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Effects of hydroxypropyl-β-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
Effects of hydroxypropyl-β-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby1, 2*, A.H. Alomrani1 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, PO Box 2457, Saudi Arabia 2 Department of Pharmaceutical Technology, College of Pharmacy, University of Tanta, Tanta, Egypt *Correspondence: [email protected]
1
This work investigated the potential of (2-hydroxy)propyl-β-cyclodextrin (HPβCD) to protect hydrocortisone against degradation by gamma radiation. Hydrocortisone stability was monitored after irradiation of aqueous solution in absence and presence of HPβCD. The study also monitored the stability after incorporation of hydrocortisone- HPβCD solution in carboxymethylcellulose gel, alginate-based and carbopol-based in situ gelling systems. Incorporation of HPβCD in hydrocortisone aqueous solution significantly increased the drug stability after gamma irradiation. The recovered chemical potency values of the drug in presence of HPβCD were significantly higher than those recovered after irradiation of drug solution in absence of HPβCD. Irradiation of the gel or in situ gelling systems containing HPβCD-drug solution resulted in a significant reduction in the viscosity with the recovered drug concentrations being similar to those recovered in absence of the gelling system. The study indicated the potential of HPβCD to protect hydrocortisone against gamma irradiation with no augmentation from the gel or gelling systems. Key words: Hydrocortisone stability – Radiosterilization – (2-hydroxy)propyl-β-cyclodextrin and gamma radiation – Irradiation of alginate solution – Irradiation of in situ gelling systems.
The sterilization process is critical in production of ocular preparations. Dry heat sterilization, steam sterilization, gas sterilization, filtration sterilization and application of ionizing radiation are the commonly used methods for sterilization [1]. Aseptic processing is usually used as an alternative for production of eye drops containing thermolabile materials in aqueous preparations. However, the aseptic processing is expensive and may fail to achieve the required levels of sterility assurance. In addition, terminal sterilization is recommended by pharmaceutical guidelines [1-3]. Radiation sterilization is the best method for terminal sterilization. It can be applied to the preparation in its final container whilst maintaining its sterilization efficiency. It can allow for continuous operation with precise measurement of the absorbed dose of radiation. In addition, radiation sterilization gives no rise in temperature providing special advantage for production of thermosensitive products [4-6]. These advantages made radiosterilization to be the first choice for thermosensitive drugs in solid state [2]. Unfortunately, the use of ionizing radiation in sterilization of liquid pharmaceuticals may induce drug degradation with the problem becoming even greater in presence of water which undergoes radiolysis creating reactive species These reactive species can attack drugs in aqueous solutions [7, 8]. Alternative strategies have been used to protect drugs against degradation by ionizing radiation. These included the use of radio-protective excipients, conducting the sterilization process with the preparation being maintained at the frozen state (cryo-irradiation) or using a combination of the two techniques [8-10]. More recently the self microemulsifying and microemulsion based systems were successfully utilized for protecting a steroidal drug against degradation by gamma radiation [11]. The use of cyclodextrins (CDs) to enhance ocular drug availability has gained interest recently. Co-administration of CDs has been reported to increase corneal penetration, ocular absorption and the efficacy of poorly water-soluble drugs such as dexamethasone, cyclosporin, acetazolamide, and others. CDs can also increase aqueous stability and bioavailability of ophthalmic drugs [12-14]. Despite of improved ocular absorption, rapid precorneal clearance caused by drainage and high tear turnover provides another source for reduced ocular availability. This problem can be solved by application of bioadhesive gels
[15] or in situ gelling systems [16]. The later systems are preferred as they can be delivered in drop form with sustained release properties [16]. Complexation of hydrocortisone with HPβCD was previously shown to protect hydrocortisone against hydrolysis [17]. The photostability of a sunscreen agent was also enhanced in presence of HPβCD [18]. In contrast, the parent cyclodextrin (βCD) was unable to provide significant protection to hydrocortisone against degradation after incubation at 60 oC. in neutral and acid solution. This was explained on the basis that the degradation of the drug under these conditions targeted the C17 side chain which was not included in the hydrophobic cavity of βCD which can hold the ring structure of the steroid [19]. Hydroxyalkylation of the primary and secondary hydroxyl groups of βCD as in case of HPβCD was suggested to provide an extension of the hydrophobic cavity to provide a protection for C17 side chain [17]. It could be thus concluded that the degree of protection of the drugs via complexation with cyclodexrins depends on the pathway of complexation between the drug and the HPβCD and the mechanism of drug degradation. The current study investigated the potential of (2-hydroxy)propylβ-cyclodextrin (HPβCD), carboxymethylcellulose gel and various in situ gelling systems as radio-protective against drug decomposition by gamma radiation. The tested in situ gelling systems included the ion activated (alginate based) system which included hydroxypropyl methylcellulose (HPMC) as viscosity modifier and the pH dependent system (carbopol based) with either HPMC or methylcellulose being used as viscosity modifiers. The study used hydrocortisone as a model drug. This selection was based on the fact that corticosteroids are widely prepared as eye drops which require sterilization.
