Excipients of preservative-free latanoprost induced ...researchpub.org/journal/jbpr/number/vol2-no4/vol2-no4-1.pdf · Excipients of preservative-free latanoprost induced inflammatory

A. Smedowski et al. / Journal of Biochemical and Pharmacological Research, Vol. 2 (4): 175-184, December 2014

ISSN 2168-8761 print/ISSN 2168-877X online 175 http://www.researchpub.org/journal/jbpr/jbpr.html

Research Article

Excipients of preservative-free latanoprost induced inflammatory response and cytotoxicity in immortalized human HCE-2 corneal epithelial cells Adrian Smedowskia,b,c, Jussi J. Paternoa,d, Elisa Toropainena, Debasish Sinhae, Edward Wylegalac, Kai Kaarnirantaa,d,* aDepartment of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland bDepartment of Physiology, Medical University of Silesia, Medykow 18, 40-752 Katowice, Poland cDepartment of Ophthalmology, Division of Medicine and Dentistry in Zabrze, Medical University of Silesia, Panewnicka 65, 40-760 Katowice, Poland dDepartment of Ophthalmology, Kuopio University Hospital, P.O. Box 100, 70210 Kuopio, Finland eWilmer Eye Institute; Johns Hopkins University School of Medicine, Baltimore, MD USA

*To whom correspondence should be addressed: Tel: +358 44 717 2485; Fax: +358 17 172486, email: [email protected] (Received July 7, 2014; Revised August 25, 2014; Accepted August 26, 2014; Published online: September 5, 2014)

Abstract: Various preservative-free eye drop formulations for glaucoma treatment have been marketed intending to decrease ocular surface side effects and improve tolerability. However, preservative-free eye drops including different solubilizers to dissolve the antiglaucoma drugs may induce detrimental effects in the eye. In this study, we exposed human corneal epithelial cells (HCE-2) for 1, 6, 12, 24 and 48 hours to the first preservative-free (PF) tafluprost (Taflotan®), the recently-launched preservative-free (PF) latanoprost (Monoprost®), preservative benzalkonium chloride (BAK) and the excipient macrogolglycerol hydroxystearate 40 (MGHS40) using dilutions 0.1%, 0.3%, 1.0%, 3.0% and 10.0% of the original products. The cells also were exposed to undiluted PF tafluprost and PF latanoprost once a day for 9 days. Cellular morphology was examined by light microscopy and cell proliferation by Ki-67 fluorescent staining with cell viability being determined by erythrosine staining and the release of lactate dehydrogenase (LDH). Mitochondrial metabolic activity was evaluated with the colorimetric MTT assay. The secretion of interleukin 6 (IL-6) was measured with ELISA. HCE-2 cells displayed no significant morphological changes after PF tafluprost treatment, but PF latanoprost caused clear cell loss. Moreover, PF latanoprost, BAK and MGHS40 evoked cellular damage and inflammation with increasing concentrations and time. Furthermore, undiluted daily PF latanoprost application significantly increased LDH release and IL-6 secretion as compared to PF tafluprost. MGHS40 was observed to be associated with the toxicity of PF latanoprost. Excipients in ocular drops should receive more attention in the future, since they seem to trigger similar detrimental effects in cells as preservatives.

Keywords: cornea, cytotoxicity, drug, excipient, glaucoma, inflammation, preservative

Introduction

The treatment of glaucoma strives to lower the intraocular pressure (IOP) e.g. by pharmacological agents, laser treatment or surgery. Good tolerability of topical

antiglaucoma drops is important for patient compliance and the success of drug therapy [1]. However, antiglaucoma medications are often associated with ocular adverse symptoms and uncomfortable signs such as itching, feelings of burning, foreign body sensations, tearing and conjunctival


ISSN 2168‐8761 print/ISSN 2168‐877X online 176 http://www.researchpub.org/journal/jbpr/jbpr.html

Fig. 1. HCE-2 cells after 48 hrs incubation in 10% drug solutions. A) Visible floating cell debris (thick arrows) is seen in all groups. Control and PF tafluprost -treated cells demonstrated no difference in morphology. The cells exposed to PF latanoprost exhibited shrunken cytoplasm and swollen nuclei (thin arrow). Bright light, scale bar = 50 μm. B) The slightly compacted cytoplasm with perinuclear granules especially in paranuclear region (arrow) were found in PF tafluprost group. The cells treated with PF latanoprost displayed shrunken cells (thin arrow) and floating cell debris (thick arrows). Bright light, scale bar = 50 μm.

