However, when administered orally it undergoes extensive hepatic first pass metabolism due to the action of enzyme cytochrome P450 3A4 which is responsible for its low oral bioavailability of 5% ^[9].

Based on the ability of lipid based drug delivery systems to enhance the bioavailability of lipophilic drugs by lymphatic transport, formulation of a solid lipid nanoparticulate delivery system that can potentially enhance bioavailability of simvastatin was aimed at. The development and optimization of solid lipid nanoparticles of simvastatin has been extensively investigated by the authors and is reported elsewhere^[^10]. Briefly, the Solid lipid Nanoparticles of simvastatin were prepared by solvent injection technique and optimized using 2³ full factorial designs. The design was validated by extra design checkpoint formulation (F9), and the possible interactions between independent variables were studied. The responses of the design were analyzed using Design Expert 7.1.6. (Stat-Ease, Inc, USA), and the analytical tools of software were used to draw Pareto charts and response surface plots. On the basis of software analysis, formulation F10 with a desirability factor of 0.611 was selected as optimized formulation and was evaluated for the independent parameters. The optimized formulation showed particle size of 258.5 nm, % EE (entrapment efficiency) of 75.81%, with of 82.67% CDR (cumulative drug release) after 55 h. The release kinetics of the optimized formulation best fitted the Higuchi model, and the recrystallization index of optimized formulation was found to be 65.51% ^[10]. Thus the present investigation reports the experimentations conducted for evaluation of pharmacokinetic parameters and organ distribution by radiolabeling technique. Biodistribution and pharmacokinetic study of nanoparticulate formulations by radiolabeling with Tc-99m are widely reported in literature ^[11-14].

In general, administration of the conventional simvastatin tablets suffer from the following limitations (i) gastric instability, because lactone form of simvastatin gets hydrolyzed in acidic/alkaline condition of GI tract; (ii) Extensive first pass metabolism of simvastatin from tablets by cytochrome 450 3A4 system and (iii) in the liver, simvastatin gets converted to its active metabolites and inhibits HMG Co A reductase enzyme. However, despite the inhibition of this enzyme, hepatic cholesterol level does not fall because hepatocytes compensate any drop in cholesterol level by increasing the synthesis of LDL receptor protein along with HMG Co A reductase ^[9]. The SLNs of simvastatin, a lipid-based drug delivery system capable of enhancing the bioavailability of lipophilic drugs by lymphatic transport is proposed to overcome these problems and was therefore investigated.

MATERIALS AND METHODS:

Materials:

Simvastatin was kind gift of Ranbaxy Lab, India. Technetium 99m (Tc-99m) was generously supplied by regional center for radio pharmaceutical, BRIT, Delhi, India. Swiss albino Mice were obtained from NICD, New Delhi, India. Stannous chloride was obtained from Sigma Chemicals, St. Louise, MO. All other chemicals are of analytical grade.

Radiolabeling of formulations:

Radiolabeling of simvastatin suspension and optimized simvastatin SLNs was done by direct method using stannous chloride as reducing agent^[^15]. Briefly, 1 ml of simvastatin suspension and 1 ml of simvastatin SLNs was separately mixed with stannous chloride. To adjust the pH of this mixture, 10μl of sodium hydrogen carbonate solution (1%) was added. Then 0.1 ml of freshly eluted Tc-99m (2 mCi) was added to each preparation, mixed well and incubated at 25˚C temperature. Final radioactivity present in the preparation was checked using gamma ray counter (Capintech, CAPRAC – R, NJ, USA). The amount of stannous chloride, pH of the final preparation and incubation time was optimized.

Optimization of Radiolabeling efficiency:

The effect of the amount of stannous chloride, the final pH of the preparation, and the incubation time on labeling efficiency was optimized by changing one parameter at a time and by performing quality-control tests for the labeled complex^[^16]. For optimizing amount of stannous chloride, a range of 25 to 400 μg of stannous chloride was used. Similarly, pH of the reaction mixture was varied from 4 to 7 and incubation time was varied between 5 to 30 minutes. Labeling efficiency was determined by as discussed in preceding section.

