Polymeric drug delivery systems are an attractive alternative to control the
release of drug substances to obtain defined blood levels over a specified time.
The patients suffering from some disease conditions such as heart disorders,
osteoporosis, tumors, often benefit from such long-term drug delivery systems
due to improved patient compliance (Schoenhammer et al.,
2009). Injectable in situ forming implants are classified into five
categories, according to their mechanism of depot formation: (1) thermoplastic
pastes, (2) in situ cross linked systems, (3) in situ polymer
precipitation, (4) thermally induced gelling system and (5) in situ solidifying
organogels. Of these, in situ polymer precipitation systems have become
commercial available so far (Hatefia and Amsdena, 2002).
The in situ Forming Implant (ISFI) systems have several advantages compared
to traditional pre-formed implant systems. Due to their injectable nature, implant
placement is less invasive and painful for the patient thereby improving comfort
and compliance. Additionally the manufacturing process required for fabrication
is relatively mild. Currently, only two FDA approved products are on the market
utilizing this type of system, Eligard® and Atridox®.
Eligard®, using the Atrigel® delivery system and
marketed by Sanofi-Aventis in the US (Medigene in Europe), is a subcutaneously
injected implant that releases Leuprolide acetate over a period of 3 months
to suppress testosterone levels for prostate cancer treatment (Sartor,
2003). Atridox® (Tolmarc Inc.) is another ISFI system that
also uses the Atrigel® delivery system to deliver the antibiotic
agent, Doxycycline to the sub-gingival space to treat periodontal disease (Buchter
et al., 2004). Some disadvantages of in situ implants are
high burst release, potential solvent toxicity and high viscosity of the polymeric
solution which may lead to problems during administration (Kranz
et al., 2001).
Dunn et al. (1999) developed an implant using
biodegradable polymer dissolved in water miscible organic solvent which undergoes
a process called liquid de-mixing when injected into aqueous phase. This technology
has been utilized for the delivery of model proteins, LHRH-antagonists, narcotic
antagonists, growth factors, anti-inflammatory agents and antibiotics (Tipton
and Fujita, 1991; Radomsky et al., 1993).
Statins like Simvastatin, Lovastatin, Fluvastatin, Rosuvastatin, Atorvastatin
are class of drugs generally used to lower blood levels by reducing the production
of cholesterol by the liver. Simvastatin is a water insoluble drug with very
poor oral absorption and a short half-life of 3 h. The bioavailability of the
drug is 5% and exhibits rapid first pass metabolism. Simvastatin is generally
administered as once daily tablet in the treatment of patients with heart disorders.
Several approaches have been investigated in order to control the levels of
cholesterol in the body. Zhang et al. (2011)
developed spherical mesocellular foam (MCF) loaded with a poorly water soluble
drug, intended to be orally administered, able to improve the dissolution rate
and enhance the drug loading capacity. They found that spherical MCF has a high
drug loading efficiency up to 37.5%. Kang et al.
(2004) prepared Self-Micro Emulsifying Drug Delivery System (SMEDDS) for
enhancing bioavailability of Simvastatin. SMEDDS form resulted in about 1.5
fold increase in bioavailability of Simvastatin compared to conventional tablet.
Bae et al. (2011) have studied the positive influences
on in vitro and in vivo osteogenesis of photo-cured Hyaluronic
Acid (HA) hydrogels loaded with Simvastatin (SVS).The results showed sustained
release of Simvastatin from these Hydrogels and had significant influence on
osteogenesis. Zhang et al. (2010) prepared Simvastatin
loaded lipid nanoparticles (SLNs) with different components to enhance its oral
bioavailability. The oral bioavailability of drug after its incorporation into
the lipid nanoparticles was improved by 3.37-fold for SLNs compared to free
drug in rats. Preparation and characterization of Polylactic acid and Polycaprolactone
nanocomposites by melt blending technique was also studied and the results showed
increase in mechanical properties and thermal stability (Hoidy
et al., 2010). White, free-flowing and spherical PLGA microspheres
were prepared using emulsion technique which showed sustained drug release (Gupta
et al., 2010; Rudra et al., 2011).