redness and these undesirable effects may lead to treatment discontinuation and reduced quality of life in patients suffering from glaucoma. The most widely used preservative in the antiglaucoma medication has been benzalkonium chloride (BAK). BAK’s cytotoxic and inflammation inducing effects on ocular surface cells have been well characterized in numerous in vitro and in vivo models [2, 3, 4, 5]. Antiglaucoma drugs containing polyquaternium-1 (PQ-1) preservative are much better tolerated compared to those with BAK solutions [6]. There has been a trend to try to develop preservative free formulations e.g. for the new prostaglandin antiglaucoma drugs [7, 8, 9].

Prostaglandin F2α analogues are currently the most widely used and effective topical antiglaucoma medications [10]. In many clinical guidelines, they have been recommended as first-line treatment of primary open-angle glaucoma [11]. Prostaglandins act by improving uveo-scleral aqueous humor outflow [12]. There are currently four

different derivative eye drops, but there does not seem to be any significant differences in their IOP lowering efficacies [13, 8, 9].

The four prostaglandin analogs, tafluprost, latanoprost, travoprost and bimatoprost are applied topically in patients with open-angle glaucoma and ocular hypertension [14]. Of these, Taflotan® (tafluprost, 2008; Santen, Osaka, Japan), Monoprost® (latanoprost, 2012; Thea, Clermont-Ferrand, France) and Lumigan®

(bimatoprost, 2013; Allergan, Irvine, CA, USA) are available as preservative free (PF) formulations. Taflotan® was the first PF prostaglandin analog for the treatment of glaucoma. It has proven to be well tolerated in randomized and multicenter clinical trials [7, 8]. However, due to the low water solubility of prostaglandin analogs, solubilizers have to be added to the formulations. Polysorbate 80 is used in PF tafluprost [15], while macrogolglycerol hydroxystearate 40 (MGHS40) is used in PF latanoprost [16]. This motivated us



Fig. 2. Erythrosine labeling of HCE-2 cells after 48 hrs of incubation with the diluted drugs. A) Considerable amounts of labeled debris were visible in all groups. PF tafluprost slightly increased cell death only at a 10% concentration, whereas cell death clearly increased with an increasing drug concentration in both PF latanoprost and BAK groups. Bright light, scale bar = 500 μm. B) The ratio of the visible debris (thick arrow) with single attached cells (thin arrows) increased slightly with increasing drug concentration in PF tafluprost group. Increasing PF latanoprost concentration evoked strong erythrosine positivity in attached cells in the background of the stained debris. Bright light, scale bar = 500 μm.

to investigate the effects of PF tafluprost and latanoprost on cytotoxicity and inflammatory response in human corneal epithelial (HCE-2) cells. Materials and methods

Human corneal epithelial cell (HCE-2) culture

Immortalized human corneal epithelial cells (HCE-2; American Type Culture Collection ATCC; [17]) were grown



Fig. 3. Immunofluorescent staining of HCE-2 cells after 48 hrs of incubation with the diluted drugs. Anti-Ki-67 (green) and DAPI (blue). PF tafluprost-treated cells displayed a slightly decreased proliferation rate only at the 10% concentration. PF latanoprost and BAK caused visible decline in the Ki-67 expression at the two lowest drug concentrations. There was no Ki-67 expression observed in the attributable to cell death. Scale bar = 500 μm.

at 37 oC in humidified air with 10% CO2 using standard culture medium which consists of Keratinocyte-Serum Free Medium (SFM) (Life Technologies, Invitrogen, Gibco, Paisley, UK) with 0.05 mg/ml bovine pituitary extract (Life Technologies), 5 ng/ml epidermal growth factor (Life Technologies), 0.005 mg/ml insulin (Sigma-Aldrich, Steinheim, Germany) and 100 U/ml penicillin/100 µg/ml streptomycin solution (Lonza, Basel, Switzerland). For cells optimal growth, 24-well plates (Greiner Bio-one BmbH, Frickenhausen, Germany) were coated with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/ml collagen and 0.01 mg/ml bovine serum albumin, and then 100 000 cells/well were seeded onto the plates. The test on concentration dependent cell toxicity

