JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS ORIGINAL ARTICLE Volume 00, Number 0, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0075

Eyelash Growth Induced by Topical Prostaglandin Analogues, Bimatoprost, Tafluprost, Travoprost, and Latanoprost in Rabbits

Ama´lia Turner Giannico,1 Leandro Lima,1 Heloisa Helena Abil Russ,2 and Fabiano Montiani-Ferreira1

Abstract

Purpose: Prostaglandin analogues (PGA) are ocular hypotensive agents used for the treatment of glaucoma. Hypertrichosis of the eyelashes has been reported in humans as a side effect. Eyelash growth was investigated with clinical trials in people using bimatoprost. Scattered reports of eyelash growth during the treatment of glaucoma with other PGA are also found in the literature. We investigated the effect of 4 different topical PGA on eyelash length. Methods: Forty New Zealand white rabbits were divided into 4 groups and received daily topical application of bimatoprost, tafluprost, travoprost, and latanoprost in the left eye for 4 weeks. The right eye received no treatment. Eyelash length was measured in both eyes before and after treatment using a stainless steel digital caliper. Results: Bimatoprost and tafluprost groups had significant increases in eyelash length. We did not observe significant eyelash growth in rabbits receiving travoprost and latanoprost after 1 month of treatment. Conclusions: Today, only bimatoprost is approved for growing eyelashes, and our research shows that ta- fluprost could be further explored by the cosmetic and pharmaceutical industry. Additional research using travoprost and latanoprost as agents for eyelash growth should be performed in the future using prolonged treatment periods to determinate whether or not these PGA induce eyelash growth, and investigate other possible side effects.

Introduction drugs for glaucoma treatment qualitatively reported eyelash growth, merely as a side effect.7–17 However, only bimato- yelashes, besides providing a natural protective barrier prost and latanoprost have been studied in clinical trials with against foreign bodies for the eyes, also possess an aes- human beings that assessed quantitatively and specifically E 1,2,18–23 thetic function.1,2 In modern society, longer and fuller eye- the eyelash growth. Body hair growth, but not eyelash 24–26 lashes are considered a desirable physical attribute on growth has been investigated in animal models. The women and a sign of femininity and beauty.3 effect of PGA specifically on eyelash growth has not been Since the beginning of the use of prostaglandin analogues studied in animals. (PGA) as an ocular hypotensive agent for the treatment of To the author’s knowledge, this is the first study com- glaucoma, hypertrichosis of eyelashes has been reported as a paring eyelash growth secondary to the use of these topical side effect.1,4,5 Currently, a synthetic PGA, bimatoprost 0.03%, PGA in a homogeneous group of animals. In this article, our besides being used to lower intraocular pressure (IOP) is also goal was to evaluate and discuss the effects of 4 topical PGA approved for cosmetic purposes to increase eyelash length, on eyelash length in New Zealand white rabbits. thickness, and darkness in normal patients and in patients with palpebral hypotrichosis. For the latter use, the drug is Methods 1,2,6 applied topically to the skin of the eyelid margins. Animals Bimatoprost, tafluprost, travoprost, and latanoprost share many similarities both structurally and pharmacologically1 The investigation was carried out using 40 clinically healthy, and these PGA may have similar effects on eyelash growth. 6-month-oldNewZealandwhiterabbits(Oryctolaguscuniculus), Several studies with human patients investigating these PGA 19 males and 21 females, weighing an average 2.66 – 0.03 kg

1Department of Veterinary Medicine, Federal University of Parana´, Curitiba, Brazil. 2Instituto Graefe de Oftalmologia, Curitiba, Brazil.

1 2 GIANNICO ET AL.

