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A potential new therapeutic system for glaucoma: Solid lipid nanoparticles containing methazolamide

Article in Journal of Microencapsulation · March 2011 DOI: 10.3109/02652048.2010.539304 · Source: PubMed

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The user has requested enhancement of the downloaded file. Journal of Microencapsulation, 2011; 28(2): 134–141 ß 2011 Informa UK Ltd. ISSN 0265-2048 print/ISSN 1464-5246 online DOI: 10.3109/02652048.2010.539304

A potential new therapeutic system for glaucoma: solid lipid nanoparticles containing methazolamide

Rui Li, Sunmin Jiang, Dongfei Liu, Xinyun Bi, Fengzhen Wang, Qing Zhang and Qunwei Xu

School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China

Abstract Methazolamide (MTA) is an antiglaucoma drug; however, there are many side effects of its systemic administration with insufficient ocular therapeutic concentrations. The aim of this study was to formulate MTA-loaded solid lipid nanoparticles (SLNs) and evaluate the potential of SLNs as a new therapeutic system for glaucoma. SLNs were prepared by a modified emulsion–solvent evaporation method and their physi- cochemical characteristics were evaluated. The pharmacodynamics was investigated by determining the percentage decrease in intraocular pressure. The ocular irritation was studied by Draize test. Despite a burst release of SLNs, the pharmacodynamic experiment indicated that MTA–SLNs had higher therapeutic efficacy, later occurrence of maximum action, and more prolonged effect than drug solution and commer- cial product. Formulation of MTA–SLNs would be a potential delivery carrier for ocular delivery, with the advantages of a more intensive treatment for glaucoma, lower in doses and better patient compliance compared to the conventional eye drops. Keywords: Methazolamide, solid lipid nanoparticles, emulsion–solvent evaporation, ocular delivery, intraocular pressure, ocular irritation For personal use only. Introduction of MTA administered systemically has poor access to the aqueous humour. The blood–aqueous and the blood– Glaucoma, one of the leading causes of irreversible ablep- retinal barriers prevent MTA from entering into the eye and sia in the world, is estimated to be suffered by 70 million reduce convection of MTA molecules (Yasukawa et al., people. It generally occurs in people aged above 40, but it 2004). Thus, a frequent administration at an extremely may also influence the visual acuity of much younger high concentration is usually needed, which limits oral people, even children (Sommer et al., 1991). Glaucoma administration by unpleasant systemic side effects is the term used for a group of ophthalmic disorders such as central nervous system depression, renal failure, characterised by an increase in intraocular pressure diuresis, vomiting, anorexia and metabolic acidosis (IOP), which results in a damage to the optic disc and (Kaur et al., 2002). visual field disturbances. Increase in IOP is a consequence The possibility of the topical administration directly of an imbalance between the production and drainage of to the eye was extensively investigated by several aqueous humour. Carbonic anhydrase inhibitors (CAIs), researchers to minimise or eliminate the systemic side Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 as a mainstay in the medical treatment of glaucoma, have effects. However, negative results had been constantly been used since 1954 (Becker, 1954), owing to their ability obtained initially. The major challenge was to improve to lower IOP by reducing aqueous humour formation in the the therapeutic effects, which was quite insufficient ciliary body. mainly because of MTA’s relatively low solubility in water As one of the most common CAIs, methazolamide (1.7 mg/mL at 25C; Maren, 1967); the pre-corneal loss fac- (MTA) has been used orally to reduce the IOP for more tors such as lacrimal secretion and nasolacrimal drainage than half a century (Zimmerman, 1978). However, most in the eye; and the relative impermeability of the corneal

Address for correspondence: Qunwei Xu, School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China. Tel/Fax: þ86 025 86862008. E-mail: [email protected]