The above approaches could increase the bioavailability of the drug and prolonged
the release of the drug for a period of 12-48 h. In situ depot systems
can thus be used for long term therapy and extended release of the drug for
a period of months up to years. Implantable technology is also preferred in
the treatment of osteoporosis, mainly in the hip joint failure (Ridzwan
et al., 2006, 2007; Zuki
et al., 2006). Some of the advancements in the implantable technology
include the implantable stimulation using an inductive power system that combines
power transfer with data transmission for implantable biomicrosystem (Hmida
et al., 2007).Ion stimulation using some rare earth ions for laser
or amplifier action was also studied (Benaissa et al.,
2007). Cochlear implants and microsensor systems are currently one of the
most preferred choices for measuring and recording neural signals from auditory
nerve (Ghorbel et al., 2006). In the present
study, Simvastatin injectable in situ implants were formulated in order
to achieve drug release up to atleast 15 days. These formulations could further
be investigated to improve the bioavailability of the drug and avoid first pass
metabolism thereby improving patient compliance in hyperlipidemic patients.
MATERIALS AND METHODS
Materials: Simvastatin (SVS) was a kind gift from Marksans Pharma Ltd, Verna, Goa, Polycaprolactone (PCL) was purchased from Hi-media, Hyderabad. Poly (D, L-Lactide-co-glycolide) (PLGA) was a gift sample from NATCO Research centre, Hyderabad. All the solvents were of HPLC grade and were purchased from SD fine chemicals, Hyderabad.
Method of preparation
PCL implants: In situ implants were prepared by polymer precipitation
method. In this method PCL is dissolved in the organic phase containing acetonitrile
(ACN) and dichloromethane (DCM). Different formulations were prepared using
5-40% of polymer. To this polymer solution, 75mg of drug was added. The polymer
drug solution was stirred vigorously until clear solution was formed.
1ml of this solution was gradually injected into 50 ml of aqueous phase containing
7.4 phosphate buffer for the formation of implant (Kranz
et al., 2008; Liu et al., 2010).
PLGA implants: In this method, PLGA was dissolved in the organic phase containing Dimethyl Sulfoxide (DMSO) and dichloromethane (DCM). Polymer concentrations of 30 and 40% were prepared. The further steps in the preparation of the implant were similar to that of PCL implant.
Characterization and evaluation of SVS in situ implants: The
prepared in situ implants were evaluated for various parameters such
as drug entrapment efficiency, evaluation of in vitro release, SEM, FTIR,
evaluation of in vivo release (Kranz et al.,
2008; Liu et al., 2010).
Drug entrapment efficiency (DEE): The amount of drug entrapped was estimated by dissolving the implant in the highly basic phase using 0.1 N NaOH under vigorous shaking for 12 h. The resultant solution was filtered using No. 1 Whatmann filter paper. The drug content in the solution was analysed spectrophotometrically using UV-VIS single beam spectrophotometer at 238 nm with further dilutions against appropriate blank. The amount of the drug entrapped in the implant was calculated using the formula:
Scanning electron microscopy (SEM): The dried in situ implants
were coated for 70 sec under an argon atmosphere with gold-palladium and then
observed under a Scanning Electron Microscope (JSM-5200 SEM, Tokyo, Japan).
FTIR: The drug-polymer containing in situ implant and the pure drug were subjected to the Fourier-transform infrared spectroscopy (Shimadzu 8400 S FTIR) in order to check the possible drug-polymer interactions.
In vitro drug release studies: In vitro release studies were performed using modified diffusion apparatus using dialysis membrane (Himedia with Mwt cut off 12,000-14,000 kDa). In situ implants were placed into conical vials open on one side and closed with dialysis membrane on other side. The formulations were placed into 50 ml 7.4 pH phosphate buffer at 37°C. At 1, 3, 5, 7, 9, 12, 15, 20, 24, 28, 32, 40, 48 and 72 h time intervals, 5 mL samples were withdrawn and assayed. Each time the vials were replaced with aliquots of fresh medium. After 48 h the complete medium was withdrawn and replaced by fresh medium to avoid saturation of the medium. The drug content was measured using UV-VIS single beam spectrophotometer at 238nm. The obtained data were fitted into mathematical equations (zero order, first order and Highuchi models) in order to describe the kinetics and mechanism of drug release from the implant formulations.
Stability studies: To assess the physical and chemical stability of the in situ implants, stability studies were conducted for 1 month under different storage conditions mentioned in ICH guidelines. The samples containing optimized formulation (F4) were packed wrapped in aluminium foil inside screw capped glass vials and stored at 5±3°C, 25±2°C/60±5% RH and 40±2°C/75±5% RH. After 30 days the formulation was checked for physical appearance and drug content.
