BACK TO MAIN PAGE

©1994-2005 All Rights Reserved. Online Journal of Pharmacokinetics. You may not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJPK publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors.

OJPKTM

Online Journal of Pharmacokinetics ©

Volume 3: 1-15, 2005.

Pharmacokinetics of flutamide liposomes

after IV and hepatotoxicity

 

Umrethia ML, Ghosh PK, Majithiya RJ, Murthy RSR

 

Drug Delivery Research Laboratory, Pharmacy Department, The M.S. University of Baroda, Vadodara, India

ABSTRACT

 

Umrethia ML, Ghosh PK, Majithiya RJ, Murthy RSR Pharmacokinetics of flutamide liposomes

after IV and hepatotoxicity Online Journal of Pharmacokinetics, 3 : 1 -15, 2005 Flutamide (FLT) is a non-steroidal anti-androgenic used for the treatment of prostate cancer: the high doses required can be hepatotoxic.  Encapsulated FLT (CL) with liposomes (SL) were thus formulatedto evaluate their blood kinetic properties, reduce RES uptake to reduce its hepatotoxicity. The size controlled FLT encapsulated CL and SL were prepared using egg phosphotidylcholine (ePC) and cholesterol by thin film hydration technique followed by high-pressure homogenization. In case of SL, methoxy polyethylene glycol 2000- phosphotidyl ethanolamine (mPEG2000-PE) was used along with lipids to provide surface hydrophiliicty to liposomes. The prepared liposomes were characterized for entrapment efficiency (EE), particle size, physical stability and percent drug retained in human plasma up to 24 hr. The CL of lower size (136 nm) with EE of 99.28% were found less stable than SL of 158 nm particle size and 98.54% EE in human plasma at 37ºC. The systemic pharmacokinetics and biodistribution of FLT encapsulated CL and SL was compared with that of free drug. The FLT-OH level in plasma and various organs of rats was determined by selective, sensitive, reproducible and validated reverse phase high-performance liquid chromatographic method. The pharmacokinetic studies showed that SL was still retained in plasma, where as CL had almost disappeared from blood circulation after 24 hr. The value of t1/2, Vss, MRT and AUC were found to be much higher for SL than that of CL and free drug. Accumulation of the FLT-OH in liver and other organ was found higher following free drug administered iv than that of CL and SL. Liver and spleen uptake of SL was 82% and 75% less than those of CL. Histopathological studies and Alanine transaminase (ALT) analysis showed that FLT encapsulated SL may help in reduction of undesired side effects associated with liver following free drug administration.

 

Keywords: Flutamide, liposomes, pharmacokinetics, biodistribution, histopathology

 

Introduction

 

While various anti-cancer agents are used to treat patients suffering from cancers, reasons for failure of chemotherapy involves daily multi-dosing therapy for several months include drug toxicity and emergence of drug resistance (Sharma et al., 1997). Increasing their amount in tumor cells, their presence in blood and reducing their exposure to normal cells can improve their therapeutic efficiency. Thus, the current strategy for enhancing the therapeutic activities of currently available drugs is to encapsulate them inside a delivery system from where they are slowly released over extended time period (Labana et al., 2002). Liposomal encapsulation reduces the toxicity and side effects associated with anti-tumor agents by altering their pharmacokinetics and distribution (Steerenberg et al., 1984: Herman et al., 1983: Gabizon et al., 1994: Newman et al., 1999: Gondal et al., 1993). These formulations have been used as carriers of cytotoxic drugs with the strategy based on reduction of toxicity and passive delivery to tumors (Pezer-soler, 1989: Gabizon, 1989) and improve the in vivo performance of these drugs as well as provide a repository for the drug to be released slowly so as to allow for prolonged therapeutic effect (Kondari et al., 2003) Although conventional liposomes can encapsulate a variety of drugs, they are recognized in-vivo by Kupffer cells of RES (Dijkstra, 1984) and cleared rapidly from the circulation so that a key obstacle to liposomes is to achieve control of their blood circulation and tissue distribution (Woodle, 1984). The term ‘stealth liposomes’ has been coined to designate long-circulating liposomes (Goren et al., 1997) which have a carefully selected size (about 100 nm) and are covered with covalently attached polyethylene glycol polymer (Lasic, 1995) that generates a steric barrier preventing hydrophobic interactions of plasma opsonins with the vesicle surface and inhibiting the uptake by RES (Torchilin, 1995). One key feature of long-circulating liposomes is their ability to accumulate in tumors, resulting in a significant selectivity in drug delivery to tumors and prevents its exposure to normal cells (Allen et al., 1991: Oku, 1994).