(Received 8 Jun 2010; accepted 1 Nov 2010) http://www.informahealthcare.com/mnc 134 A potential new therapeutic system for glaucoma 135

epithelial membrane (Le Bourlais et al., 1998; Sultana et al., Hanbon Science and Technology Co., Ltd. (Jiangsu, China). 2006). To overcome these problems, various ophthalmic Distilled water was purified by Hitech-K flow Water vehicles such as -cyclodextrin or its derivative Purification System (Hitech Instruments Co. Ltd., (Fridriksdottir et al., 1997; Jonathan et al., 1996), and Shanghai, China). All other chemicals and solvents were ocular implants (Chang, 2006) had been investigated. of analytical grade or higher. These dosage forms offered numerous advantages over conventional oral forms, yet they were not devoid of draw- backs including poor patient compliance (discomfort, dif- ficulty of insertion and blurring of vision), as ocular Animals implants and tissue irritation, as well as damage and toxi- cological complications caused by cyclodextrin (Kaur et al., New Zealand albino rabbits of either sex, weighing 2004). 2.5–3.0 kg with normotensive eyes, were used in this Considering these points, solid lipid nanoparticles study. They were provided by Animal Experimental (SLNs) as a drug delivery system were utilised in Center of Nanjing Medical University. Rabbits were kept our study for formulating MTA as ocular eye drops. in individual cages with free access to standard food and The potential use of SLNs as ocular drug carriers is followed water, under controlled environmental conditions ¼ ¼ by the development of various colloidal dispersion systems (Ta 24 C; RH 60%; 12 : 12 h light : dark cycle). All the such as liposomes (Hathout et al., 2007), microemulsion procedures were conducted in accordance with the (Vandamme, 2002), nanoemulsion (Ammar et al., 2009), Principles of Laboratory Animal Care (NIH publication polymer nanoparticles (or nanosuspension; Pignatello no. 92-93, revised in 1985) and approved by the local et al., 2002; Kassem et al., 2007) and nanoparticles ethics committees for animal experimentation. (Gupta et al., 2010) that many published articles have reported as effective ophthalmic drug carriers. SLNs Quantitative determinations are defined as particles with the average particle size of 10–1000 nm, composed of lipids which are solid both at The quantitative determinations were performed by HPLC body and room temperatures, being stabilised by a suitable using a LC-10 ATvp pump, a SPD-10Avp UV–Vis surfactant. To develop efficient drug ocular delivery sys- detector (Shimadzu Co., Tokyo, Japan) with a Shim-pack tems, various matrices like natural or synthetic solid VP-ODS-2C18 column (5 mm, 4.6 150 mm2; Shimadzu lipids, phospholipids and polymers (Muller et al., 1995) Co., Tokyo, Japan) at 40C. The mobile phase was a mixture can be used. SLNs combine the advantages and avoid the of methanol andwater (30 : 70, v/v) with a flow rate at disadvantages of other colloidal dispersion systems, and 1.0 mL/min. The detection wavelength was set at 290 nm.

For personal use only. are regarded as their alternative for ophthalmic use. Aliquots of 20 mL of each sample were injected into the The advantages of the SLNs include low irritation, high column and MTA could be detected at a retention time penetration into the deeper layers of the ocular structure of about 5.0 min. Each sample was analysed in triplicate. and aqueous humour, ease of sterilisation as well as The MTA calibration curve was linear from 1 to 30 mg/mL patient-friendly use. All these properties make SLNs (r ¼ 1.0000). The limits of detection and quantisation were a promising avenue to achieve therapeutic action with 10 and 30 ng, respectively. No interference from the com- a smaller dose and fewer systemic and ocular side effects. position of the formulation was observed. All samples were This study focused on the preparation, characterisation filtered through a 0.45 mm pore size membrane filter to of SLNs, in vitro and in vivo performance of MTA-loaded protect the column. SLNs; and some comparison studies were also performed with reference eye drops. SLNs preparation