On the third day post-seeding, the cells were exposed

to PF tafluprost (Taflotan®; Santen Oy, Tampere, Finland), PF latanoprost (Monoprost®; Théa, Clermont-Ferrand, France), benzalkonium chloride (BAK) (FeF Chemicals, Copenhagen, Denmark) and macrogolglycerol hydroxystearate 40 (MGHS40) (Croda INC, Edison, NJ, USA) at the following dilutions: 0.1%, 0.3%, 1.0%, 3.0% and 10.0% of the original products and for the following times: for 1, 6, 12, 24 and 48 hours. The stock solutions of BAK and MGHS40 were 0.02% and 50 mg/ml, respectively. The concentrations used represent the concentration of BAK in commercially available antiglaucoma eye drops (Xalatan® and Xalcom®, Pfizer, New York, USA) and MGHS40 in Monoprost®. The drugs were diluted in the cell culture medium. HCE-2 cells cultured in standard medium were used as a control group. Osmotic pressure using Auto-Osmometer Osmostat (DIC, Kyoto, Japan) and pH of the applied drugs and the dilutions were measured prior to the



treatments. Staining for cell viability and proliferation

The cells were stained with 0.1% erythrosine diluted 1:1 ratio in culture medium at one to three different time points. The red color labels dead cells, due to membrane barrier integrity impairment, while it cannot enter into living cells. Erythrosine stained cells were observed under bright light and fluorescent microscope with detection at 568 nm (Zeiss Axio Scope.A1, Oberkochen, Germany).

After erythrosine staining, the cells were fixed with ice-cold 4% paraformaldehyde (PFA), which de-stained cells of erythrosine, and processed for immunostaining. Nonspecific binding was eliminated by blocking with 20% normal goat serum (NGS) in 0.05% TBS / 0.5% Triton X-100 for 15 min followed by incubation with primary antibody (polyclonal mouse anti-Ki67, dilution 1:100, DAKO, Glostrup, Denmark) for 1 hour at room temperature (RT) and with secondary antibody (anti-mouse Alexa Fluor 488, dilution 1:500, Life Technologies, Carlsbad, CA, USA) for 30 min at RT. After TBS washing twice, the samples were incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI 0.5 µM/ml, dilution 1:50 000, Sigma, St. Louis, MO, USA). Primary antibody was omitted from the negative controls. The cells were visualized on the fluorescent microscope (Zeiss Axio Vert.A1, Oberkochen, Germany). The test modeling the drainage of the drug away from the eye

On the third day after seeding, the cells were exposed to undiluted PF-tafluprost and PF- latanoprost once a day for 9 days. Prior to the exposure, the cells were washed with excipient-free, full supplemented SFM medium. The applied volume of the drug per well was calculated to mimic the volume of one eye drop considering the ratio of the growth area of the cells in the well (1.911 cm2) and intact human cornea (1.539 cm2). When applied topically, most of the drug is rapidly eliminated from the eye within the first 5 minutes, and ultimately only a small amount (1-7%) of the instilled dose actually penetrates through the cornea and reaches the anterior chamber [18, 19]. This situation was mimicked in vitro by diluting the drug applied to the cells with SFM. The dilution rate followed the drainage of the drug away from the rabbit’s eye in vivo [18]. After seven minutes, the cells were washed (excipient-free SFM) and fresh cell culture medium was added onto the cells. The medium was collected once a day prior to each new drug application. The samples were stored at -20 oC. The control cells were treated in the same way, but excipient-free SFM was used instead of the drugs.

Colorimetric cytotoxicity assays

Cellular viability was analysed by the MTT assay. The assay was performed as previously described [20]. The absorbance was measured at a wavelength of 595 nm. The cell viability was described as the percentage of the control group values. The release of lactate dehydrogenase (LDH) due to the cell membrane damage was detected by a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. LDH activity was quantified using a plate reader (BIO-RAD Model 550, BIO-RAD, Hercules, CA, USA) with a measurement wavelength of 490 nm and a reference wavelength of 655 nm. Results were presented as optical densities (OD). IL-6 secretion analysis

Interleukin 6 (IL-6) is a pro-inflammatory cytokine and its production and release increases in response to inflammation and cell/tissue injury [21]. The concentration of IL-6 (pg/ml) in the cell culture medium was measured by a commercial enzyme-linked immunosorbent assay (ELISA) using OptEIATM sets (BD Pharmingen, San Jose, CA, USA). The method was performed according to the manufacturer's instructions. Absorbance was measured at the wavelength of 450 nm with a reference wavelength of 655 nm. Statistics