(mean – standard deviation). The animals were selected ran- performed with the rabbits manually restrained by the same domly from a commercial breeder collection. All procedures investigator taking care to keep the animals comfortable usingliverabbitswereconductedinaccordancewiththeFederal during the measurements. University of Parana Animal Use Committee (Curitiba city, Eyelash lengths of both the left and right eyes were mea- Parana´ state,Brazil)andwiththeARVOStatementfortheUseof sured using a stainless steel digital caliper with an LCD Animals in Ophthalmic and Vision Research. display (PD-150, 0-150 mm, accuracy: 0.01 mm; Vonder, Physical examinations were performed before ocular exam- Curitiba, PR, Brazil) from the base of the eyelid margin to the inations to exclude animals with any indication of systemic end of the cilia. The central region of the upper eyelid was disease. The anterior ocular structures of all animals were determined by measuring the entire horizontal length of the evaluated using a transilluminator and a slit lamp biomicro- eyelid, dividing the resulting number by 2, thus locating the scope (Hawk Eye; Dioptrix, L’Union, France). Rabbits with any center of the lid. The central region was 6 mm in length with evidence of ocular or systemic diseases were excluded from this 3 mm from the center of the superior eyelid to each side, research. To avoid inter-investigator discrepancies, the same nasal, and temporal (Fig. 1). Eyelashes in this central area are masked investigator performed all the ocular tests. Another naturally bigger than the other ones located at the nasal and investigator (not masked) instilled the eyedrops. The humidity temporal region. The 5 longer eyelashes were selected at the (70%) and temperature (22.5C) conditions were controlled. central palpebral region of the superior eyelid on each rabbit. Top 5 eyelash lengths were then measured pre- and post- Treatments treatment.

Rabbits were divided into 4 groups of 10 animals using the Statistical analysis random function = RANDBETWEEN (1,40) on Microsoft Office Excel (Excel version in Microsoft Office 2007 for Windows) after The Shapiro–Wilk normality test demonstrated that the all animals received a number from 1 to 40. The left eyes were data errors were normally distributed. Data were statistically treated daily for 4 weeks with one drop of topical PGA eye- analyzed using the computer software StatView (SAS In- drops applied to the conjunctival fornix. The ophthalmic drugs stitute, Cary, NC) applying the 2-way analysis of variance used in each group were, respectively: Group 1—bimatoprost and Tukey–Kramer post hoc test to compare pre- and post- 0.03% containing benzalkonium chloride (BAK) as preservative treatment results. Values of P < 0.05 were considered signif- (Lumigan colı´rio; Allergan Indu´stria Farmaceˆutica Ltda., icant. Guarulhos, SP, Brazil); Group 2—tafluprost 0.0015% without preservative (Saflutan colı´rio; Merck Sharp & Dohme Ltda., Results Guarulhos, SP, Brazil); Group 3—travoprost 0.004% with BAK (Travatan colı´rio; Alcon Laborato´rios do Brasil Ltda., Sa˜o Mean, standard deviation, and standard error of the eye- Paulo, SP, Brazil); Group 4—latanoprost 0.005% with BAK lash length values expressed in millimeters (mm) in pre- and (Xalatan colı´rio; Pfizer Indu´stria Farmaceˆutica, Sa˜oPaulo,SP, post-treatment are demonstrated in Table 1. Brazil). All groups are homogenous in the way that they have Comparisons of the pretreatment eyelash length values of almost the same proportion of males and females (5 males and 5 the left eye of all groups were not significantly different > females) each, with the exception of Group 2 (tafluprost) that (P 0.05), showing that the groups were homogeneous be- presented 4 males and 6 females. This was the product of the fore treatment. After PGA treatment, all groups demon- random selection applied. The right eyes remained untreated strated a tendency to increase mean eyelash length values, throughout the study. however, only bimatoprost and tafluprost groups had a significant increase (P < 0.0001 and P = 0.0361, respectively). Eyelash evaluation No significant differences were found on eyelash length on travoprost and latanoprost groups comparing pre- and post- The left and right eyes were evaluated 1 day before the treatment values (P = 0.1330 and P = 0.0581, respectively) start of the treatment and 30 days after. The evaluation was (Table 1). Additionally, comparison between bimatoprost

FIG. 1. Representative eye- lash measuring procedure being demonstrated on the left eye of a New Zealand white rabbit. (A) Five eye- lashes were randomly se- lected measured from the central region of the superior eyelid (6 mm in length—black arrow). (B) Measuring proce- dure being performed on one eyelash at the superior central palpebral region, with a stainless steel digital caliper. EYELASH GROWTH INDUCED BY PROSTAGLANDIN ANALOGUES 3