Materials and methods The MTA-loaded SLNs were prepared according to Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 a modified emulsion–solvent evaporation method Materials (Ye et al., 2008). In brief, MTA, Precirol ATO5 (lipid component) and phospholipids (emulsifier) were MTA, of 99% purity, was purchased from Hangzhou dissolved in ethanol at 70C to form an oil phase. The aque- Aoyipollen Pharmaceutical Co. Ltd. (Hangzhou, China); ous phase (0.5% Tween 80 and 1.0% PEG 400 used as sur- 1% w/v brinzolamide eye drops (AZOPTÕ) were purchased factant and co-surfactant, w/v) was heated to the same from Alcon (Puurs, Belgium). Glyceryl palmitostearate temperature of oil phase. The oil phase was instilled into powder (Precirol ATO5Õ) was a kind gift from Gattefosse the hot aqueous phase under mechanical stirring at sas Co. Ltd (Saint-Priest, France). Phospholipids (Lipoid 1200 rpm (RET Control-visc C, IKA, Germany) at 70C S100) were provided by Lipoid (Ludwigshafen, Germany). for dispersion. After removing the organic solvent, the Tween 80 was purchased from JiuJiu Biotech. Co. Ltd. pre-emulsion was poured into the cold continuous phase (Jiangsu, China). Polyethylene glycol 400 (PEG 400) was (2–3C) under stirring at 1000 rpm and SLNs were obtained supplied by the Dow Chemical Company (Shanghai, after continual stirring for 2 h. The continuous phase con- China). HPLC grade methanol was obtained from Jiangsu tained certain volume of 5.0% (w/v) mannitol, 1.9% boric 136 R. Li et al.

acid and 0.01% (w/v) benzalkonium bromide, which were St. Louis, MO). The amount of initial drug for assay and used as isotonicity agent, pH adjustor and bacteriostatic free non-entrapped drug in the aqueous phase after isola- agent, respectively. The resulting suspensions were steri- tion of the system was detected by the HPLC method. The lised by filtration through a membrane with 0.22 mm pore amount of incorporated drug was calculated by subtracting size and kept at 4C for use. Blank SLNs were prepared in the amount of free drug in the aqueous phase from the total a similar way without the drug. amount of the drug in the suspension. Drug loading (DL) All glassware was sterilised by autoclaving and the entire was calculated as drug analysed in the nanoparticles versus procedure mentioned above was done under aseptic the total amount of drug inside the particles and the lipid conditions. matrix (Precirol ATO5 and phospholipids) added during preparation. The EE and DL of MTA in SLNs were calcu- lated according to the following equations (Souto et al., SLNs characterisation 2004; Luo et al., 2006):

Winitial drug Wfree drug Osmotic pressure and pH EE ð%Þ¼ 100%, ð1Þ Winitial drug Osmotic pressure was determined by the freezing-point method using a Model FM-9X Osmometer (Instrumental W W Factory of Shanghai Medical University, Shanghai, China). ð Þ¼ initial drug free drug ð Þ DL % 100%, 2 The pH was determined at 25 C using a Model PHS-3C Winitial drug Wfree drug þ Wlipid pH-meter (Shanghai precision & Scientific Instrument Co., Ltd, China). where ‘‘Winitial drug’’ is the amount of drug used for the assay and ‘‘Wfree drug’’ is the amount of free drug detected in the aqueous phase after isolation of the suspension. Particle size and zeta potential analysis ‘‘W ’’ stands for the weight of lipid matrix The mean particle size and polydispersity index (PI) of lipid (Precirol ATO5 and phospholipids). the formulations were determined by photocorrelation spectroscopy with a Zetasizer 3000 HAS (Malvern Instruments Ltd, Malvern, UK). Every sample was appropriately diluted with distilled water and the reading In vitro release studies was carried out at a 90. The zeta potentials were deter- mined by a laser Doppler anemometer using the same The drug release from SLNs was performed using the instrument. A suitable amount of sample (50–100 mL) dialysis bag method (Ye et al., 2008) and an RC Drug was diluted with 5 mL of filtered water and injected in