Flutamide (FLT) (3-trifluoromethyl-4-nitro methyl propionylanilide) is a non-steroidal pure anti-androgen (Baker et al., 1967) indicated for palliation of advanced prostate cancer. It acts by inhibiting the uptake and/or binding of di-hydro testosterone to the target cell receptor and affecting Prostate Specific Antigen (PSA) level in blood, thus interfering with androgen action which requires its stability in blood for enough time (Goldspiel et al., 1990). Its pharmacokinetics and dosage characteristics (usually three doses per day of 250 mg each) make it suitable candidate for design of controlled release delivery systems. Flutamide is rapidly and completely absorbed. The biologically active alpha-hydroxylated metabolite (FLT-OH) reaches maximum plasma concentrations in about 2 hours, indicating that it is rapidly formed from flutamide following 250 mg of oral dose in humans Reported FLT toxicity includes diarrhoea and hepatotoxicity (Chabner et al., 1996).   

Attempts have been made to formulate FLT liposomes and compared the pharmacokinetic, bio-distribution, histopathology and biochemical analysis (ALT level measurement) of its liposomal preparations with that of pure drug in rats specially to reduce hepatotoxicity associated with FLT as well as to increase therapeutic efficacy.

 

Materials and Methods

 

Materials

 

Flutamide was kindly supplied as a gift sample by Coral Drugs Pvt. Ltd., New Delhi; India. Egg Phosphotidylcholine (ePC), Methoxy Polyethylene Glycol (M.Wt 2000)  (mPEG 2000), Phosphotidylethanolamine (PE) and Cholesterol (CHOL) were purchased from Sigma Chemical Co., St. Louis, M.O.; USA. mPEG2000-PE was synthesized and characterized in house. ALT kit was purchased from Span diagnostic Ltd., Gujarat, India. All other chemicals and solvents were of analytical reagent grade and were used without further purification. 

 

Animals

 

Male Wistar-Albino Rats of both sex weighing 200-300 gm obtained from Zydus Research Center, Gujarat, India. The animals were housed in departmental animal house under natural light conditions, fed a standard pellet diet and water ad libidium. Social Justice and Empowerment Committee approved all animal experiments conducted for purpose of control and supervision on experiments on animals, Ministry of Government of India, New Delhi.  

 

Preparation of FLT conventional and stealth liposomes

 

The FLT loaded conventional (CL) and sterically stabilized liposomes (SL) were prepared by thin film hydration method (Umrethia et al., 2004) using rotary vacuum evaporator (Superfit, Rotovap). Drug and lipid mixtures as shown in Table 1 were accurately weighed and dissolved in organic phase in round bottom flask (RBF). The RBF was attached to the rotary evaporator and rotated at definite RPM at 370C under vacuum (250 mmHg) until uniform film was deposited in flask. The film was hydrated by adding an aqueous phase in the flask and rotated at the same RPM and temperature for 20 min or until lipid film had completely dispersed to produce suspension of multi-lamellar vesicles. Small and homogenous vesicles were obtained by sonication for 15 min in Probe Sonicator (Ralsonic, Mumbai, India) at reduced temperature. Prepared dispersion was allowed to hydrate again for 2 hr at 2-8°C. The liposomal dispersion was taken and passed through high-pressure homogenizer (Microfluidizer, Avestin, Canada) at15, 000 psi pressure for 3 cycles to reduce the size of vesicles below 200 nm and subjected to centrifugation on laboratory centrifuge (Sigma, 3K30) at 15, 000 RPM for 30 min at 0OC to separate liposomal vesicles. The vesicles were diluted with sucrose solution, frozen using liquid nitrogen and lyophilized in Heto Drywinner (Heto-Holten, Allerod, Denmark) for 24 hr under vacuum. The lyophilized products were stored at 4°C till further use.