Table 1. Mean, Standard Deviation, Standard Error, and Coefficient of Variation of the Superior Eyelid Central Region Eyelash Length Values Expressed in Millimeters with P Value of the Comparisons Between Pre- and Post-Treatment (OS) with Prostaglandin Analogues and Untreated Eye (OD) in New Zealand White Rabbits

Mean SD SE CV P value Prostaglandin Group Eye analogues drugs Pre Post Pre Post Pre Post Pre Post Pre vs. Post

1 OS Bimatoprost 0.03% 9.50 11.99 1.09 1.02 0.35 0.32 0.11 0.09 < 0.0001a OD Untreated 9.31 9.38 1.04 1.03 0.33 0.32 0.11 0.11 0.8830 2 OS Tafluprost 0.0015% 9.97 11.03 1.01 1.08 0.32 0.34 0.10 0.10 0.0361a OD Untreated 10.08 10.11 1.04 0.87 0.33 0.28 0.10 0.09 0.9470 3 OS Travaprost 0.004% 10.16 11.32 1.57 1.70 0.50 0.54 0.15 0.15 0.1330 OD Untreated 9.90 9.92 1.63 1.73 0.51 0.55 0.16 0.17 0.9822 4 OS Latanoprost 0.005% 9.70 10.51 0.97 0.81 0.31 0.26 0.10 0.08 0.0581 OD Untreated 9.65 9.77 0.94 0.93 0.30 0.29 0.10 0.10 0.7807

aSignificant difference. SD, standard deviation; SE, standard error; CV, coefficient of variation; OS, oculus sinister (left eye); OD, oculus dexter (right eye). and tafluprost post-treatment values were not statistically treated with latanoprost also showed a slight tendency to- significant (P = 0.3820). There was no significant difference in ward an increase in eyelash length, however, with no sig- eyelash length between males and females in pre- and post- nificant differences.8,15 These references corroborate our treatment (P > 0.05). The mean eyelash length values from the result. Nevertheless, other studies with human patients right eye (that did not receive treatment) did not show sig- showed that latanoprost significantly increased eyelash nificant difference between pre- and post-treatment for all length.18,27 Perhaps, the differences in experimental condi- groups (P > 0.05) (Table 1). tions, species studied (human vs. animals), and duration of No clinical ophthalmic signs such as mucous secretion, the treatment might be responsible for these discrepancies blepharospasm, blepharoedema, conjunctival and palpebral and should be taken into account. congestion or pigmentation were observed after treatment in Possibly, 1 month of treatment was time enough to dem- any group. onstrate differences in eyelash growth in rabbits receiving