I. MATERIALS AND METHODS 1. Materials
Hydroxypropylmethylcellulose (HPMC, 100 cp, low viscosity), hydrocortisone and prednisolone acetate were obtained from Sigma Chemical Co., St Louis, United States. HPβCD was obtained from Acros Organics, New Jersey, United States. Sodium carboxymethylcellulose (CMC), methylcellulose (MC), methanol and acetonitrile 385
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
Table I - The composition of the tested formulations. Code AqSol CDAqSol CMC gel Alginate-HPMC Carbopol-HPMC Carbopol-MC
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Drug solution in water (0.02 % w/v) Drug solution in 5 % w/v HPβCD in water CMC (1.5 % w/v) in CDAqSol Sodium alginate (0.6 % w/v) and HPMC (2 % w/v) in CDAqSol Carbopol (0.5 % w/v) and HPMC (1.5 % w/v) in CDAqSol Carbopol (0.3 % w/v) and MC (1.5 % w/v) in CDAqSol
Aqueous solution HPbCD aqueous solution Hydrogel In situ gelling system In situ gelling system In situ gelling system
sampling system (Waters 717 Plus Autosampler, United States) was used. Separation was achieved on a C18 reversed phase column of (150 mm × 4.6 mm) C18, μ Bondapak, Waters, with an average particle size of 10 μm. The system was under computer control with the Millennium software being used for HPLC data analysis. The mobile phase was a mixture of acetonitril and water (40:60) flowing at 1 mL/min. The column effluent was monitored at 238 nm and the chromatographic data analysis was performed with the Millinium Program (Waters, United States). The samples were transferred to volumetric flasks previously spiked with prednisolone acetate (internal standard) which was included to provide a concentration of 10 μg/ mL after dilution. These samples were suitably diluted with methanol before loading into the HPLC vials and injecting 30 μL into the HPLC. The method was validated for linearity, limit of quantitation (LOQ), accuracy, precision and selectivity. The selectivity was conducted by comparing the chromatogram of the intact drug with that obtained after exposing the drug solution to gamma radiation.
(HPLC grade) were purchased from BDH, Poole, United Kingdom. Sodium alginate (high viscosity) was obtained from MP Biomedicals, LLC, Ohio, United States. Carbopol 934 was a gift from Spimaco, Al-Qassim, Saudi Arabia. All other chemicals were of analytical grade. Water was double distilled.
2. Preparation of the tested formulations
Table I presents the composition of the tested formulations. Aqueous drug solutions were prepared by simply dissolving known amount of the drug in water with the aid of sonication. HPβCD (5 % w/v) was dissolved in distilled water before adding the drug to produce drug-HPβCD aqueous solution (CDAqSol). The CMC gel was prepared simply by sprinkling the polymer on the CDAqSol solution while stirring. This was allowed to hydrate overnight. The carbopol based in situ gel-forming systems was prepared by sprinkling the HPMC or MC on 75 % of the CDAqSol while stirring at 80 °C. Carbopol was sprinkled on this solution while stirring before adding the rest of CDAqSol which was cold. The pH was adjusted to 5. The sodium alginate-HPMC in situ-gelling system was prepared by dispersing the polymers in 75 % of the CDAqSol, with continuous stirring until complete hydration before adding the rest of the aqueous vehicle.
6. Data analysis
The percent of the intact drug remaining (chemical potency) was calculated from the peak area ratios of irradiated samples relative to that of the corresponding non-irradiated samples.[10] The percent of intact drug remaining was plotted as a function of the radiation dose to produce the radiation stability profile of the drug. One-way analysis of variance (ANOVA) with Tukey`s multiple comparison was performed to test for significance. The differences were considered significant at a level of P < 0.05.
3. Rheological studies
The rheological properties of the gel and in situ gelling formulations were determined using a DV II+ rotating Brookfield viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA, United States). Spindles number 15 and 21 were used depending on the viscosity of the formulation with the later being adopted for the less viscous system. The angular velocity was increased gradually from 0.5 to 50 rpm. The viscosity and shear stress of the formulations were measured at various shear rates before and after irradiation. To investigate the effect of gamma irradiation on the in situ gelling capacity of the in situ gelling systems the rheological characteristics were determined under non-physiological condition (storage conditions) and at physiological condition (pH 7.4 and 37 °C for carbopol-based systems and in presence of calcium ions in case of alginate based system).
II. RESULTS AND DISCUSSION 1. HPLC analysis of hydrocortisone
Figure 1 shows representative chromatograms obtained for intact hydrocortisone or after exposure to increasing doses of gamma radiation in presence and absence of HPβCD. The drug was eluted after a retention time of 3.7 min with the internal standard being eluted after 6.2 min (Figure 1). The method was linear in the range of 0.5 to 50 μg/mL with LOQ of 0.5 μg/mL. The drug recovery was in the range of 97-103 % of the nominal values indicating the accuracy of the method. The method was precise as the relative standard deviation values determined after replicate analysis of calibration curves constructed in the same day and in different days did not exceed 2.1 %. To ensure the suitability of the assay for monitoring the stability of hydrocortisone after exposure to gamma radiation, the selectivity of the assay was determined by comparing the chromatograms of intact drug with that of irradiated drug. This comparison indicated the selectivity of the method as evidenced by the absence of any interference between the peaks of degradation products and that of the drug or the internal standard (Figure 1). The suitability of the assay for this study was evidenced further by the purity of HPLC peak of hydrocortisone after irradiation. The peak was baseline to baseline with no tailing. Two major degradation pathways were identified for corticosteroids after irradiation [20]. The major degradation processes included loss of the corticoid side chain to form C-17 ketone and the conversion of the C-11 alcohol to ketone. Other minor degradation products resulting from changes affecting the side chain were also recorded [20]. The representative chromatograms reveal the stabilizing effect of HPβCD
4. Radiation study
Samples (10 g) of each formulation were loaded into neutral glass vials stoppered with rubber closure, before being subjected to different doses of gamma radiation (2, 5, 10, 15, 20 and 25 KGy). The irradiation was achieved using Cobalt-60 source in a Gammacell-220 (Nordion International Inc., Kanata, Canada) at a rate of 1.15 Grays/s. The remaining concentration of intact drug was then determined using HPLC analysis (see below).