In vivo drug release studies: In vivo animal studies were done in accordance with CPCSEA Guidelines after due approval by Sri Venkateshwara Institutional ethical committee (Protocol no. SVCP/IAEC/2011/14). Drug release from in situ forming implants was examined in 12 week old male rats. The animals were divided into two groups. One group of rats was administered with 50 μL of the injectable implant containing 20%PCL solution. Other group of rats was administered with 1 mL of oral suspension of marketed tablet (ZOCOR 10mg). Blood samples were collected at predetermined time intervals at the end of 1, 7 and15 days. The blood samples were centrifuged immediately after collection and the resultant plasma was stored at -20°C for analysis. The drug content was analysed using High Performance Liquid Chromatography (HPLC).
Chromatographic system and conditions: HPLC (Shimadzu Co. Kyoto, Japan) equipped with LC-10 AT solvent delivery unit, SPD-10 AVP UV-Spectrophotometric detector, Spinchrom software, Rheodyne injector of 25 μL capacity was used. The separation was performed on C18 analytical column (250x4.6 mm, i.d.,). The mobile phase consisted of a degassed mixture of acetonitrile and pH 4.6 phosphate buffer (65:35).The mobile phase was freshly prepared, sonicated and filtered before use and delivered at a flow rate of 1.5 mL min-1. The column was maintained at ambient temperature (20°C) and the compounds eluted were recorded by the detector at 238 nm.
Sample extraction procedure: Heparinized blood samples from the animals
were centrifuged and plasma was collected into eppendorf tubes. The samples
were frozen at -20°C for storage and analysed within 7 days. At the time
of analysis Plasma (1.0 mL) was mixed with 50 μL of a mixture of acetonitrile-water
(60:40 v/v) in a 3 mL centrifuge tube. Separation of the phase from precipitate
was achieved by centrifugation at 1500 g for 3 min. The supernatant was transferred
to another centrifuge tube. Fresh acetonitrile (400 μL) was then added
to the first tube and the same extraction procedure was repeated twice. The
supernatants thus collected from the extractions of the same sample were pooled.
This fraction was finally centrifuged and evaporated to dryness under vacuum.
The samples were filtered through a Millipore filter (0.45 μm) and then
reconstituted with (200 μL) acetonitrile-water (25:75, v/v). Aliquots of
each sample (20 μL) were analyzed using HPLC (Carlucci
et al., 1992).
RESULTS AND DISCUSSIONS
Formulation and optimization: Formulations were prepared using different
concentrations of polymer PCL and two different ratios (2:1 ACN:DCM and 1:2
ACN:DCM) of solvent. Formulations before injection into buffer are clear and
transparent as shown in Fig. 1a. Upon injection of the polymer
solutions into the phosphate buffer medium, the polymer solidified as the solvent
dissipated into the aqueous medium and formed implants (Kranz
et al., 2008). The scanning electron microscopy (Fig.
7) revealed hard rod shaped structures. Based on the results, Acetonitrile
and dichloromethane in the ratio of 2:1 was selected as a solvent system as
it showed faster implant formation within 20 sec when injected into the aqueous
In situ implants containing polymer PLGA were prepared in two different concentrations of 30 and 40% in the solvent ratio 2:1 DMSO:DCM. The in situ implants using PLGA polymer formed within 20 sec when the solution was injected into the aqueous phase. The formed solid implants were soft and slightly porous in nature as shown in Fig. 1d. This ratio was found to be best suitable for quick implant formation.
|| Formation of implant using different polymers and solvent
ratios. (a) PCL and PLGA formulations before injection into aqueous buffer,
(b) PCL-SVS implant with solvent ratio ACN:DCM in ratio 2:1 after injection
into buffer, (c) PCl implant containing solvent ratio DCM:ACN in ratio 2:1
after injection into buffer and (d) PLGA implant formed after injected into
|| Formulations of SVS in situ implants using two different
|| Drug entrapment efficiency of different formulations
Effect of solvents: When water miscible solvent acetonitrile is used in higher concentration, soft rod-shaped solid implant formed within seconds as the solvent dissipated into the aqueous phase as shown in Fig. 1b. Formulations F1 to F7 were prepared by using the same solvent system.
When the water immiscible dichloromethane was used in higher amounts, solid implant was formed after 2-3 h depending on the concentration of the polymer. The implant formed was porous and patch-like solid as shown in Fig. 1c. Formulations F8 to F10 were prepared using this solvent system. Different formulations using different polymers and solvents are shown in Table 1.
Evaluation of in situ implants: The prepared in situ implants were evaluated for various parameters such as drug entrapment, in vitro drug release and in vivo drug release.