bimatoprost and tafluprost groups, but not enough time to Discussion promote a difference in eyelash length in rabbits receiving travoprost and latanoprost. In some studies of PGA use in There are several studies that aimed to evaluate the effect humans, treatments were longer than 3 months.7,9–12,14–16 of PGA in human patients with glaucoma that mentioned as Considering this, possibly one of the limitations of our in- a curiosity or merely as a subjective observation some side vestigation was a short duration of treatment. effects on the eyelashes, such as increased growth, number, Other side effects reportedly caused by PGA drugs in length, thickness, curvature, and pigmentation.12–14,17 Since humans include conjunctival hyperemia, increase of iris in these articles, the main objective was the evaluation of the pigmentation, eyelid pigmentation and, in rare cases, perio- main action of these aforementioned PGA drugs, which is cular pigmentation.5,7,28 Hyperpigmentation can occur lowering the IOP, the experimental design and statistical within 3 to 8 weeks after treatment initiation in human pa- analysis used to demonstrate eyelash growth were unclear or tients.7,29 Some researchers suggest that a faster onset of not mentioned at all. Previously, eyelash growth was de- periocular skin hyperpigmentation may occur with direct scribed qualitatively and never quantitatively.9–11,16 application of PGA to the eyelid.29 Conjunctival hyperemia, No other study has detailed the method used to measure skin hyperpigmentation, and erythema of the eyelid are the the eyelash growth in rabbits. We have chosen the 5 longer most common adverse events caused by the bimatoprost eyelashes of the central palpebral region for both, pre- and solution 0.03% (Latisse)6 and these side effects were observed post-treatment because we have noticed that this region al- in another study with human patients diagnosed with ocular ready has longer cilia. We assumed that most if not all the hypertension and treated with latanoprost, travoprost, ta- cilia chosen on both occasions were most likely the same. We fluprost, and bimatoprost eye drops for more than 3 believe that even if different cilia were measured in the post- months.30 Probably, all PGA used in our study may cause treatment period, these would be the longest ones and it still similar effects if used for a long time, as eyedrops and as a would be a strong indicative of eyelash growth. solution applied to the upper eyelid margin. No other signs The bimatoprost solution 0.03% (Latisse)6 is the only such as erythema of the eyelids or conjunctiva hyperemia prescription product approved by the US Food and Drug were observed in our study. Changes in pigmentation could Administration for growing eyelashes.6 Our research shows not be evaluated in this study because New Zealand white that tafluprost 0.0015% has similar effects as bimatoprost rabbits are albinos and do not have any pigmented cells. The 0.03% on eyelash growth of rabbits after 30 days of daily next logical step for further studies would be developing treatment. new solutions to be applied to the upper eyelid margins In the conditions of the present investigation, travoprost containing tafluprost and observing possible side effects. and latanoprost did not significantly increased eyelash Based on the general desire for longer, thicker, and darker length even though higher values were found in the post- eyelashes as a sign of femininity and beauty in most cultures, treatment period. Human patients affected by glaucoma and we tested other PGA as alternatives for those seeking to 4 GIANNICO ET AL. augment their lash appearance. Tafluprost 0.0015% eye 15. Stecchi, G., Saccucci, S., Molinari, S., et al. Eyelash hyper- drops can promote similar eyelash growth as bimatoprost trichosis induced by topical latanoprost: 6-month follow-up. 0.03% in rabbits when used once daily for 30 days. Travo- Acta. Ophthalmol. Scand. 236:56–57, 2002. prost 0.004% and latanoprost 0.005% did not affect eyelash 16. Uusitalo, H., Pillunat, L.E., and Ropo, A. Efficacy and safety length in this study. of tafluprost 0.0015% versus latanoprost 0.005% eye drops in open-angle glaucoma and ocular hypertension: 24-month Acknowledgments results of a randomized, double-masked phase III study. Acta. Ophthalmol. 88:12–19, 2010. The authors thank Dr. Gillian Shaw, Johns Hopkins Uni- 17. Swymer, C., and Neville, M.W. Tafluprost: the first preser- versity, Baltimore, MD, for her invaluable help in the prep- vative-free prostaglandin to treat open-angle glaucoma and aration of this manuscript. ocular hypertension. Ann. Pharmacother. 46:1506–1510, 2012. 