Determination of labeling efficiency:

The labeling efficiency of simvastatin suspension and simvastatin SLNs was determined by developing ascending thin layer chromatography using instant thin-layer chromatography (ITLC) strips coated with silica gel (Gelman Science Inc, Ann Arbor, MI). The ITLC strips were used to determine free technetium and percentage of radio colloids in the preparation. ITLC strips were spotted with 1 to 2 μl of labeled complex at 1 cm above the bottom. These strips were developed using acetone and a solvent system to determine free Tc-99m pertechnetate and reduced/hydrolyzed (R/H) Tc-99m. The solvent front was allowed to reach to a height of approximately 6 to 8 cm from the origin and the strip was cut horizontally into two halves. Radioactivity in each half was determined by well-type gamma ray spectrometer. The free pertechnetate present in the preparation migrates to the top portion of the ITLC strip, leaving the radio colloids (reduced/hydrolyzed technetium) along with the labeled complex at the point of application. The presence of radio colloids was determined by developing ITLC strip using pyridine: acetic acid: water in ratio of 3:5:1.5. Reduced/hydrolyzed Tc- 99m present in the preparation will remain at point of application, while both the free Tc-99m-pertechnetate as well as labeled complex migrates with the solvent front. Thus ITLC strips were used to determine free Tc -99m and reduced/ hydrolyzed technetium, and based on these two parameters labeling efficiency was determined ^[17].

Stability of labeled complex:

Stability of the Tc-99m labeled simvastatin suspension and SLNs was determined in vitro in mice serum by ascending TLC technique. The labeled complex (0.1 ml) was incubated with freshly collected mice serum (0.9 ml) at room temperature. Aliquots were taken at time intervals of 0.5, 1, 2, 4, 6, 24 and 48 hours and ITLC was performed. These strips were counted for radioactivity in gamma ray counter and percentage labeling efficiency was calculated for simvastatin suspension and SLNs.

Preparation of simvastatin suspension:

Simvastatin suspension, of strength 1.04 mg/mL was prepared (using 0.5 % w/v methyl cellulose as suspending agent). This preparation was used as reference formulation that contained an amount of simvastatin equivalent to amount of simvastatin in optimized test SLN.

Animal Experimentation:

Swiss albino mice (25-30 gm) were used for pharmacokinetic and biodistribution studies. Before experimentation, they were divided in two groups each containing 21 mice. Each group was further divided in three subgroups consisting seven mice each to maintain n= 3. All surgical and experimental procedures were reviewed and approved by the animal and ethics review committee of Rajiv Academy for Pharmacy, Mathura, India (IAEC//RAP/2554/2009 dated 23.07.09). Euthanasia and disposal of carcass was in accordance with the ethical committee guidelines. The mice were fasted for 12 h before the experiments but had free access to water. The radiolabelled simvastatin suspension and SLNs having final radio activity of 2mCi/ml was prepared and the formulations in a dose of 5 mg/kg were administered to the respective groups/subgroups, using 16 gauge cannula, while the leftover formulation was kept aside as standard solution.

Pharmacokinetic evaluation:

For pharmacokinetic evaluation, the mice were anesthetized using chloroform at 0.5, 1, 2, 4, 6, 24 and 48 h post-administration and blood was collected via cardiac puncture. Blood samples were placed into pre-weighed test tubes and weighed. The samples were analyzed for radioactivity by gamma ray counter. Radioactivity in various samples was determined in the unit of counts. Along with the blood samples, a standard solution was also checked for its radioactivity that accounted to standard counts. From these data, percent activity/gram (% A/G) was calculated by using Eq. (1).

% --- ={( Counts/ Weight)/ Standard counts} 100 ……..…….Eq.1

Area under the curve (AUC), relative bioavailability, elimination half-life, volume of distribution and other pharmacokinetic parameters were calculated by one compartmental open model using QuickCal software (developed by Dr. Shivaprakash, Plexus, Ahmadabad, India). Relative bioavailability of simvastatin from SLNs can be calculated by following equation;

Relative bioavailability ={AUC0-∞/AUC0-∞_suspension}X 100 …..Eq.2

AUC 0 - ∞ _suspension: Area under the curve for simvastatin suspension

AUC 0 - ∞_SLNs: Area under the curve for simvastatin SLNs

Biodistribution study:

After collecting blood, each mice was sacrificed humanely and various organs including the heart, liver, lungs, kidneys, spleen, stomach and intestine were then isolated. Each organ was weighed and radioactivity was determined using gamma ray counter.