5. Chromatography
The drug concentrations in all samples were determined using HPLC analysis. A high pressure liquid chromatograph (Waters 600 controller, United States) equipped with a variable wavelength UVVis detector (SPD-10 AV, Shimadzu, Kyoto, Japan) and an automatic 386
Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
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Figure 2 shows the effect of gamma radiation on the viscosity and flow behaviour of CMC gel. CMC gel showed a shear thinning pseudoplastic flow with the viscosity being reduced with increasing the rate of shear. Exposing this system to ionizing radiation resulted in significant reduction in the viscosity. This effect was clear even at the lowest radiation dose (2 KGy) (Figure 2). The viscosity values recorded at an angular velocity of 0.5 rpm was decreased from 28,000 cp before irradiation to 200 cp after exposure to 2 KGy. The viscosity showed gradual reduction with increasing the radiation dose. A reduction of the viscosity of CMC solution was recorded after exposure to gamma radiation [21]. This reduction was attributed to cleavage of the glucosidic bonds [21]. Similarly, irradiation of low concentration of CMC resulted in degradation but irradiation of CMC of higher degree of substitution resulted in crosslinking which started at a concentration of 5 % w/v and increased with increasing the concentration and the radiation dose [22]. More recent investigations revealed that crosslinking can occur only at 20 % w/v of CMC with the gel fraction being lower than 40 % [23]. Figure 3 shows the effect of gamma irradiation on the viscosity and flow behavior of the alginate-HPMC based in situ gelling system. The data revealed significant reduction in the viscosity of the system after exposure to gamma radiation (Figure 3a). To further verify the effect of radiation, the in situ gelling capacity of the system was monitored before and after radiation. This was conducted by addition of calcium chloride to produce a concentration of 8 mg/L with the system being maintained at 37 °C, before monitoring the viscosity. The recorded viscosity profile of this revealed a reduction in the gelling capacity of the system after exposure to radiation. This is evidenced by reduction in the viscosity of the gel after irradiation compared to the corresponding non-irradiated system (Figure 3b). The reduced in situ gelling capacity of the alginate system after irradiation can be explained on the basis of
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possible degradation of alginate. This degradation reduced the amount of intact alginate which produces the gellation process after interaction with calcium. Reduction in the gel strength of calcium alginate produced after irradiation of alginate solution was recorded by other researchers. This effect was attributed to the reduction in the degree of polymerization of alginate after sterilization [24]. Figures 4 and 5 show the effect of gamma irradiation on the viscosity and flow behavior of the Carbopol based in situ gelling systems in which either HPMC or MC were used as viscosity modifiers. In case of carbopol-HPMC system, carbopol was included at 0.5 % w/v 387
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
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Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
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Figure 4 - Effect of gamma radiation on the viscosity of carbopol-HPMC in situ gelling system. The viscosity was monitored at storage conditions (a) and at physiologic conditions (b).
but in case of carbopol-MC system, lower concentration of carbopol (0.3 % w/v) was effective for the in situ gelling. This can be attributed to the higher viscosity obtained from the MC compared to the low viscosity HPMC. The data in Figures 4 and 5 showed shear thinning flow behavior with the viscosity being reduced by increasing the shear rate. This was the case both at storage (ambient) and physiological (pH 7.4 and 37 °C) conditions. Exposure of these systems to gamma radiation resulted in a reduction in the viscosity with effect increasing with increasing the radiation dose. This was the case at both storage and physiological conditions (Figures 4 and 5). The extent of reduction was greater in case of carbopol-MC system which comprised lower concentration of carbopol with greater contribution from the viscous MC solution. This suggests that the cellulose derived polymers are subject to degradation by irradiation. A reduction in the viscosity of poly-acrylic acid solutions was recorded after exposure to gamma radiation. This reduction was attributed to possible scission of the polymer chains [25].
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3. Effect of gamma radiation on the stability of hydrocortisone in various formulations
Figure 6 shows the radiation stability profiles of hydrocortisone in different formulations. Irradiation of simple aqueous solution of the drug resulted in significant loss in the chemical potency even at small doses of radiation with solution loosing more than 83 % of its potency after being subjected to 2 KGy. The loss exceeded 95 % after exposure to 5 KGy (Figure 6). Similar radiation stability profile was recorded for corticosteroids in aqueous solution [11, 26]. The degradation of the drug in aqueous solution was attributed to the attack of the drug by the free radicals which were generated by the radiolysis of water [8].
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Figure 6 - Radiation stability profiles of hydrocortisone in different formulations. The formulation details are presented in Table I.