 

Determination of % Entrapment Efficiency

 

From each prepared batch, a definite amount of liposomal dispersion was taken and subjected to centrifugation on laboratory centrifuge (Sigma, 3K30) at 15, 000 RPM for 15 min at 0OC after mixing with 50 ml protamine solution (10 mg/ml) using micropipette. The clear supernatant and sediment were separated. The definite amount of supernatant was diluted to 5 ml with methanol and the absorbance was recorded at 295 nm on Shimadzu 1601 UV-Visible Spectrophotometer (Kyoko, Japan) (Schulz et al., 1988). The sediment was resuspended in 1.5 ml distilled water and aliquot was diluted to 10 ml with methanol to lyse the liposomes and the absorbance was recorded at 295 nm. Each time blank containing blank liposome was treated in the same manner to account for any absorbance due to lipid components. Each experiment was repeated three times and average values were calculated (Umrethia et al., 2004).

 

The entrapment efficiency (% EE) was calculated as follows:

 

 % EE =         Amount of FLT in Sediment *100                               Equation (1)

                Total Amount of FLT added in sample

 

Determination of particle size of liposomes

 

The particle size distribution of the FLT liposomes was estimated by laser light scattering on a Malvern particle size analyzer (Malvern Master Sizer 2000 SM, U.K.). (Umrethia et al., 2004) Diluted liposome suspension was added to the sample dispersion unit containing stirrer and stirred in order to reduce the inter-particle aggregation, and laser obscuration range was maintained between 10 and 15 %. The average particle size was measured after performing the experiment in triplicate.

 

HPLC method

 

The samples of varied concentration of FLT-OH were chromatographed on reverse phase 250 × 4 mm C18 Hypersil® column provided by (Thermo electron company, Bellefonte, North America). A 40 × 4 mm precolumn of the same material was also used. The mobile phase consisted of methanol and water (80:20, v/v).  Mobile phase, filtered and degassed before use, was pumped at 1 ml /min flow-rate for runtime of 8 min. The column was thermostated at an ambient temperature. An injection volume of 20 μl was used. 6-Mercaptopurine was used as an internal standard (IS).

 

Stability of liposomes

 

To determine the stability of liposomes, the lyophilized product of CL and SL were stored at various temperature viz. 2-8°C, 37°C and in human plasma at 37°C. 10 mg of lyophilized powder was sampled at regular time intervals (0, 2, 4, 6, 8 and 12 weeks) and diluted to 2 ml with phosphate buffer saline (PBS) pH 7.4. The liposomal dispersion was centrifuged in a laboratory centrifuge (Remi) at 3000 RPM for 15 min; in this condition the liposomes remained suspended and the leaked drug sedimented. Amount of FLT in supernatant and sediment was measured spectrophotometrically at 295 nm (Shimadzu 1601 UV-Visible Spectrophotometer, Kyoko, Japan) and % drug retained was calculated. To evaluate FLT leakage from liposomes in plasma, liposomal dispersion was diluted 1:2 with human plasma and incubated at 37°C. The leakage drug was separated, extracted with methanol and estimated by HPLC method as described above.

 

Pharmacokinetic Study

 

Pharmacokinetic studies were performed as described elsewhere (Dosio et al., 1997) using male wisart rats. The animals were treated with either conventional or PEGylated liposomes and compared with the free drug solution (drug dissolved in mixture of 0.9%w/v of NaCl-ethanol-PEG-200 [2/0.18/3.82, v/v/v]) (Zhong et al., 2002). The different formulations were injected through the tail vein at the FLT dose of 25 mg/kg in rat (groups of three animals per group). Blood samples were taken from the retro-orbital plexus at various time intervals (30 min, 1, 2, 4, 6, 8, 12, 24 and 36 hr) in centrifuge tube containing 0.1 ml of 3.3 % w/v of sodium citrate solution as an anti-coagulant and centrifuged immediately at 5000 RPM for 10 min. The plasma samples were spiked with IS, diluted to 5 ml with methanol, vortexed (10 min) and centrifuged at 8000 RPM for 20 min. Supernatant was collected and 20 µl was injected in to HPLC column as described above. Pharmacokinetic parameters were calculated using the software Winlonlin® (Version 3.0, Pharsight Corporation Ltd., USA). Elimination, distribution and disposition were represented by the following parameters: Area under curve (AUC); mean residence time (MRT); total body clearance (Clt); volume of distribution at steady state (Vss); plasma half life (t1/2).