For personal use only. Dissolution Tester (Tianjin Medical Instrumental Factory, the electrophoretic cell of the instrument, the obtained Tianjin, China). The diffusion medium used was freshly electrophoretic mobility values were used to calculate the prepared simulated tear fluid (STF) which was prepared

zeta potentials. In each case, the experiment was carried using NaCl 0.678 g, NaHCO3 0.218 g, CaCl22H2O 0.0084 g, out three times. KCl 0.138 g and water up to 100 g (Hagerstrom et al., 2000). The dialysis bag (12 000 MWCO, Sigma) could retain nano- Particle morphology particles and allow the diffusion of free drug into dissolu- The shape and morphology of SLNs were characterised tion media. The bags were soaked in STF for 12 h before using a transmission electron microscopy (TEM, JEM-200 use. The formulation (2 mL) to be studied were pipetted CX, JEOL, Tokyo, Japan) with an accelerating voltage into the bag and immersed in 100 mL STF, and stirred at of 100 kV. One drop of the SLNs suspension was deposited 50 rpm in a 35 0.5C water bath. Aliquots (4 mL) were on a carbon-coated copper grid. The excess staining withdrawn at each time interval (5, 10, 20, 30, 40, 60, 120, solution was removed with filter paper in 60 s and 180, 240, 360 and 480 min) and immediately replaced with the sample was allowed to dry before examined. an equal volume of STF to maintain a sink condition (Huo

Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 All the data presented were the mean values of at least et al., 2005). Samples were analysed by the HPLC method three independent samples produced under identical as described above. production conditions. The cumulative amount of drug (Qn, mg/mL) was plotted as a function of time (t, min) and calculated based on the following equation: Entrapment efficiency and drug loading Xn1 Qn ¼ Cn V0 þ Ci Vi, ð3Þ In this study, the entrapment efficiency (EE) of MTA- i¼1 loaded SLNs was determined by an ultrafiltration method. One millilitre of SLN dispersion was transferred where Cn stands for the drug concentration of the dissolu- to the upper chamber of centrifuge tubes fitted with an tion medium at each sampling time, Ci for the drug con- ultrafilter (Amicon Ultra 15, MWCO 100 KD, Millipore, centration of the ith sample, and V0 and Vi stand for the Ireland). The unit was centrifuged at 21 000 g for 10 min volumes of the dissolution medium and the sample, at 4C using a Sigma-3k30 Centrifuges (Sigma-Aldrich, respectively. A potential new therapeutic system for glaucoma 137

In vivo studies Statistical analysis

IOP measurements All experiments were performed in replicates for validity of This study was of a single-dose crossover design and was statistical analysis. Results were expressed as mean SD. performed on blank solution, MTA solution (0.1%, w/v), ANOVA was performed on the data sets generated by the the commercial product (AZOPTÕ, 1% w/v brinzolamide, SPSSÕ software. Differences were considered significant for as a reference standard), the blank SLNs in addition p 5 0.05. to the MTA-loaded SLNs formulations. Sterility of formulations was achieved by filtration through sterile 0.22 mm pore size pyrogen-free cellulose filters. Results and discussion The rabbits were acclimatised before the beginning of the study. An indentation tonometer (YJI, Suzhou Mingren Characterisation of the SLNs Medical Apparatus and Instruments Co. Ltd., Suzhou, China) was used to determine IOP measurements In this study, we have developed an economical, simple after instillation of one drop of 0.2% (w/v) lidocaine and reproducible method for the preparation of SLNs. hydrochloride as local anaesthetic. Eye drops were instilled Some essential characteristics of the SLNs were given topically into the lower cul-de-sac of the eye and the in Table 1. eye was manually blinked three times; one eye received 50 mL of the preparation and the other was not treated Osmolality and pH as a control. In all cases, the IOP was measured with The pH of ophthalmic preparations can vary from 4 to 9. the interval of 1 h after the treatment. All the measurements A pH exceeding this range is irritant and causes a painful were done three times at each interval and mean sensation. The osmolality of lacrimal fluid is between 280 values were taken. The percentage decrease in IOP was and 293 mOsm/L and solutions with an osmolality determined according to the following equation lower than 100 mOsm/L or higher than 640 mOsm/L are (Ammer et al., 2009): considered as irritants (Ammar et al., 2009). Unsuitable pH value and osmotic pressure can stimulate tear secretion IOP control eye IOP dosed eye and accelerate the outflow of therapeutic substances. %Decrease in IOP ¼ 100: IOP control eye In this study, formulations were prepared without ð4Þ adding any buffer, because in this case the limited buffer- ing capacity of the tears was able to adjust the pH to phys- The experiments were carried out in the same laboratory iological levels on administration and differing pH values