Incorporation of HPβCD with the drug in the aqueous solution resulted in a significant protection of the drug from degradation by gamma radiation. The drug retained 96, 85, 83, 73, 70 and 59 % of
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Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
its chemical potency after exposure to radiation doses of 2, 5, 10, 15, 20 and 25 KGy, respectively. These values were significantly higher than that retained after exposure of the drug to the same doses in absence of HPβCD. This can be explained on the basis that inclusion of hydrocortisone in the hydrophobic cavity of HPβCD can provide some protection of the drug from being attacked by the free radicals. The success of HPβCD may be attributed to the presence of hydroxyalkyl groups which provide an extension of the hydrophobic cavity to provide a protection for C17 side chain as well as the steroidal rings [17]. Taking into consideration the fact that irradiation-induced degradation of corticosteroid can affect both the C17 and the steroidal rings, inclusion complexation with HPβCD can provide some protection against degradation by ionizing radiation which explains the recoded data in this study. The current study introduced HPβCD as a potential radioprotective excipient but it should be considered as case by case basis for each drug as the efficacy depends on both the complexation and degradation pathways. Radioprotective excipients were used to protect drugs against degradation during radiosterilization. These excipients included mannitol, nicotinamide and pyridoxine [10]. Incorporation of the HPβCD-hydrocortisone solution in CMC gel, alginate-based or carbopol-based in situ gelling systems did not show any significant augmentation of the radioprotective effect of HPβCD. This is evidenced by the recovered intact drug concentrations after irradiation in presence of the gel or in situ gelling system. The values were statistically similar to the recovered chemical potency from the HPβCD-hydrocortisone solution (Figure 6). Cryo-irradiation was used as a tool for radio-sterilization while maintaining the integrity of the drug in solutions. The protective effect of freezing was attributed to the reduction of the diffusion of the reactive species on the frozen state [8-9]. It was thus expected that the viscosity of the gel and in situ gelling systems could reduce the diffusion of the active species with the result that, the stability of hydrocortisone was further improved compared to that recorded in case of HPβCD-hydrocortisone solution. The recorded results here were against this expectation. This can be attributed to the fact that gamma irradiation resulted in reduction of the viscosity of the gel and the gelling systems which was shown in the above sections.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21.
* Incorporation of HPβCD in the aqueous solution of hydrocortisone significantly improved the drug stability against degradation by gamma irradiation. The study thus introduced HPβCD as a potential radioprotective excipient but it should be considered as case by case bases for each drug. The gel and in situ gelling systems were unable to enhance the radioprotective effect of HPβCD towards hydrocortisone.
22.
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United States Pharmacopoeia National Formulary, 24th Ed. United States Pharmacopoeial Convention, Rockville, 2000. European Agency for the Evaluation of Medicinal Products (EMEA). - Decision trees for the selection of sterilization methods (CPMP/QWP/054/98 corr). - London, 2000. European Pharmacopoeia, 5th ed. - Council of Europe, Strasbourg, 2005. Langley R., Edwards R., Reuter G. - Specifying a gamma irradiation plant for safe and cost-effective sterilization. - Med Device Tech., 5, 23-27, 1994. Reid B.D. - Gamma processing technology: an alternative technology for terminal sterilization of parenterals. - PDA J. Pharm. Sci. Technol., 49, 83-89, 1995.