 

Biodistribution study

 

Either conventional or stealth liposomal dispersion equivalent to 25 mg of FLT/kg was injected through tail vein to rats. The rats were sacrificed and organs (liver, heart, lung, kidney, and spleen) were collected at different time intervals (30 min, 4, 8 and 24 hr) of i.v injection of drug and liposomes. The organs were washed twice with 0.9 % w/v of NaCl, wiped and weighed. Approximately 500 mg of organ slices were excised, minced, homogenized with 10 ml of methanol in a tissue homogenizer (Ultra-Turrax, T25, Germany) and centrifuged immediately at 8000 RPM for 20 min. The supernatant was transferred and evaporated to dryness. The residues were reconstituted with 1 ml of mobile phase and 20 µl of sample was injected in to HPLC column as described above.

 

 

 

Histopathological studies and ALT estimation

 

Each animal was treated with 25 mg/kg/day intravenously with control, free drug and encapsulated drug in CL and SL for 7 days and was sacrificed by cervical dislocation and dissected to collect liver after one week of last injection. The liver was fixed in 10% neutral buffered formalin. Sections of 3-5 mm thickness were stained with hematoxylin and eosin (H&E) for microscopical examination under Olympus (BX 40F4, Tokyo, Japan) microscope. An alternative estimation method is the measurement of ALT level to probe liver damage. After three days of injection of all formulations during histopathological studies, 1 ml of blood from each rat was collected, serum was separated by centrifuged at 1500 RPM for 10 min at 4°C and ALT level was measured using standard diagnostic kit in pathology laboratory. Statistical analysis of ALT estimation was performed by analysis of variance (ANOVA), difference was determined by applying student ‘t’ test and a statistical probability of p < 0.01 was considered to be significant. All results were expressed as mean ±standard deviation.

 

Results and Discussion

 

Characterization of liposomes

 

Flutamide could be entrapped into CL and SL by thin film hydration technique with the % EE of 99.32% and 98.56%, respectively. The relatively narrow particle size distributions were achieved with an average particle size of CL and SL formulation was 136±5.8 nm and 158±6.3 nm as mentioned in Table 1. The concentration of polymer necessary to produce steric stabilization was determined in vitro by electrolyte-induced flocculation test (Subramanian et al., 2004) at different mole % of mPEG2000-PE and was found to be 5 mole% which gave better stabilization (data not shown).

 

Physical stability of liposomes

 

Figure 2 indicates the % drug retained in lyophilized CL and SL in different conditions. It showed that liposomal preparations were physically stable for more than 3 months in lyophilized form at 2-8°C and retained 90% of their initial content over that period. Leakage of FLT from CL was faster than from SL in human blood plasma at 37ºC. The % drug retained in CL (48.12 %) was less than that of SL (66.38%) after 24 hr and no more drugs was retained after 2 days. Maximum instability was observed at 37ºC in presence of plasma of CL and SL. Thus, from these studies, it was observed that liposomes remained more stable at refrigeration temperature.

  

Pharmacokinetic study

 