18. Sugimoto, M., and Uji, Y. Quantitative analysis of eyelash Author Disclosure Statement lengthening following topical latanoprost therapy. Can. J. Ophthalmol. 37:342–345, 2002. No competing financial interests exist. 19. Akhyari, M., Jafari, A.K., Seyrafi, H., et al. Latanoprost for the treatment of alopeci areata of eyelashes. Ir. J. Dermatol. References 1:7–10, 2008. 20. Faghihi, G., Andalib, F., and Asilian, A. The efficacy of la- 1. Law, S.K. Bimatoprost in the treatment of eyelash hypo- tanoprost in the treatment of alopecia areata of eyelashes trichosis. Clin. Ophthalmol. 4:349–358, 2010. and eyebrows. Eur. J. Dermatol. 19:586–597, 2009. 2. Smith, S., Fagien, S., Whitcup, S.M., et al. Eyelash growth in 21. Roseborough, I., Lee, H., Chwalek, J., et al. Lack of efficacy subjects treated with bimatoprost: a multicenter, random- of topical latanoprost and bimatoprost ophthalmic solutions ized, double-masked, vehicle-controlled, parallel-group in promoting eyelash growth in patients with alopecia study. J. Am. Acad. Dermatol. 66:801–806, 2012. areata. J. Am. Acad. Dermatol. 60:705–706, 2009. 3. Shaikh, M.Y. and Bodla, A.A. Hypertrichosis of the eye- 22. Fagien, S. Management of hypotrichosis of the eyelashes: Focus lashes from prostaglandin analog use: a blessing or a bother on bimatoprost. Clin. Cosmet. Investig. Dermatol. 3:39–48, 2010. to the patient? J. Ocul. Pharmacol. Ther. 22:76–77, 2006. 23. Wester, S.T., Lee, W.W., and Shi, W. Eyelash growth from 4. Wand, M. Latanoprost and hyperpigmentation of eyelashes. application of bimatoprost in gel suspension to the base of Arch. Ophthalmol. 115:1206–1208, 1997. the eyelashes. Ophthalmology 117:1024–1031, 2010. 5. Hollo´, G. The side effects of the prostaglandin analogues. 24. Uno, H., Zimbric, M.L., Albert, D.M., et al. Effect of lata- Expert. Opin. Drug. Saf. 6:45–52, 2007. noprost on hair growth in the bald scalp of the stump-tailed 6. Allergan, Inc. LatisseTM (Bimatoprost Ophthalmic Solution) macacque: a pilot study. Acta. Derm. Venereol. 82:7–12, 2002. 0.03% Prescribing Information. Irvine, CA: Allergan, Inc.; 25. Sasaki, S., Hozumi, Y., and Kondo, S. Influence of prosta- 2009. glandin F2alpha and its analogues on hair regrowth and 7. Johnstone, M.A. Hypertrichosis and increased pigmentation follicular melanogenesis in a murine model. Exp. Dermatol. of eyelashes and adjacent hair in the region of the ipsilateral 14:323–328, 2005. eyelids of patients treated with unilateral topical latano- 26. Woodward, D.F., Tang, E.S., Attar, M., et al. The biodispo- prost. Am. J. Ophthalmol. 124:544–547, 1997. sition and hypertrichotic effects of bimatoprost in mouse 8. Noecker, R., Bulau, S., and Schwiegerling, J. Xalatan-in- skin. Exp. Dermatol. 22:145–148, 2013. duced changes in periocular skin pigmentation and lash 27. Casson, R.J., and Selva, D. Lash ptosis caused by latano- dimensions measured using a digital imaging technique. prost. Am. J. Ophthalmol. 139:932–933, 2005. Invest. Ophthalmol. 40:S832, 1999. 28. Doshi, M., Edward, D.P., and Osmanovic, S. Clinical course 9. Gandolfi, S., Simmons, S., Sturm, R., et al. Three-month of bimatoprost-induced periocular skin changes in Cauca- comparison of bimatoprost and latanoprost in patients with sians. Ophthalmology 113:1961–1967, 2006. glaucoma and ocular hypertension. Adv. Ther. 18:110–121, 29. Priluck, J.C., and Fu, S. Latisse-induced periocular skin hy- 2001. perpigmentation. Arch. Ophthalmol. 128:792–793, 2010. 10. Netland, P., Landry, T., Sullivan, K., et al. Travoprost com- 30. Inoue, K., Shiokawa, M., Higa, R., et al. Adverse periocular pared with latanoprost and timolol in patients with open- reactions to five types of prostaglandin analogs. Eye (Lond) angle glaucoma or ocular hypertension. Am. J. Ophthalmol. 26:1465–1472, 2012. 132:472–484, 2001. 11. Sherwood, M., and Brandt, J. Six-month comparison of bi- Received: April 6, 2013 matoprost once-daily and twice-daily with timolol twice- Accepted: July 25, 2013 daily in patients with elevated intraocular pressure. Surv. Ophthalmol. 45:S361–S368, 2001. Address correspondence to: 12. Easthope, S., and Perry, C. Topical bimatoprost: a review of Fabiano Montiani-Ferreira, DVM, MSc, PhD its use in open-angle glaucoma and ocular hypertension. Department of Veterinary Medicine Drugs Aging. 19:231–248, 2002. Federal University of Parana´ 13. Eisenberg, D., Toris, C., and Camras, C. Bimatoprost and Rua dos Funciona´rios, 1540 travoprost: a review of recent studies of two new glaucoma Juveveˆ drugs. Surv. Ophthalmol. 47:S105–S115, 2002. Curitiba City CEP 80035-050 14. Higginbotham, E., Feldman, R., Stiles, M., et al. Latanoprost Brazil and timolol combination therapy vs monotherapy. One-year randomized trial. Arch. Ophthalmol. 120:915–922, 2002. E-mail: [email protected]