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Assemand E., Lacroix M., Mateescu M. - Protective role of ltyrosine in the sterilization of ceruloplasmin therapeutic protein by gamma irradiation. - Radiat. Phys. Chem., 71, 403-407, 2004. Tilquin B. - Radiostérilisation des medicaments. - J. Pharm. Belg., 46, 396-398, 1994. Terryn H., Maquille A., Levin C.H., Tilquin B. - Irradiation of human insulin in aqueous solution, first step towards radiosterilization. - Int. J. Pharm., 343, 4-11, 2007. Maquille A., Jiwan J.L.H., Tilquin B. - Cryo-irradiation as a terminal method for the sterilization of drug aqueous solution. - Eur. J. Pharm. Biopharm., 69, 358-363, 2008. Maquille A., Jiwan J.L.H., Tilquin B. - Radiosterilization of drugs in aqueous solutions may be achieved by the use of radioprotective excipients. - Int. J. Pharm., 349, 74-82, 2008. El Maghraby G.M., Bosela A. - Investigation of self-microemulsifying and microemulsion systems for protection of prednisolone from gamma radiation. - Pharm. Devel. Tech., 2010, in press. Loftsson T., Järvinen T. - Cyclodextrins in ophthalmic drug delivery. - Adv. Drug Deliv. Rev., 36, 59-79, 1999. Aktas Y., Unlü N., Orhan M., Irkeç M., Hincal A.A. - Influence of hydroxypropyl beta-cyclodextrin on the corneal permeation of pilocarpine. - Drug Dev. Ind. Pharm., 29, 223-230, 2003. Loftsson T., Jarho P., Másson M., Järvinen T. - Cyclodextrins in drug delivery. - Exp. Opin. Drug Deliv., 2, 335-351, 2005. Ludwig A. - The use of mucoadhesive polymers in ocular drug delivery. - Adv. Drug Deliv. Rev., 57, 1595-1639, 2005. Liu Z., Li J., Nie S., Liu H., Ding P., Pan W. - Study of an alginate/HPMC-based in situ gelling ophthalmic delivery system for gatifloxacin. - Int. J. Pharm., 315, 12-17, 2006. Davies N.M., Wang G., Tucker I.G. - Evaluation of a hydrocortisone/hydoxypropyl-b-cyclodextrin solution for ocular drug delivery. - Int. J. Pharm., 156, 201-209, 1997. Scalia S., Villani S., Casolari A. - Inclusion complexation of the sunscreen agent 2-ethylhexyl-p-dimethylaminobenzoate with hydroxypropyl-b-cyclodextrin: effect on photostability. - J. Pharm. Pharmacol., 51, 1367-1374, 1999. Andersen F.M., Bundgaard H. - The influence of b-cyclodextrin on the stability of hydrocortisone in aqueous solutions. - Arch. Pharm. Chem. Sci. Ed., 11, 61-66, 1983. Kane M.P., Tsuji K. - Radiolytic degradation scheme for 60Coirradiated corticosteroids. - J. Pharm. Sci., 72, 30-35, 1983. Lee H.S., Choi J.I., Kim J.H., Lee K.W., Chung Y.J., Shin M.H., Byun M.W., Shin M.G., Lee J.W. - Investigation on radiation of carboxymethylcellulose by ionizing irradiation. - Applied Radiation and Isotopes, 67, 1513-1515, 2009. Fei B., Wach R.A., Mitomo H., Yoshii F., Kume T. - Hydrogel of biodegradable cellulose derivatives. I. Radiation-induced crosslinking of CMC. - J. Appl. Polym. Sci., 78, 278-283, 2000. Liu P., Zhai M., Li J., Peng J., Wu J. - Radiation preparation and swelling behaviour of sodium carboxymethyl cellulose hydrogels. - Radiat. Phys. Chem., 63, 525-528, 2002. Leo W.J., McLoughlin A.J., Malone D.M. - Effects of sterilization treatments on some properties of alginate solutions and gels. - Biotech. Progess, 6 (1), 51-53, 1990. Sakurada I., Ikada Y. - Effects of gamma radiation on polymer in solution. V. Intra-molecular crosslinking of poly(acrylic acid) in dilute aqueous solution. - Bulletin of the Institute for Chemical Research, Kyoto University, 41 (1), 114-122, 1963. Bosela A.A., Al-Bagory I.M. - Sensitivity of hydrocortisone to gamma radiation in topical preparations. - J. Drug Del. Sci.Tech., 18, 209-213, 2008.
Manuscript Received 18 May 2010, accepted for publication 28 June 2010.
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Effects of hydroxypropyl-β-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby1, 2*, A.H. Alomrani1 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, PO Box 2457, Saudi Arabia 2 Department of Pharmaceutical Technology, College of Pharmacy, University of Tanta, Tanta, Egypt *Correspondence: [email protected]
1
This work investigated the potential of (2-hydroxy)propyl-β-cyclodextrin (HPβCD) to protect hydrocortisone against degradation by gamma radiation. Hydrocortisone stability was monitored after irradiation of aqueous solution in absence and presence of HPβCD. The study also monitored the stability after incorporation of hydrocortisone- HPβCD solution in carboxymethylcellulose gel, alginate-based and carbopol-based in situ gelling systems. Incorporation of HPβCD in hydrocortisone aqueous solution significantly increased the drug stability after gamma irradiation. The recovered chemical potency values of the drug in presence of HPβCD were significantly higher than those recovered after irradiation of drug solution in absence of HPβCD. Irradiation of the gel or in situ gelling systems containing HPβCD-drug solution resulted in a significant reduction in the viscosity with the recovered drug concentrations being similar to those recovered in absence of the gelling system. The study indicated the potential of HPβCD to protect hydrocortisone against gamma irradiation with no augmentation from the gel or gelling systems. Key words: Hydrocortisone stability – Radiosterilization – (2-hydroxy)propyl-β-cyclodextrin and gamma radiation – Irradiation of alginate solution – Irradiation of in situ gelling systems.