With stealth liposomes, drug release mechanisms often remain undefined but circulation profile and drug pharmacokinetics are often comparable (Allen, 1998: Gabizon et al., 1994). Figure 2 shows the plasma concentration-time profile of FLT-OH after i.v injection of free drug, CL and SL in a dose equivalent to 25 mg/kg/day of FLT in rats. The pharmacokinetic parameters of free drug, CL and SL are represented in Table 2 which indicated that there was a rapid elimination of the free drug from the blood circulation and only 3.6 % of initial drug level per ml in blood was remaining after 4 hr. There was marked increase in Cmax of FLT-OH in blood following of the administration of SL (15.54 µg/ ml) relative to CL (7.88 µg/ml) and free drug (5.48 µg/ml) as well as plasma concentration of FLT-OH was significantly high even after 24 hr (1.908µg/ml) when compared with CL (0.129µg/ml). It indicated that the SL showed much longer circulation time with half-life of about 17.4 hr after i.v administration comparatively, CL was distributed to the tissue in few short times and was cleared from circulation with in 24 hr. The increase in circulation time may be due to the surface modification made on liposomes made by PEG which are of particle size less than 200 nm. It is reported that bare liposomes circulate for only about few hrs half-life in rats. With small amounts of PEG-2000 (7-10%), liposome circulation half-life can be extended to about 10-15 hr (Liu et al., 1995: Efremova et al., 2000). The AUC of SL was 85.12 (µg h ml-1), which was five and fourteen times greater than CL and free drug, respectively. The half-life of SL (17.4 hr) resulted from the slower release of FLT from liposomes, as it remained circulated in the body as reported.  The denser hydrophobic core yields vesicles that will (Woodle, 1993) retain their content in plasma for longer time (Lee et al., 2001) which may help to reduce the level of PSA in blood and as reported previously, there was an inverse relationship between liposome clearance by the RES and prolonged circulation time of liposomes which may reduce side effects associated with hepatic system (Gabizon et al., 1988). The mean clearance value of FLT-OH was 6 and 16 folds greater than CL and SL, respectively. It, therefore, appears that the longer half-life of SL and a pronounced increase in the blood residence time was the results of a reduced clearance rate.

 

Biodistribution Study

 

Hepatotoxicity is the limitation of therapeutic potential of FLT (Ozonol, 2001). In this study, it was proven that stealth liposomes were shown fewer uptakes by RES (liver and spleen) as compared to conventional liposomes and free drug as well as it exhibited longer half-life. To facilitate a comprehensive analysis of liposomes and free drug distribution, the distribution of FLT-OH in different organs at different time intervals after intravenous injections of free drug, CL and SL are shown in Figure 3. It indicated that the distributions of SL in liver, spleen and blood were significantly different form those of CL and free drug (P>0.05) throughout the period of experimentation. It showed that FLT-OH was more concentrated in liver and spleen as compared to other organs in case of free drug than that of CL and SL. RES also continued to accumulate liposomes with in 4 hr and then after the biodistribution of SL showed considerable decrease in drug uptake by liver in comparison to CL and free drug. The ratio of uptake between free drug: CL: SL was found to be (1:0.297:0.07) at 4 hr, (1:0.298:0.05) at 8 hr and (1:0.766:0.106) at 24 hr in liver and was (1:0.34:0.22) at 4hr, (1:0.375:0.06) at 8 hr and (1:0.39:0.05) at 24 hr in case of spleen. It has been postulated that the decreased uptake of sterically stabilized liposomes by RES is possibly due to the presence of steric barrier, which decreases the adsorption of plasma proteins (opsonins) on the surface of the liposomes (Huang, 1992). Initial uptake of SL in liver, together with increase in AUC and the decreased in Clt (Table 3) suggest rapid uptake of SL by the liver may be due to extravasation of liposomes to interstitial spaces that well re-enter in to blood stream with initial decrease of the blood level (Kim et al., 1994) In contrast, CL was accumulated intensely (9.06 µg/g) in liver and (7.51 µg/g) in spleen, though the size of liposomes was only 150 nm. The localization of FLT-OH in lung is less as compared to other organs and may be due to endocytosis mechanism in all formulation (Oku, 1999). It indicated that SL could remain in blood for prolonged time and reduce the uptake of liposomes to RES and thereby reduce the possibility of the risk of toxicity to RES generally seen with free FLT.

 

Histopathological studies and ALT estimation

 

Each drug produces different morphological and functional alterations and therefore different clinical manifestations. Reported FLT toxicity includes diarrhoea and hepatotoxicity. Figure 4 shows microscopic examination of liver collected after treated animals as described above with (a) control, (b) free drug, (c) CL and (d) SL. It is evident that free drug showed patchy marked necrosis, fatty degeneration changes and eccentrically situated nuclei with bile duct proliferation. CL showed cloudy degeneration and patchy necrosis, while SL showed no changes in hepatocytes and any other liver structure the same as control. ALT level in free drug (453±27.51U/L with P=0.0048) was also higher than those in CL (213.3±18.58 U/L with P=0.011) as shown in Figure 5. ALT level in SL (125±10.81 U/L with P=0.0248) was insignificant different from control (110.33±14.01 U/L). The increase or decrease of enzyme activity is related to the intensity of cellular damage. Therefore, increase of transaminase activity may be the consequence of FLT induced pathological changes of the liver.