Drug entrapment efficiency: The entrapment efficiency of various formulations was studied. Drug loading percentage in the range of 70-80% was observed for F4, F5, F6 and F7. With increase in the drug to polymer ratio, the percentage drug encapsulated was also found to increase as seen in the Fig. 2. In case of F1, F2, F3 formulations, as the polymer concentration is less, only 50-60% of drug was found to be entrapped.
In F7 as the polymer concentration is higher, 83% drug entrapment was observed. In the case of F8, F9, F10 which were prepared using more proportion of DCM (ACN:DCM 1:2) the drug entrapment was found to be lower i.e; 55-65% owing to the leakage of drug into the organic phase.
The entrapment efficiency of PLGA formulations was studied. From the results it can be seen that drug loading percentage of F11 and F12 formulations was in the range of 75-79%. By increasing the drug to polymer ratio percentage of drug encapsulated also increased.
In vitro drug release studies of in situ implants: In
vitro diffusion studies were performed using dialysis membrane with 7.4
pH phosphate buffer. Comparison of in vitro release studies of various
formulations are shown in Fig. 3 and 4.
As the polymer concentration is decreased, more burst release is seen.
|| Comparative dissolution profile of F1-F7 prepared with PCL
and solvent (2:1 ACN:DCM)
|| Comparative dissolution profiles of F8-F10 prepared with
PCL and solvent (1:2 ACN:DCM)
Formulation with 20% polymer concentration (F4) showed a sustained release
of drug for 15 days and it has shown 25% of drug release at 72 h equals the
therapeutic dose for 3 days. On the 15th day, 71% of drug release was achieved
indicating that more sustained drug levels are possible in a period of one month.
The graph representing the sustained release of F4 is shown in Fig.
More prominent burst release was observed in case of F1, F2, F3 formulations. Although, F5, F6 and F7 formulations showed sustained action with less burst release, they could not reach therapeutic drug levels as shown in Fig. 3. This could be due to the fact that increased polymer concentration retarded the drug release. The formulations prepared using DCM in more amounts (F8, F9 and F10) showed clear burst effect and lesser drug release as shown in the Fig. 4. From the above results, F4 was found to be the most suitable formulation and hence was optimized for the conduct of further studies.
|| Drug release of the optimized F4 in situ implant
|| Comparison of dissolution profiles of PLGA formulations FI
In the case of formulations prepared using PLGA (F11 and F12), as the polymer concentration is decreased more burst release was observed. F12 showed more sustained release (11% at 72 h) when compared to the F11 (15% at 72 h) and could not reach therapeutic levels of drug as shown in Fig. 6.
Prediction of drug release mechanism: The optimized formulation was fitted into different drug release plots. The formulation showed first order drug release pattern and Fickian diffusion.
Stability studies: The stability studies of PCL - SVS in situ implant (F4) at different temperatures as per ICH guidelines like 5±3°C, 25±2°C/60±5%RH, 40±2°C/75±5%RH was studied for 30 days. The physical appearance of the formulation was clear and transparent and it was observed that there was no colour change indicating physical stability. The drug content was analyzed and the data is presented in Table 2. From the data, it is observed that there was negligible change in the drug content indicating chemical stability.
Characterization of in situ implants
Surface morphology by scanning electron microscopy (SEM): SEM analysis
was performed to understand the surface Morphology of the implant. The cross
linking and the rod shaped structures are clearly seen as shown in Fig.
|| Interpretation of stability studies
|| SEM photograph of PCL in situ implant
Drug interaction studies by FTIR: FTIR spectra of pure drug and drug
loaded PCL in situ implants F4 were studied to understand any possible
interactions. It was observed that the main functional group peaks are in the
range of reported values in both the pure drug and the formulation indicating
no drug-polymer interactions. The main functional groups in the drug are OH
group and C = O group. The reported frequencies are 3200-3650 cm and 1700-1725
cm-1. The observed values for OH group are 3550 cm-1 (drug)
and 3518 cm-1 (implant). The observed values for C = O group are
1724 cm-1 (drug) and 1712 cm-1 (implant) as shown in Fig.
8 and 9.
In vivo pharmacokinetic studies: The drug release kinetics in rats was investigated for a period of 15 days and the drug content data of the test and the control are shown in the Table 3 and 4. Sustained release of drug for 15 days was observed and Cmax was achieved on the 15th day. When compared to control, test formulation has shown 3 fold increase in the bioavailability. On the 15th day, the drug concentration of test and control were found to be 37.30±25.13 μg mL-1 and 1.02±0.58 μg mL-1, respectively indicating there is clear sustained release of the drug from the implant. The chromatograms indicating the peaks of test and control with retention time 15 min are shown in Fig. 10 and 11.
|| FTIR spectrum showing drug functional groups-Simvastatin
|| FTIR spectra of Simvastatin in situ implant
|| Chromatogram showing the test peaks of simvastatin
|| Data for in vivo drug release of test containing injectable
|| Data for in vivo drug release of control containing
|| Chromatogram showing control peaks of Simvastatin
Simvastatin is a lipid lowering drug known for anti-hyperlipidemic activity. It is commercially available as tablet dosage form. The objective of the work was to formulate injectable in situ implant of the drug with two different polymers to sustain drug release for atleast 15 days.