RESULTS:

The optimized SLN formulation with a particle size of 258.5 ± 5.38 nm, polydispersity Index of 0.245, zeta potential of -22.34 mV and percent EE of 75.71 ± 3.78%, that was able to provide 82.61 ± 6.92% cumulative drug release after 55 hr was selected for pharmacokinetic and biodistribution studies.

The pharmacokinetic studies were carried out using Tc-99m radiolabeled formulations. The amount of stannous chloride (SnCl₂) required to reduce Tc-99m, pH of the final preparation and incubation time of radiolabeled complex were considered as determinants of the labeling process. Higher amount of SnCl₂ is reported to result in formation of radiocolloids that is undesirable and lower amounts, result in poor labeling efficiency. Maximum labeling efficiency was observed with 200 mcg/ml SnCl₂ in the pH range of 6 to 6.5 and incubation time of 25 minutes (Table 1a- c). Radiolabeled preparations were found to be stable in mice serum (Table 2). All preparations were stable in mice serum for 48 hours as at all time points more than 93 % labeling efficiency was observed. This indicates the usefulness of the label Tc-99m as a marker for the pharmacokinetic and biodistribution studies. ^[13]

Table 1 (a): Optimization of amount of stannous chloride

Amount of Stannous chloride (mcg)	% Drug labeled ± S.D.
Amount of Stannous chloride (mcg)	Simvastatin suspension	SLNs
25	74.36±1.39	79.56±1.45
50	79.56±1.67	84.43±1.78
100	95.14±1.29	93.85±1.62
200	98.69±1.36	98.38±1.56
400	94.18±1.59	94.72±1.51

Table 1 (B): Optimization of pH of the final preparation

pH of preparation	% Drug labeled ± S.D.
pH of preparation	Simvastatin suspension	SLNs
4.0 - 4.5	75.39 ± 1.83	74.12 ± 1.95
4.5 - 5.0	81.11 ± 1.46	82.15 ± 2.11
5.0 - 5.5	87.73 ± 2.06	87.44 ± 1.09
5.5 - 6.0	91.61 ± 2.19	94.19 ± 2.08
6.0 - 6.5	98.46 ± 1.67	97.51 ± 1.76
6.5 - 7.0	93.41 ± 2.44	95.22 ± 1.53

Table 1 (C): Optimization of incubation time

Incubation time (min)	% Drug labeled ± S.D.
Incubation time (min)	Simvastatin suspension	SLNs
5	78.32 ± 2.06	80.54 ±1.63
10	86.32 ± 1.95	84.12 ± 2.31
15	84.21 ± 2.66	87.35 ± 1.85
20	89.51 ± 1.41	92.86 ± 2.47
25	95.53 ±1.98	97.43 ± 1.93
30	89.65 ± 2.16	94.31 ± 2.45

Table 2: Stability study of radiolabeled complexes in mice serum

Time (hr)	Labeling efficiency (Mean ± S.D.)
Time (hr)	Simvastatin suspension	SLNs
0	98.39 ± 1.56	98.76 ± 1.23
0.5	98.11 ± 1.21	98.67 ± 1.07
1	97.86 ± 1.41	98.21 ± 0.89
2	97.42 ± 0.97	97.79 ± 1.07
4	97.25 ± 1.03	97.67 ± 1.15
6	97.16 ± 1.13	97.43 ± 0.89
24	96.89 ± 1.09	95.88 ± 1.17
48	94.38 ± 0.89	93.76 ± 0.83

Following administration of the stable radiolabeled simvastatin suspension and simvastatin SLNs, the blood samples collected at various time intervals were analyzed for % A/G. % A/G versus time data are shown in Fig. 1.

Figure 1: Percent activity per gram versus time profile of radiolabelled simvastatin suspension and SLNs in blood after oral administration to mice (n=3).