   

Conclusion

 

The present study showed that conventional and sterically stabilized liposomes with particle size less than 200 nm and high entrapment efficiency can be prepared by thin film hydration technique followed by high-pressure homogenization. The liposomal formulations are stable in lyophilized form and retained enough amount of drug for 24 hr in human plasma. The pharmacokinetic parameters and biodistribution in different organs of sterically stabilized liposomes are significantly different than that of conventional liposomes and the free drug. Sterically Stabilized liposomes exhibited long circulation and fewer uptakes by RES and less change in ALT level, which may help to reduce side effects associated particularly with liver and improve efficacy of the flutamide.

 

Acknowledgement

 

The authors are thankful to TIFAC- CORE for providing the infrastructure, to B.V.Patel PERD Center for HPLC analysis, to Dr. Shailesh Patel (MD) for helping in histopathology and biochemical analysis and to AICTE for financial support as National Doctoral Fellowship to Manish Umrethia

 

 References:

 

1. Sharma A and Sharma US. (1997) Liposomes in drug delivery: progress and limitations. International Journal of Pharmaceutics 154:123-140.

 

2. Labana S and Pandey R. (2002) Chemotherapeutic activity against murine tuberculosis of once weekly administered drugs (isoniazid and rifampicin) encapsulated in liposomes. International Journal of Antimicrobial Agents 20: 301-304.

 

3. Steerenberg PA and Crommelin DJ. (1984) Reduced cardiotoxicity and nephrotoxicity with preservation of antitumor-activity of doxorubicin entrapped in stable liposomes in the LOU/M Wsl rats. Cancer Research 44: 3698-705.

 

4. Herman EH and Schein PS. (1983) Prevention of chronic doxorubicin cardiotoxicity in beagles by liposomal encapsulation. Cancer Research 43: 5427-32.

 

5. Gabizon A and Catane R. (1994) Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene glycol coated liposomes. Cancer Research 54: 987-92.

 

6.  Newman MS and Colbern GT. (1999) Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long circulating, pegylated liposomes (SPI-077) in tumor-bearing mice. Cancer Chemotherapy and Pharmacology 43: 1-7.

 

7. Gondal JA and Preuss HG. (1993) Comparative pharmacological, toxicological and antitumoral evaluation of free and liposome-encapsulated cisplatin in rodents. European Journal for Cancer 29A: 1536-42.

 

8. Pezer-soler R. (1989) Liposomes as carriers of anti-tumor agents; towards a clinical reality. Cancer Treatment Review 16:67-82.

 

9. Gabizon A. (1989) Liposomes as a drug delivery system in cancer chemotherapy. In: Drug Carrier Systems, Horizons in Biochemistry and Biophysics Roerdinck F, Kroon A. John Wiley and sons. Vol IX, 185-211.

 

10. Kondari KS and Suzara V. (2003) Efficacy of  liposomal budesonide in experimental ashthma. Journal of  Allergy and Clinical Immunology 111 (2):321-327.

 

11. Dijkstra J and Kalicharan D. (1984) Interaction of liposomes with Kupffer cells in vitro: Experimental Cell Research. 150:160-176.

 

12. Woodle MC. (1998) Controlling liposomes blood clearance by surface grafted polymers. Advanced Drug Delivery Review 32:139-152.

 

13. Goren D and Lossos A. (1997) Long-circulating liposomes for drug delivery in cancer therapy: A review of biodistribution studies in tumor-bearing animals. Advanced Drug Delivery Review 24:337-344.