The injectable in situ implants of Simvastatin were prepared using polymers PCL and PLGA using different solvent ratios like ACN:DCM in the ratio 2:1 and DCM:ACN in the ratio 2:1 for PCL formulation and DMSO:DCM in the ratio of 2:1 for PLGA formulation. All the formulations were tested for drug content, SEM, in vitro drug release, stability, effect of solvents, in vivo drug release. Injectable in situ implants with 15 days release of drug (through dialysis membrane) could be successfully formulated in PCL 20% with ACN:DCM in the ratio 2:1. So, it is selected as optimized formulation of injectable in situ implants.
The drug content of the implants was checked and it was observed that with increase in the drug to polymer ratio, the percentage drug encapsulated was also found to increase and less burst effect was seen. With decrease in the polymer ratio, the percentage of drug encapsulated was found to decrease and more burst effect was observed.
In situ implants were prepared using two different ratios of solvents. It was observed that the implants prepared with polymer PCL and the solvent ratio ACN:DCM in the ratio of 2:1 formed in 20 sec when the polymer solution was injected into the buffer solution and the implants formed were soft. The implants with polymer PCL and the solvent ratio DCM:ACN in the ratio of 2:1 has taken 4 h to form the implant and the texture of the implant is brittle and porous.
The prepared injectable in situ implant possessed satisfactory physicochemical
characteristics. In vitro release studies were conducted and the optimized
formulations followed first order kinetics and Fickian transport mechanism.
The surface morphology of the implant carried out by SEM showed crosslinking
and rod shaped structures as against porous implants resulted in previous studies
(Kranz and Bodmeier, 2008). The drug excipient interactions
analysis proved that there is no chemical interaction between the drug and the
The drug release from all the implants was found to follow diffusion-controlled
mechanism. Dialysis membrane was used as the diffusion barrier for the drug-release
studies. From the results it was observed that Simvastatin in situ implant
using 20% PCL concentration showed higher drug release than the formulations
using other polymers. The drug release was found to have sustained up to 15
days as compared to PLGA-Secnidazole implants prepared in previous studies with
97% drug release within 24 h and 92% drug release in case of PLGA-doxycycline
implants with in 24 h (Gad et al., 2008). Some
extended release studies showed a 9% drug release after 8 days from Poly (sebacic-co-ricinoleic-ester-anhydride)-gentamicin
implants (Krasko et al., 2007).
Stability studies were performed and the results showed that the formulation
is stable at different temperatures. It was observed that there is no physical
change in the formulation. In vivo studies were performed on Wistar rats
using 2 groups (n = 6) as a test and control. Studies showed that injectable
in situ implant containing PCL 20% was able to provide sustained release
of drug for 15 days. When compared to marketed formulation (Zocor®
10mg) which showed a release up to 3 h, the drug release of the test formulation
was found to have sustained up to 15 days. Bioavailability of Simvastatin was
found to improve by 3-fold when compared to control. Maximum amount of drug
(37.3 μg mL-1) was released from the implant on 15th day. This
indicated sustained action of drug when formulated as an implant. The in
vivo studies performed in the previous studies showed Cmax of
5.57 mg mL-1 for the Bupivacaine Hydrochloride in situ implant
containing PLGA and reached after 1 h (Kranz et al.,
2008). In another study there was continuous release seen up to 28 days
with initial release up to 29% in case of thymosin alpha1 in situ implants
(Liu et al., 2010).
Rationale of the present study was to prevent first pass metabolism of the drug, to increase the bioavailability, to decrease the frequency of administration and to sustain the drug release atleast for 15 days. The in vitro release studies suggest that release rate was related to drug: polymer ratio. There are no Drug-Excipient interactions and formulation is stable for 30 days. Pharmacokinetic studies suggest that formulation has sustained the release for 15 days and there is 3 fold increase in bioavailability. From all the optimized parameters, we can conclude that injectable in situ implants can be successfully administered for the chronic disease conditions which need a long term therapy with less therapeutic dose.