The sterilization process is critical in production of ocular preparations. Dry heat sterilization, steam sterilization, gas sterilization, filtration sterilization and application of ionizing radiation are the commonly used methods for sterilization [1]. Aseptic processing is usually used as an alternative for production of eye drops containing thermolabile materials in aqueous preparations. However, the aseptic processing is expensive and may fail to achieve the required levels of sterility assurance. In addition, terminal sterilization is recommended by pharmaceutical guidelines [1-3]. Radiation sterilization is the best method for terminal sterilization. It can be applied to the preparation in its final container whilst maintaining its sterilization efficiency. It can allow for continuous operation with precise measurement of the absorbed dose of radiation. In addition, radiation sterilization gives no rise in temperature providing special advantage for production of thermosensitive products [4-6]. These advantages made radiosterilization to be the first choice for thermosensitive drugs in solid state [2]. Unfortunately, the use of ionizing radiation in sterilization of liquid pharmaceuticals may induce drug degradation with the problem becoming even greater in presence of water which undergoes radiolysis creating reactive species These reactive species can attack drugs in aqueous solutions [7, 8]. Alternative strategies have been used to protect drugs against degradation by ionizing radiation. These included the use of radio-protective excipients, conducting the sterilization process with the preparation being maintained at the frozen state (cryo-irradiation) or using a combination of the two techniques [8-10]. More recently the self microemulsifying and microemulsion based systems were successfully utilized for protecting a steroidal drug against degradation by gamma radiation [11]. The use of cyclodextrins (CDs) to enhance ocular drug availability has gained interest recently. Co-administration of CDs has been reported to increase corneal penetration, ocular absorption and the efficacy of poorly water-soluble drugs such as dexamethasone, cyclosporin, acetazolamide, and others. CDs can also increase aqueous stability and bioavailability of ophthalmic drugs [12-14]. Despite of improved ocular absorption, rapid precorneal clearance caused by drainage and high tear turnover provides another source for reduced ocular availability. This problem can be solved by application of bioadhesive gels
[15] or in situ gelling systems [16]. The later systems are preferred as they can be delivered in drop form with sustained release properties [16]. Complexation of hydrocortisone with HPβCD was previously shown to protect hydrocortisone against hydrolysis [17]. The photostability of a sunscreen agent was also enhanced in presence of HPβCD [18]. In contrast, the parent cyclodextrin (βCD) was unable to provide significant protection to hydrocortisone against degradation after incubation at 60 oC. in neutral and acid solution. This was explained on the basis that the degradation of the drug under these conditions targeted the C17 side chain which was not included in the hydrophobic cavity of βCD which can hold the ring structure of the steroid [19]. Hydroxyalkylation of the primary and secondary hydroxyl groups of βCD as in case of HPβCD was suggested to provide an extension of the hydrophobic cavity to provide a protection for C17 side chain [17]. It could be thus concluded that the degree of protection of the drugs via complexation with cyclodexrins depends on the pathway of complexation between the drug and the HPβCD and the mechanism of drug degradation. The current study investigated the potential of (2-hydroxy)propylβ-cyclodextrin (HPβCD), carboxymethylcellulose gel and various in situ gelling systems as radio-protective against drug decomposition by gamma radiation. The tested in situ gelling systems included the ion activated (alginate based) system which included hydroxypropyl methylcellulose (HPMC) as viscosity modifier and the pH dependent system (carbopol based) with either HPMC or methylcellulose being used as viscosity modifiers. The study used hydrocortisone as a model drug. This selection was based on the fact that corticosteroids are widely prepared as eye drops which require sterilization.
I. MATERIALS AND METHODS 1. Materials
Hydroxypropylmethylcellulose (HPMC, 100 cp, low viscosity), hydrocortisone and prednisolone acetate were obtained from Sigma Chemical Co., St Louis, United States. HPβCD was obtained from Acros Organics, New Jersey, United States. Sodium carboxymethylcellulose (CMC), methylcellulose (MC), methanol and acetonitrile 385
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
Table I - The composition of the tested formulations. Code AqSol CDAqSol CMC gel Alginate-HPMC Carbopol-HPMC Carbopol-MC
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Type
Drug solution in water (0.02 % w/v) Drug solution in 5 % w/v HPβCD in water CMC (1.5 % w/v) in CDAqSol Sodium alginate (0.6 % w/v) and HPMC (2 % w/v) in CDAqSol Carbopol (0.5 % w/v) and HPMC (1.5 % w/v) in CDAqSol Carbopol (0.3 % w/v) and MC (1.5 % w/v) in CDAqSol
Aqueous solution HPbCD aqueous solution Hydrogel In situ gelling system In situ gelling system In situ gelling system
sampling system (Waters 717 Plus Autosampler, United States) was used. Separation was achieved on a C18 reversed phase column of (150 mm × 4.6 mm) C18, μ Bondapak, Waters, with an average particle size of 10 μm. The system was under computer control with the Millennium software being used for HPLC data analysis. The mobile phase was a mixture of acetonitril and water (40:60) flowing at 1 mL/min. The column effluent was monitored at 238 nm and the chromatographic data analysis was performed with the Millinium Program (Waters, United States). The samples were transferred to volumetric flasks previously spiked with prednisolone acetate (internal standard) which was included to provide a concentration of 10 μg/ mL after dilution. These samples were suitably diluted with methanol before loading into the HPLC vials and injecting 30 μL into the HPLC. The method was validated for linearity, limit of quantitation (LOQ), accuracy, precision and selectivity. The selectivity was conducted by comparing the chromatogram of the intact drug with that obtained after exposing the drug solution to gamma radiation.
(HPLC grade) were purchased from BDH, Poole, United Kingdom. Sodium alginate (high viscosity) was obtained from MP Biomedicals, LLC, Ohio, United States. Carbopol 934 was a gift from Spimaco, Al-Qassim, Saudi Arabia. All other chemicals were of analytical grade. Water was double distilled.
2. Preparation of the tested formulations
Table I presents the composition of the tested formulations. Aqueous drug solutions were prepared by simply dissolving known amount of the drug in water with the aid of sonication. HPβCD (5 % w/v) was dissolved in distilled water before adding the drug to produce drug-HPβCD aqueous solution (CDAqSol). The CMC gel was prepared simply by sprinkling the polymer on the CDAqSol solution while stirring. This was allowed to hydrate overnight. The carbopol based in situ gel-forming systems was prepared by sprinkling the HPMC or MC on 75 % of the CDAqSol while stirring at 80 °C. Carbopol was sprinkled on this solution while stirring before adding the rest of CDAqSol which was cold. The pH was adjusted to 5. The sodium alginate-HPMC in situ-gelling system was prepared by dispersing the polymers in 75 % of the CDAqSol, with continuous stirring until complete hydration before adding the rest of the aqueous vehicle.