Statistical analysis was performed using IBM SPSS STATISTICS 20 (IBM, Armonk, NY, USA). Descriptive statistical results are presented as the mean value ± 1 SD or 2 SE. Mann-Whitney U-test was used to compare differences between the cell culture groups. Probability values less than 0.05 were considered statistically significant. Results The concentration-dependent cell toxicity

No differences in morphology were observed in control or PF tafluprost groups, while clear cytotoxicity was detected in the PF latanoprost group (Fig. 1A). Additional features were observed at higher magnification, including slightly compacted cytoplasm with granules especially in the perinuclear region in the PF tafluprost -treated cells and shrunken cells with highly compacted cytoplasm in the PF latanoprost group (Fig. 1B).



Fig. 4. Viability (MTT) (A), cellular damage (LDH) (B) and inflammation (IL-6) (C) levels of the human corneal epithelial cells (HCE-2) with increasing concentrations of PF latanoprost, PF tafluprost, 0.02 % benzalkonium chloride (BAK) and 5 % macrogolglycerol hydroxystearate 40 (MGHS40). Incubation times 1, 6, 12, 24 and 48 hours (n = 4, mean ± 1 SD). Statistical analysis compared to untreated controls. * indicates p < 0.05, ns “not significant” (Mann-Whitney U-test).

Erythrosine staining revealed labeled debris in all groups. The ratio between dead and healthy cells was similar in both control and PF tafluprost groups, whereas PF

latanoprost and BAK evoked increased cell death dependent on the concentration gradient (Fig. 2A). The fluorescent microscope examination illustrated that mostly it was cell



Fig. 5. Release of lactate dehydrogenase (LDH) (A) and inflammation marker IL-6 (B) of HCE-2 cells during treatment with PF latanoprost and PF tafluprost (n = 6 – 12, mean ± 2 SE). The cells were treated with one eye drop once a day for nine days. While on the top of the cells, the drops were diluted so as to mimic the drainage of the drug away from the eye. * indicates p < 0.05, ** p < 0.01, *** p < 0.001 and ns “not significant” (Mann-Whitney U-test).

debris that was labeled and only single attached cells were erythrosine positive in PF tafluprost groups (Fig. 2B), whereas in PF latanoprost groups, increased concentrations caused erythrosine positive staining in most of the attached cells.

A slightly decreased proliferation rate was observed when the cells were treated with 10% PF tafluprost (Fig. 3). The cells which survived exposure to lower concentrations of PF latanoprost and BAK revealed a visible defect in Ki-67 expression, while the observations could not be reliably performed at higher concentrations because there was such severe cell death.

The mitochondrial metabolic activity and cell membrane damage of HCE-2 cells are illustrated in Fig. 4A and B. Overall, the cell viability decreased and the

cytotoxicity increased as either the concentration or the incubation time increased, resulting in severe cell damage and even total cell death. However, the two lowest concentration of PF tafluprost maintained 100% cell viability with all exposure times. The cells treated with BAK underwent severe progressive cell death, and with as little as 12 hrs of exposure, all the cells died even with the two lowest concentrations. The changes in cellular damage and the reduced cell viability of MGHS40-treated cells were significant at all studied concentrations. IL-6 secretion increased with PF latanoprost and MGHS40 in a concentration dependent manner (Fig. 4C). PF tafluprost was well tolerated in HCE-2 cells. BAK-treated cells induced only a mild IL-6 response due to the rapid cell death.



There was no significant difference in pH and osmotic pressure values in pure drug or drug dilutions. The values of pH ranged between 7.43 and 7.67 (mean 7.59 ± 0.06) and osmotic pressure 281 and 302 mOsm/kg (mean 288.8 ± 8.22 mOsm/kg). The test modelling the drainage of the drug away from the eye

The MTT detected no statistical significant difference in mitochondrial metabolic activity between control and drug groups (PF latanoprost and PF tafluprost) at the end of the study on day nine (data not shown). LDH release from the cells in every group remained at similar levels until day five. However, at later time points, PF latanoprost caused significantly elevated LDH leakage as compared to either control group or PF tafluprost-treated cells (Fig. 5A). IL-6 secretion varied 1.4 – 2.0 times higher in PF latanoprost comparison to the control culture, whereas PF tafluprost did not increase the inflammation level (Fig. 5B). Discussion