 

14. Lasic DD. (1995) The stealth liposomes: a prototypical biomaterial. Chemical Review 95:2601-2634.

 

15. Torchilin VP. (1995) Long-circulating drug delivery systems. Advanced Drug Delivery Review 16:125-348.

 

16.  Allen T and Mathhay K. (1991) Sterically stabilized liposomes improvements in pharmacokinetics and anti-tumor therapeutic efficacy. Proceedings of the National Academy of Sciences USA. 88:11460-11464.

 

17. Oku N and Namba Y (1994) Long-circulating liposomes. Critical Reviews in Therapeutic Drug Carrier Systems 11: 231-274.

 

18. Baker JW and Bachman, GL. (1967) Synthesis and Bacteriostatic Activity of Nirtrofluromethylenilides. Journal of Medicinal Chemistry. 10:93-95

.

19. Goldspiel BR and Kholer DR. (1990) Flutamide: an antiandrogen for advanced prostate cancer Drug Intelligence and Clinical Pharmacy 24:611-623.

 

20.  Chabner BA and Calabresi P. (1996) in: The Pharmacological Basis of Therapeutics. A. Goodman, J.G. Hardman, L.E. Limbird, P.B. Milinoff, R.W. Ruddan , 9th ed., Mc-Graw Hill, Mexico, 1309-1367.

 

21. Umrethia ML and Murthy RSR. (2004) Optimization of formulation parameters of flutamide liposomes by 33 factorial 26-term logit model. Pharmaceutical Development Technology 9(4):369-378.

 

22. Schulz M and Schmoldt A. (1988) The Pharmacokinetics of Flutamide and Its Major Metabolites after a Single Oral Dose and During Chronic Treatment. European Journal of Clinical Pharmacology 34:633-636.

 

23. Dosio F, Brusa P, Crosasso P, Arpicco S. (1997) Preparation, Characterization properties, in vitro and in vivo of paclitaxel albumin conjugate. J. Control. Release 47:293-304.

 

24. Zhong Z and Diakur J. (2002) Hydroxypropryl-B-cycledextrin-flutamide inclusion complex II. Oral and intravenous pharmacokinetics of flutamide in the rat.  Journal of Pharmacy and Pharmaceutical Sciences 5(3):292-298.

25. Subramanian N and Murthy R.S.R. (2004) Use of electrolyte induced flocculation technique for in-vitro steric stability of sterically stabilized liposomal formulation. Die Pharmazie 1:69-74.

 

26. Allen TM. (1998) Liposomal drug formulations- rationale for development and what we can expect from the future? Drugs 56(5):747-756.

 

27. Gabizon A and Catane R. (1994) Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene glycol coated liposomes Cancer Research 54 (4):987-992.

 

28. Liu DX and Liu F. (1995) Recognition and clearance of liposomes containing phosphotidylserine are mediated by serum opsonins. Biochimica et Biophysica Acta-Biomembranes 1235:140-146.

 

29. Efremova NK and Bondurant B. (2000) Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly (ethylene glycol) chains. Biochemistry 39:3441-3451.

 

30. Woodle MC. (1993) Surface-modified liposome-assessment and characterization for increased stability and prolong blood circulation. Chemistry and Physics of Lipids 64(1-3):249-262.

 

31. Lee JCM and Bermudez H. (2001) Preparation, stability and in-vivo performance of vesicles made with diblock copolymers. Biotechnology and Bioenergetics 73 (2):135-145.

 

32. Gabizon G and Papahadjopoulos D. (1988) Liposomes formulation with prolonged circulation time in blood and enhance uptake by tumors. Proceedings of the National Academy of Sciences USA. 85:6949-6953.

 

33. Ozonol S and Kawakami1 T. (2002) Caffeine test in predicting flutamide-induced hepatic injury in patients with prostate cancer. Prostate Cancer and Prostatic Diseases 5:128–131.

 

34. Huang L. (1992) Covalently attached polymers and glycans to alter the biodistribution of liposomes. Journal of Liposome Research 2:289-291.

 

35. Kim CK and Han JH. (1994) Pharmacokinetics and tissue distribution of methotrexate after intravenous injection of differently charged liposome-entrapped methotrexate to rats. International Journal of Pharmaceutics 108:21-29.

 

36. Oku N. (1999) Anti-cancer therapy using glucuronate modified long-circulating liposomes. Advanced Drug Delivery Review 40:63-73.