6. Data analysis
The percent of the intact drug remaining (chemical potency) was calculated from the peak area ratios of irradiated samples relative to that of the corresponding non-irradiated samples.[10] The percent of intact drug remaining was plotted as a function of the radiation dose to produce the radiation stability profile of the drug. One-way analysis of variance (ANOVA) with Tukey`s multiple comparison was performed to test for significance. The differences were considered significant at a level of P < 0.05.
3. Rheological studies
The rheological properties of the gel and in situ gelling formulations were determined using a DV II+ rotating Brookfield viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA, United States). Spindles number 15 and 21 were used depending on the viscosity of the formulation with the later being adopted for the less viscous system. The angular velocity was increased gradually from 0.5 to 50 rpm. The viscosity and shear stress of the formulations were measured at various shear rates before and after irradiation. To investigate the effect of gamma irradiation on the in situ gelling capacity of the in situ gelling systems the rheological characteristics were determined under non-physiological condition (storage conditions) and at physiological condition (pH 7.4 and 37 °C for carbopol-based systems and in presence of calcium ions in case of alginate based system).
II. RESULTS AND DISCUSSION 1. HPLC analysis of hydrocortisone
Figure 1 shows representative chromatograms obtained for intact hydrocortisone or after exposure to increasing doses of gamma radiation in presence and absence of HPβCD. The drug was eluted after a retention time of 3.7 min with the internal standard being eluted after 6.2 min (Figure 1). The method was linear in the range of 0.5 to 50 μg/mL with LOQ of 0.5 μg/mL. The drug recovery was in the range of 97-103 % of the nominal values indicating the accuracy of the method. The method was precise as the relative standard deviation values determined after replicate analysis of calibration curves constructed in the same day and in different days did not exceed 2.1 %. To ensure the suitability of the assay for monitoring the stability of hydrocortisone after exposure to gamma radiation, the selectivity of the assay was determined by comparing the chromatograms of intact drug with that of irradiated drug. This comparison indicated the selectivity of the method as evidenced by the absence of any interference between the peaks of degradation products and that of the drug or the internal standard (Figure 1). The suitability of the assay for this study was evidenced further by the purity of HPLC peak of hydrocortisone after irradiation. The peak was baseline to baseline with no tailing. Two major degradation pathways were identified for corticosteroids after irradiation [20]. The major degradation processes included loss of the corticoid side chain to form C-17 ketone and the conversion of the C-11 alcohol to ketone. Other minor degradation products resulting from changes affecting the side chain were also recorded [20]. The representative chromatograms reveal the stabilizing effect of HPβCD
4. Radiation study
Samples (10 g) of each formulation were loaded into neutral glass vials stoppered with rubber closure, before being subjected to different doses of gamma radiation (2, 5, 10, 15, 20 and 25 KGy). The irradiation was achieved using Cobalt-60 source in a Gammacell-220 (Nordion International Inc., Kanata, Canada) at a rate of 1.15 Grays/s. The remaining concentration of intact drug was then determined using HPLC analysis (see below).
5. Chromatography
The drug concentrations in all samples were determined using HPLC analysis. A high pressure liquid chromatograph (Waters 600 controller, United States) equipped with a variable wavelength UVVis detector (SPD-10 AV, Shimadzu, Kyoto, Japan) and an automatic 386
Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
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Figure 1 - Representative chromatograms of hydrocortisone aqueous solution after exposure to increasing doses of gamma radiation, in presence (top 7 chromatograms) and absence (bottom 3 chromatograms) of HPbCD. The peak of the drug is at 3.7 min and that of the internal standard is at 6.1 min.
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Figure 2 shows the effect of gamma radiation on the viscosity and flow behaviour of CMC gel. CMC gel showed a shear thinning pseudoplastic flow with the viscosity being reduced with increasing the rate of shear. Exposing this system to ionizing radiation resulted in significant reduction in the viscosity. This effect was clear even at the lowest radiation dose (2 KGy) (Figure 2). The viscosity values recorded at an angular velocity of 0.5 rpm was decreased from 28,000 cp before irradiation to 200 cp after exposure to 2 KGy. The viscosity showed gradual reduction with increasing the radiation dose. A reduction of the viscosity of CMC solution was recorded after exposure to gamma radiation [21]. This reduction was attributed to cleavage of the glucosidic bonds [21]. Similarly, irradiation of low concentration of CMC resulted in degradation but irradiation of CMC of higher degree of substitution resulted in crosslinking which started at a concentration of 5 % w/v and increased with increasing the concentration and the radiation dose [22]. More recent investigations revealed that crosslinking can occur only at 20 % w/v of CMC with the gel fraction being lower than 40 % [23]. Figure 3 shows the effect of gamma irradiation on the viscosity and flow behavior of the alginate-HPMC based in situ gelling system. The data revealed significant reduction in the viscosity of the system after exposure to gamma radiation (Figure 3a). To further verify the effect of radiation, the in situ gelling capacity of the system was monitored before and after radiation. This was conducted by addition of calcium chloride to produce a concentration of 8 mg/L with the system being maintained at 37 °C, before monitoring the viscosity. The recorded viscosity profile of this revealed a reduction in the gelling capacity of the system after exposure to radiation. This is evidenced by reduction in the viscosity of the gel after irradiation compared to the corresponding non-irradiated system (Figure 3b). The reduced in situ gelling capacity of the alginate system after irradiation can be explained on the basis of
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possible degradation of alginate. This degradation reduced the amount of intact alginate which produces the gellation process after interaction with calcium. Reduction in the gel strength of calcium alginate produced after irradiation of alginate solution was recorded by other researchers. This effect was attributed to the reduction in the degree of polymerization of alginate after sterilization [24]. Figures 4 and 5 show the effect of gamma irradiation on the viscosity and flow behavior of the Carbopol based in situ gelling systems in which either HPMC or MC were used as viscosity modifiers. In case of carbopol-HPMC system, carbopol was included at 0.5 % w/v 387
J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
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Effects of hydroxypropyl-b-cyclodextrin, gel and in situ gelling systems on the stability of hydrocortisone after gamma irradiation G.M. El Maghraby, A.H. Alomrani
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Figure 4 - Effect of gamma radiation on the viscosity of carbopol-HPMC in situ gelling system. The viscosity was monitored at storage conditions (a) and at physiologic conditions (b).
