polymer degradation and as a result, a slower drug release (15).
The technique of defining and investigating all possible conditions in an experiment involving multiple factors is known as the design of experiments (16).
A common approach to optimize operating parameters of a particular process is to perform all or some of the possible experiments employing one at a time, i.e. varying a parameter while keeping the others constant, or trial and error methods with the design variables to find a feasible or optimum condition. To avoid spending a lot of time and money when the variables are numerous, a method called factorial design is used. This technique aims to discover the combinations of factors that give the best combination. However, when many factors and levels are being studied, it is difficult to employ a full factorial experimental design (17).
As an in situ forming formulation in this study, parameters which affected release rate of Triamcinoline Acetonide loaded on PLGA in the presence of other additives are evaluated. The process parameters are optimized using the Taguchi method; a method providing a complete system for improving and optimizing product and process quality (18).
Triamcinolone Acetonide (TR) is a steroidal anti-inflammatory drug usually administered by the parenteral route (Fig. 2).
Drug release from biodegradable PLGA polymer can be evaluated by burst release rate and period of release which are two key Parameters. These Parameters are affected by the drug loading and type of additives which are considered as main objectives for optimization the burst and long term release.
Materials and methods
The following chemicals were used as received: PLGA, Resomerƒ’ RG 504H (MW=48 KD) (Boehringer Ingelheim Int. Co., Germany) as polymer, Triamcinolone Acetonide (TR, Pharmabios, Italy) as drug substance, 1-methyl-2-pyrrolidone (NMP, Merck Co.) as solvent, Methanol (HPLC Gradient Grade, Merck Co.) Acetonitrile(HPLC Gradient Grade, Merck Co.), NaOH (Merck Co.), Tetrahydrofurfuryl alcohol, Polyethylene glycol ether (Glycofurol, Merck Merck Co.), and Hydroxyapatite nanoparticles (HA, Sigma-Aldrich) as additives.
Preparation of In Situ Gel
Homogeneous polymer solutions (33% w/w) were prepared from PLGA in NMP. Furthermore different amounts of drug were added and dissolved completely in all solutions and then the additives were added in accordance with Table 1. The efficiency of prepared formulations was calculated based on drug release. In order to study drug release properties the experiments were carried out in glass vials type ‰ (15 ml) due to the lowest drug absorption of these kinds of vials. For this purpose, the vials were charged by the polymer solutions and accurately weighted right away and then the vials filled by 10 ml phosphate buffer (PB) (0.03 M, pH= 7.4) to avoid solvent exchange by air. All vials incubated at (37±0.5) °C. The entire receptor phase (10 ml) was withdrawn at predetermined time intervals and was replaced by the same amount of freshly prepared receptor medium.
The drug release was determined by a validated high performance liquid chromatography (HPLC) method. The reverse phase C-18 column, 4.6´250 mm (NovaPakƒ’, Waters) was used. The mobile phase was an isocratic elusion of a mixture of deionized water (62), acetonitrile (33), and methanol (5). UV detection was achieved at 254 nm. Column temperature was at 25 °C.
Analytical method was developed using changes in mobile phase composition to obtain the best resolution between Triamcinolone Acetonide, Poly Lactide, and Glycolic acid peak. Afterwards the method was validated. Each experiment was performed in triplicate.
The experiments were designed by Taguchi method. In order to determine the best formulation compositions, three Parameters including Triamcinolone Acetonide, Hydroxyapatite nanoparticles and Glycofurol, as well as three levels for each Parameter (Table 1), and also the fractional factorial design, namely, a standard L9 orthogonal array (19) were employed (Table 2). To reduce interaction among the Parameters, this orthogonal array was selected. Each row of the matrix represents one run. However, the sequence of these runs is randomized. As shown in Table 1 three different levels related to each Parameter are represented as "1", "2" and "3".
The parameters and their levels are shown in Table 1. The standard L9 orthogonal array is given in Table 2. The QUALITEK-4 software was used for design of experiments and results analysis. For each run, three samples were prepared so the results are the average of three individual analyses.
Results and Discussion
The release profile of Triamcinolone Acetonide (TR) in PLGA for different formulation compositions are shown in Fig. 3. A literature survey illustrated that special aspects of drug release through PLGA medium have been studied. In the present study, to reduce burst release associated with PLGA-based delivery systems in situ formulation, we have prepared 9 different PLGA-based drug formulations in the presence of the above mentioned additives according to Table 1. The experiments were run based on Taguchi design in Table 2.
The release rates of a variety of formulations containing different levels of additives then were measured (Table 3). We found that the presence of these additives especially Hydroxyapatite improved the period of release. Hydroxyapatite is a naturally occurring mineral form of calcium apatite with the formula of Ca5(PO4)3(OH), but is usually written as Ca10(PO4)6(OH)2 to denote that the crystal unit cell comprises two entities. Hydroxyapatite is the hydroxyl endmember of the complex apatite group (Fig. 4).
Most of the drug loaded formulations show a biphasic release pattern (Fig. 5) wherein there is an initial burst followed by a sustained release (20, 21). The high initial release may be due to the presence of free and weakly bound drug on the surface of carriers.