Figure 1: Percent activity per gram versus time profile of radiolabelled simvastatin suspension and SLNs in blood after oral administration to mice (n=3).

Table 3: Pharmacokinetic parameters of Simvastatin formulations after oral administration to mice (n=3)

Parameter	Simvastatin suspension	SLNs
Elimination half life (hr)	3.44	5.72
Absorption constant (hr^-1)	0.193	0.126
Elimination constants (hr^-1)	0.201	0.121
Volume of distribution (L)	6.59	6.89
Clearance (L /hr)	1.33	0.71
AUC _{0 - ∞} (% A/G. hr)	3.77	7.01
Relative bioavailability (%)	100	186

The data was subjected to calculations of pharmacokinetic parameters based on one compartment open model (Table 3). The area under the curve (AUC 0 - ∞) for SLN(7.014 % A/G.hr) was found to be 1.86 times higher as compared to simvastatin suspension (3.77 % A/G.hr). The relative bioavailability of simvastatin from SLNs calculated by Eq. (2) was 186 % (Table 3).

The biodistribution of Tc-99m labeled formulations in heart, lungs, liver, spleen, kidney, stomach and intestine were studied and it was found that depending on the formulation, the distribution patterns varied. Fig. 2, Fig 3 and Fig 4 respectively show the distribution of drug in the stomach, intestine and liver following oral administration of SLNsand simvastatin suspension with respect to time. Initially, radioactivity of 37% to 45% respectively for simvastatin suspension and simvastatin SLNs was observed in stomach and gradually decreases as solutions passes the stomach. In case of intestine, no activity was seen initially, however after 2 hrs 17% of %A/G was observed for simvastatin suspension and 35% was observed for SLNs. Results of %A/G for liver are more important than other organs. It has been shown that initially 0.4% to 0.5% activity was observed in case of Simvastatin SLNs while 1.1% to 1.6% was observed for simvastatin Suspension. Activity was seen up to 24 hours in case of Simvastatin SLNs (0.2% A/G). Organ distribution of simvastatin in lungs, heart, spleen and kidney were higher from SLNs as compare to suspension.

Figure 2: Percent activity per gram versus time histograms for simvastatin suspension and SLNs in stomach after oral administration to mice (n=3)

Figure 3 : Percent activity per gram versus time histograms for simvastatin suspension and SLNs in small intestine after oral administration to mice (n=3).

Figure 4: Percent activity per gram versus time histograms for simvastatin suspension and SLNs in liver after oral administration to mice (n=3)

DISCUSSION:

The optimized SLN formulation upon pharmacokinetic evaluation revealed an area under the curve (AUC 0 - ∞) of 7.014 % A/G.hr that was 1.86 times higher as compared to simvastatin suspension. This clearly indicated an increase in bioavailability of simvastatin from SLNs that may attributed to minimized hepatic first pass metabolism of simvastatin. Lipids can enhance lymph formation and simultaneously promote lymph flow rate ^[5]. It is suggested that the transport of drugs through the intestinal lymphatics via the thoracic lymph duct to the systemic circulation at the junction of the jugular and left subclavian vein avoids presystemic hepatic metabolism and therefore enhances bioavailability. Increased stability of simvastatin to hydrolytic degradation in GI tract ^[18] might be another reason for increasing bioavailability as acidic/alkaline condition of GI tract hydrolyzes lactone form of simvastatin to its hydroxyl acid derivative. In case of SLNs, surrounded solid lipid coat offers protection to simvastatin against hydrolytic degradation in GI tract consequently an increase in t_1/2 is expected. Low aqueous solubility (4.3 X 10^-7 mg/ml) and High partition coefficient of Simvastatin may also enhance distribution of Simvastatin in Lymphatic circulation. This was confirmed when the elimination half life of simvastatin from SLNs was found to be 1.9 times higher than from suspension. As mentioned earlier that the in vitro release data fitted Higuchi model it can be interpreted that the SLN acted as matrix system from which the drug was gradually released thus contributing to extension in t_1/2.