but in case of carbopol-MC system, lower concentration of carbopol (0.3 % w/v) was effective for the in situ gelling. This can be attributed to the higher viscosity obtained from the MC compared to the low viscosity HPMC. The data in Figures 4 and 5 showed shear thinning flow behavior with the viscosity being reduced by increasing the shear rate. This was the case both at storage (ambient) and physiological (pH 7.4 and 37 °C) conditions. Exposure of these systems to gamma radiation resulted in a reduction in the viscosity with effect increasing with increasing the radiation dose. This was the case at both storage and physiological conditions (Figures 4 and 5). The extent of reduction was greater in case of carbopol-MC system which comprised lower concentration of carbopol with greater contribution from the viscous MC solution. This suggests that the cellulose derived polymers are subject to degradation by irradiation. A reduction in the viscosity of poly-acrylic acid solutions was recorded after exposure to gamma radiation. This reduction was attributed to possible scission of the polymer chains [25].
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3. Effect of gamma radiation on the stability of hydrocortisone in various formulations
Figure 6 shows the radiation stability profiles of hydrocortisone in different formulations. Irradiation of simple aqueous solution of the drug resulted in significant loss in the chemical potency even at small doses of radiation with solution loosing more than 83 % of its potency after being subjected to 2 KGy. The loss exceeded 95 % after exposure to 5 KGy (Figure 6). Similar radiation stability profile was recorded for corticosteroids in aqueous solution [11, 26]. The degradation of the drug in aqueous solution was attributed to the attack of the drug by the free radicals which were generated by the radiolysis of water [8].
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Figure 6 - Radiation stability profiles of hydrocortisone in different formulations. The formulation details are presented in Table I.
Incorporation of HPβCD with the drug in the aqueous solution resulted in a significant protection of the drug from degradation by gamma radiation. The drug retained 96, 85, 83, 73, 70 and 59 % of
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J. DRUG DEL. SCI. TECH., 20 (5) 385-389 2010
its chemical potency after exposure to radiation doses of 2, 5, 10, 15, 20 and 25 KGy, respectively. These values were significantly higher than that retained after exposure of the drug to the same doses in absence of HPβCD. This can be explained on the basis that inclusion of hydrocortisone in the hydrophobic cavity of HPβCD can provide some protection of the drug from being attacked by the free radicals. The success of HPβCD may be attributed to the presence of hydroxyalkyl groups which provide an extension of the hydrophobic cavity to provide a protection for C17 side chain as well as the steroidal rings [17]. Taking into consideration the fact that irradiation-induced degradation of corticosteroid can affect both the C17 and the steroidal rings, inclusion complexation with HPβCD can provide some protection against degradation by ionizing radiation which explains the recoded data in this study. The current study introduced HPβCD as a potential radioprotective excipient but it should be considered as case by case basis for each drug as the efficacy depends on both the complexation and degradation pathways. Radioprotective excipients were used to protect drugs against degradation during radiosterilization. These excipients included mannitol, nicotinamide and pyridoxine [10]. Incorporation of the HPβCD-hydrocortisone solution in CMC gel, alginate-based or carbopol-based in situ gelling systems did not show any significant augmentation of the radioprotective effect of HPβCD. This is evidenced by the recovered intact drug concentrations after irradiation in presence of the gel or in situ gelling system. The values were statistically similar to the recovered chemical potency from the HPβCD-hydrocortisone solution (Figure 6). Cryo-irradiation was used as a tool for radio-sterilization while maintaining the integrity of the drug in solutions. The protective effect of freezing was attributed to the reduction of the diffusion of the reactive species on the frozen state [8-9]. It was thus expected that the viscosity of the gel and in situ gelling systems could reduce the diffusion of the active species with the result that, the stability of hydrocortisone was further improved compared to that recorded in case of HPβCD-hydrocortisone solution. The recorded results here were against this expectation. This can be attributed to the fact that gamma irradiation resulted in reduction of the viscosity of the gel and the gelling systems which was shown in the above sections.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21.
* Incorporation of HPβCD in the aqueous solution of hydrocortisone significantly improved the drug stability against degradation by gamma irradiation. The study thus introduced HPβCD as a potential radioprotective excipient but it should be considered as case by case bases for each drug. The gel and in situ gelling systems were unable to enhance the radioprotective effect of HPβCD towards hydrocortisone.
22.
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Manuscript Received 18 May 2010, accepted for publication 28 June 2010.
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