Cytotoxicity in the ocular surface cells is a well-known detrimental effect induced by BAK-containing antiglaucoma agents. There are numerous studies describing the advantages of PF formulation in eye drops, but usually studies do not focus on other excipients besides BAK [5, 22, 23]. Conjunctival hyperaemia due to vessel dilatation is one of the most common side effects caused by prostaglandins and prostamids [24, 25], but otherwise, these compounds are well tolerated in long-term use. Prostaglandins have even been claimed to act as non-specific cytoprotectors against BAK toxicity [26]. BAK induces time- and concentration dependent negative effects on ocular surface cells that can be observed as increased cytokine production and induction of apoptotic and necrotic cell death [27, 28]. Our present observations on HCE-2 cells are in line with the previous reports that show a massive detachment of cells from the culture dish, increases in the secretion of proinflammatory cytokines and cell death in response to BAK containing solutions [29, 30, 31, 32, 33, 34, 35, 36, 37].

Recent reports investigating PF formulations in glaucoma drugs have clearly indicated the good tolerability of prostaglandins in both in vivo and in vitro conditions [7, 8, 9]. Therefore, it was an unexpected observation that PF latanoprost induced clear signs of cellular damage in HCE-2 cells. Pauly et al. [38] recently reported that PF latanoprost (Monoprost®) had a good tolerability, when applied topically in a rabbit model or in 3D HCE cell construct model, although mildly increased apoptotic cells were detected. Our recent publication of PQ-1 containing drugs revealed only a trend towards elevated caspase-3 activity after 24 hours follow-up time. We estimated that exposure and follow-up time was too short to detect clear apoptotic cell death [6]. That was one reason why it was decided to study drugs for

longer time periods in the present study. Indeed, we could see many detrimental effects, including decreased cellular proliferation, increased cytokine expression and cytotoxicity in the PF latanoprost rather than PF tafluprost -treated cells with the longer time exposures. Moreover, we exposed non-confluent HCE-2 cells to undiluted drugs mimicking natural eye clearance. Interestingly, PF latanoprost treatment provoked the release of LDH and IL-6 in comparison to control or PF tafluprost -treated HCE-2 cells. Thus, the differences in cytotoxicity results between the Payly et al. [38] report and our present data for PF latanoprost may be due to the different exposure times and cell culture model i.e. their 3D cell culture model versus our non-differentiated layers of HCE-2 culture. Similarly as in previous reports, the hypersecretory stimulation of HCE-2 cells led to the appearance of intracellular granulations and vacuoles, what may also explain the changes in cellular ultrastructure observed with high magnification [39].

The tested prostaglandin agents influenced not only the cell viability but also inflammatory stimulation in different ways. It was demonstrated that the cells treated with PF tafluprost in contrast to PF latanoprost and BAK treatments, were able to retain their proliferation ability and this resulted in better cellular viability after 12 hours of exposure. In both PF latanoprost and BAK groups the loss of all viability was in agreement with the reports of others [38, 22]. Strikingly, the MGHS40 concentration (50 mg/ml) is one thousand times higher than that of latonoprost (50 µg/ml) in Monoprost® formulation. In the Travatan® formulation, the MGHS40 concentration is only 2 mg/ml, which is only 5% of the amount present in Monoprost® (SmPCs of the products). Taken together, it seems that it is the high concentration of the solubiliser MGHS40 in Monoprost® which is responsible for causing many of the detrimental cellular effects in HCE-2 cells.

Our data reveals that the concentration of MGHS40 present in PF latanoprost formulation was associated with IL-6 -mediated inflammatory response and increased cytotoxicity in HCE-2 cell cultures. Currently, popularity of PF formulations in ocular drops has increased in the clinics due to their lower side effects and better patient compliance. In addition to preservatives, the other excipients in ocular drop formulations should be evaluated, since some of these agents exert detrimental effects on cells. Acknowledgements

The authors thank Mrs Anne Seppänen for technical assistance and Dr Ewen MacDonald (PhD) for revising the language. This work was supported by the VTR grants of Kuopio University Hospital (KK), the Finnish Eye Foundation (KK), and the Päivikki and Sakari Sohlberg Foundation (KK). DS was supported by EY019037-S from the National Institutes of Health, USA.



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