For personal use only. by the same investigator using the same instrument. On could be tolerated (Fialho and da Silva-Cunha, 2004). The any given day, only a single dose was tested on a single pH values of the prepared formulations were within the animal, which was washed out at least 2 days between acceptable range. The osmolality of the prepared SLNs experiments. The pharmacodynamic parameters taken ranged from 274 to 280 mOsm/L (Table 1). into consideration were maximum percentage decrease

in IOP, time for maximum response (Tmax), area under per- Particle size and zeta potential analysis centage decrease in IOP versus time curve (AUC0–8h) and Particle size is a crucial parameter for absorption or perme- mean residence time (MRT). These parameters were ation through the ocular barriers which should not exceed calculated using the 3P87/97 software. 10 mm (Zimmer and Kreuter, 1995). The mean particle size Statistical analysis of the results was performed using of all batches obtained was about 200 nm (Table 1). one-way analysis of variance (ANOVA) (Palma et al., All SLNs exhibited slightly negative zeta potential values 2009). Statistical analysis was computed with the SPSSÕ (Table 1). This fact could be explained by the shielding þ þ 2 software. effect of ions (H ,Cl,Na and CO3 ) present in the particle dispersion, adsorbing to the particle surface and

Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 compensating for charge.

Ocular irritation test Particle morphology The ocular tolerance and potential irritation of the SLN Transmission electron micrograph of SLNs showed that the dispersions were evaluated according to a modified particles had nearly spherical shape (Figure 1). Draize test (Draize et al., 1944), following single application of 50 mL of test products to one eye, while the other eye EE% and DL% served as control was treated with physiological saline or As shown in Table 1, the EE% increased while DL% blank SLNs. Observation of the ocular tissue condition was decreased with the reduction of MTA amount. This result performed at the 8 h after application. The congestion, was in good agreement with that reported by Liu et al. swelling, discharge and redness of the conjunctiva were (2010). The solubility of MTA in oil phase and the overall graded on a scale from 0 to 3, 0 to 4, 0 to 3 and 0 to 3, content of lipid matrix were constant. The decreased respectively. Irritation and corneal opacity were graded on amount of MTA could let less number of drug molecules a scale from 0 to 4 (Das et al., 2010). incorporate into the lipid matrix, while it served the matrix 138 R. Li et al.

Table 1. Essential physicochemical characteristics of the SLNs investigated in the study.a

Batch of SLNs Particle PI Zeta potential Osmotic pH EE (%) DL (%) size (nm) (mV) pressure (mOsm/L)

Blank–SLNs 199.5 2.6 0.226 0.015 8.4 2.1 280 5.6 – – 0.05% MTA–SLNs 191.6 3.8 0.211 0.014 8.1 1.9 278 5.4 63.7 3.3 3.42 0.17 0.03% MTA–SLNs 188.2 2.3 0.188 0.009 10.7 2.5 274 5.5 70.4 3.8 2.29 0.12

Note: aThe results are the means SD (n ¼ 3).

Figure 2. In vitro release profiles of MTA solution and MTA from SLNs (mean SD, n ¼ 6). For personal use only. The lipid might start crystallising first and formed an inner Figure 1. TEM photomicrograph of SLN dispersions containing MTA. core, leading to more MTA enriching around the lipid (Bar ¼ 200 nm). core and aqueous medium than in the core of the lipid nanoparticles (Zur Muhlen and Mehnert, 1998). Another reason was that the large specific surface of the small particles could increase the initial drug release. with more space for the incorporation of MTA thus leading In this formulation of SLNs, the presence of phospholipids to the consequence of less amount of MTA escaping into and surfactants provided an additional specific surface area the external phase, that was why DL% decreased and EE% and smaller particle size (Schubert and Muller-Goymann, increased. 2005). Thus, MTA–SLNs had a large total external surface and high diffusion coefficient allowing for a more intense interaction with the medium in which they were dispersed, In vitro release studies resulting in a fast drug release, as was demonstrated

Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 by Wakayima et al. (1981). The obtained release curves (Figure 2) revealed that MTA–SLNs exhibited slightly sustained drug release behav- iour compared with that of MTA solution. For MTA solu- In vivo studies tion, almost all drugs were released within 3 h. The amount of drug released was about 92.64% to the medium after 1 h IOP measurements for the solution, compared to 77.34% for the SLNs. Also, Table 2 and Figure 3 demonstrated the results of therapeu- there was a burst release pattern of SLNs which possibly tic efficacy after administration of a single dose of MTA– could be explained by two reasons. SLNs, drug solution and the commercial product. It could One possible explanation of the burst release was be seen that blank solution and blank SLNs were devoid of mainly because of the free drug dispersed in external any significant change in IOP. MTA–SLNs showed pro- phase or the encapsulated drug concentrated in the outer nounced decrease in IOP in contrast to drug solution shell of the SLNs and/or on the surface of the particles. (p 5 0.05). It was also observed that the mean maximum Such enrichment might occur during the cooling process. percentage decrease in IOP occurred 2 h after instillation A potential new therapeutic system for glaucoma 139

Table 2. Pharmacodynamic parameters after administration of MTA solution, SLNs and the commercial product (mean SD).

Formula Pharmacodynamic parameters

Maximum Tmax (h) AUC0–8h MRT percentage decrease in IOP

0.1% MTA solution 12.63 3.58 c 2 0.45 46.19 5.82 c 3.99 0.08 d AZOPTÕ 33.09 1.60 a 2 0.55 148.83 3.64 a 4.08 0.15 b 0.03% MTA–SLNs 35.69 1.98 a,d 3 0.20 196.48 4.17 a,c 5.16 0.14 a,c 0.05% MTA–SLNs 36.66 5.96 a,d 4 0.20 186.11 7.21 a,c 5.28 0.06 a,c

Notes: AUC0–8h: area under the percentage decrease in IOPtime curve); Tmax, time required to reach the peak effect, MRT: mean residence time. ‘‘a’’ and ‘‘b’’ denote significant difference and no significant difference from the group of MTA solution, respectively (p 4 0.05). ‘‘c’’ and ‘‘d’’ denote significant difference and no significant difference from the group of AZOPTÕ, respectively (p 5 0.05). For personal use only.

Figure 3. Percentage decrease in IOP after administration of MTA–SLNs, drug solution and commercial product (mean SD, n ¼ 6).

of drug solution, or the commercial product compared Table 3. Data for Draize eye irritation test. to MTA–SLNs which occurred at 3–4 h. With respect to

Degree of irritationa the duration of drug action, it was evident that the effect of SLNs continued over 8 h, while those of the drug solution AZOPTÕ MTA–SLNs Journal of Microencapsulation Downloaded from informahealthcare.com by National Library Health Sciences on 11/26/11 and the commercial product lasted for only 6 and 8 h, Control Test Blank SLNs Test respectively (Figure 3). This would indicate that SLNs exhibited a more prolonged effect compared to Cornea Corneal opacity 0 2 0 0 drug solution and commercial product. Iris Table 2 demonstrated some pharmacodynamic param- Irritation value 0 0 0 1 eters for tested formulations. Concerning the parameter of Conjunctiva area under the percentage decrease in IOP–time curve Degree of flare 0 1 0 1 (AUC ), MTA–SLNs showed higher values compared to Degree of swelling 0 0 0 0 0–8h Degree of redness 0 0 0 0 that of drug solution and commercial product (p 5 0.05). Congestion 0 0 0 0 It can be seen from Table 2 that SLNs had a longer MRT for Secretion (discharge) 0 2 0 0 percentage decrease in IOP in contrast to drug solution and commercial product (p 5 0.05). This would also indicate Notes: aIrritation and corneal opacity were graded on a scale from 0 to 4. Congestion, flare, swelling, discharge and redness of the conjunctiva were that SLNs exhibited a more prolonged effect compared graded on a scale from 0 to 3, 0 to 4, 0 to 4, 0 to 3 and 0 to 3, respectively. to drug solution and commercial product. 140 R. Li et al.

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