To identify the main Parameters influence on burst release the 1.5 days release data (Fig. 6) were considered as the basis for calculation. The analysis of the results (Table 3) carried out using QUALITEK-4 software. In Taguchi method the main effect of control Parameters indicates the trend of influence of a Parameter. The key effects were calculated using average release. The results indicate the drug loading effect on the release profile (Fig. 7).
Data analyses based on analysis of variance (ANOVA) are given in Table 4. ANOVA is another technique, suggested by Taguchi method, to optimize the results. These data display relative influence of factor and interaction to the variation of results. ANOVA is similar to regression which is used to investigate and figure the relationship between a response variable and one or more independent variables (22).
The optimal combination of process parameters can be predicted with the performance characteristics and ANOVA analyses (23). The contribution of each parameter on burst release optimization and expected result in the optimum condition are listed in Table 5.
According to the Taguchi method, Triamcinolone Acetonide has the highest contribution in decrease of burst release. The best percentage for control Parameters are obtained as Triamcinolone Acetonide (15 %), Hydroxyapatite (1%), and Glycofurol (3%).
At the optimum conditions, burst release is decreased to 1.3%. The drug contents in PLGA- and PLGA-HA- based formulations were 5, 10 and 15% (drug/PLGA). These starting values of TR for PLGA and PLGA-HA formulations were taken as the 100% starting values of the released drug. The drug release of TR from PLGA (Fig. 3) and PLGA-HA (Fig. 6) were carried out in PB (pH 7.4). As can be seen the release profiles of PLGA and PLGA-HA formulations are different. PLGA release profile is characterized by a typical biphasic drug release and normally exhibited by biodegradable polymers, an initial burst followed by a sustained release. However, PLGA-HA is characterized by triphasic drug release. During the first 1.5 days, a small 'burst' release was observed for PLGA (8%) and PLGA-HA (4.5%) based on the total amount of loaded drug. The initial burst is caused by the rapid release of the near surface drugs. The second plateau phase for PLGA-HA lasted around 20 days with a low dose of TR released. The plateau-phase was followed by a third stage with decreasing drug release lasting for over 100 days. A gradually increased release during the first 20 days was followed by a slow release at a nearly constant rate, at 0.05% per day for PLGA-HA. The cumulative drug release profile of TR (15%) from PLGA is as well as PLGA-HA whereas these release profiles are 1.5 and 2 times of TR (10%) and TR (5%) respectively (Fig. 3, 6). These results shows independent behavior of TR (15%) from additives roles but in TR (5% and 10%) the HA can control the release rate. The triphasic drug release is a result of the following stages: the initial burst is followed by drug diffusion, and the polymer degradation is occurred at the third phase. The second phase is governed by swelling of the PLGA by internal diffusion of water during dissolution of TR and diffuses out in low dose. This drug release phase continues about 20 days. The precipitation of PLGA followed a two phasic mechanism. After injection a thin layer PLGA is formed immediately in the injection site. Dissolved NMP and TR are diffused through this polymer shell into aqueous environment, and water diffused inside at the same time. As hardening of PLGA is increased due to further precipitation of the polymer, the diffusion distances are increased. According to the Einstein-Smoluchowski equation (1) (24), where D, d, and t are diffusion coefficient, distance and time respectively, as diffusion distances inside the implant are increased, the diffusion time for NMP outside and water inside is increased (26).
During the incubation of PLGA and PLGA-HA in PB, pH was decreased to about 5 and 6 respectively. In the presence of HA in drug formulation the decrease of pH value was more slowly (Fig. 8).
Compared to PLGA, PLGA-HA demonstrated a significant pH buffer capability. That is, the media were buffered in agreement with other studies (25). After 20 days, even though the PLGA was greatly degraded (Fig. 9a), the PLGA-HA was only partially broken (Fig. 9b). More slowly degradation of PLGA-HA is due to the presence of HA as a bisphosphonated compound which is a chelating component and can be encapsulated into PLGA matrix. Meanwhile this additive causes low burst and controls the overall rate of drug release as shown in Fig. 5.
In this study we applied the Taguchi method to optimize the in situ gel formulation of Triamcinolone Acetonide and PLGA in the presence of Hydroxyapatite and Glycofurol as additives. The drug release followed a linear pattern throughout the dissolution after 1.5 days using PLGA and PLGA-HA. The amount of released TR after 1.5 days, irrespective the type of formulation, was less than 8%. Different formulations were achieved by Taguchi method and interestingly we found that burst release was decreased to 1.3% using TR (15 %), HA (1%), and GL (3%) formulation.
Analysis of variance (ANOVA) showed that TR concentration has the highest contribution in the control of burst release. The study of drug release continued over 125 days in phosphate buffer. This prolonged delivery periods will decrease body drug dosage and possible undesirable side effects of Triamcinolone Acetonide. PLGA-HA combination demonstrated a significant pH buffering capability that prevents tissue inflammation at the injection site which is the result of acidic degradation products of PLGA. The drug release pattern developed during this study can be considered as a novel formulation in drug delivery systems. This is useful for routine drug delivery research particularly in situ gel formulation and burst control for high potent drugs.
The authors would like to thank the Exir Pharmaceutical Co. (Boroujerd, Iran) for financial support.