Modulation of pharmacokinetic parameters can also be analyzed by biodistribution studies. The biodistribution of Tc-99m labeled formulations in heart, lungs, liver, spleen, kidney, stomach and intestine were studied and it was found that depending on the formulation, the distribution patterns varied. Fig. 2 shows the distribution of drug in the stomach following oral administration of SLNsand simvastatin suspension with respect to time.

As the suspensions have short gastric residence time, these rapidly entered into the intestine. This was proved by the radioactivity measurements of intestine (Fig. 3). Maximum activity was observed at the second hour and at each time point, the activity of simvastatin suspension was significantly less than SLNs and was not observed beyond24 hr but activity of SLNswas seen even at 48th hour. This may be attributed to the protective effect of SLNs against hepatic metabolism and also to the villae present in the intestine, in which nanoparticles can be easily entrapped for longer period of time.

Fig. 4 shows comparative distribution of simvastatin in liver. It was found that simvastatin from SLNs was poorly accumulated in the liver as compared to simvastatin from suspension within 2 hour of administration. This was because of the lymphatic uptake of SLNs. The lymphatic transport of simvastatin incorporated into SLNs can be attributed to two possible mechanisms. First, exogenously administered triglycerides are digested by the action of pancreatic lipase/co-lipase digestive enzymes present in the small intestine and absorbed into enterocytes. After absorption, long-chain fatty acids or lipids are biosynthesized into triglyceride-rich lipoprotein particles (chylomicrons) which are secreted into intestinal lymph. The size of intestinal lipoproteins precludes their absorption into the blood capillaries and therefore they are secreted into the lymph. Secondly, the cellular lining of the gastrointestinal tract is composed of absorptive enterocytes interspersed with membranous epithelial (M) cells. M cells that cover lymphoid aggregates, known as Payer’s patches, take up nanoparticles by a combination of endocytosis or transcytosis.

It was shown that even after 2 to 4 hr of administration, simvastatin from SLNs continuously entered into the liver. This was because of the long circulatory time of simvastatin - SLNs in blood and higher liver extraction ratio of drug (80%) ^[18]. This is advantageous because the liver is the target organ to reduce the cholesterol level in blood. Transport of simvastatin from blood to liver is proposed to occur mainly via a Na⁺-independent anion transporter in the sinusoidal membrane by passive diffusion ^[19]. In liver, simvastatin is hydrolyzed in its active hydroxyl acid derivative and is acted upon the HMG coenzyme A reductase enzyme present in the liver and thus inhibiting the cholesterol synthesis. It is reported that despite the inhibition of HMG coenzyme A reductase, hepatic cholesterol level does not fall. This is because hepatocytes compensate any drop in cholesterol level by increasing the synthesis of LDL receptor protein along with HMG Co A reductase^[^9]. In this investigation, because of the sustained delivery of simvastatin, the newly synthesized HMG Co A reductase inhibited too, and hence the hepatocytes must meet its cholesterol demand by uptake of LDL from the blood and finally removing cholesterol from blood. Simvastatin from prepared formulation (SLN) continuously entered in the liver and expressed its therapeutic activity by proposed manner and thus potentially lowering blood cholesterol level for prolonged period.

Organ distribution of simvastatin in lungs, heart, spleen and kidney were higher from SLNs as compare to suspension as shown in table 4. However, this may relate to adverse effect of drug in these tissues. The higher distribution was because of the long circulating time of drug in the blood which led to more partition of drug in these tissues due to its high partition coefficient value. Simvastatin was highly distributed to lungs and spleen, which might be because of the higher perfusion rate of these organs than other.

CONCLUSION:

The solid lipid nanoparticulate drug delivery system was identified as preferred drug carrier for transport of Simvastatin through the intestinal lymphatics via the thoracic lymph duct to the systemic circulation as it minimize presystemic hepatic metabolism and therefore enhances oral bioavailability and achieves lowering of blood cholesterol level in sustained manner with the help of hepatocytes.

ACKNOWLEDGMENT:

Authors are highly thankful to All India Council of Technical Education, New Delhi, India for providing financial support.

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Received on 21-10-2012

Modified on 27.10.2012

Accepted on 01.11.2012

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(6): November–December, 2012, 336-342