Gelling hypotonic polymer solution for extended topical drug delivery to the eye

doi:10.1038/s41551-020-00606-8

. 2020 Nov;4(11):1053-1062.

doi: 10.1038/s41551-020-00606-8. Epub 2020 Sep 7.

Gelling hypotonic polymer solution for extended topical drug delivery to the eye

Yoo Chun Kim^{1

2}, Matthew D Shin^{1

2}, Sean F Hackett², Henry T Hsueh^{1

3}, Raquel Lima E Silva², Abhijit Date^{1

2

4}, Hyounkoo Han^{1

2}, Byung-Jin Kim², Amy Xiao^{1

5}, Youngwook Kim^{1

6}, Laolu Ogunnaike^{1

3}, Nicole M Anders⁷, Avelina Hemingway⁷, Ping He⁷, Albert S Jun², Peter J McDonnell², Charles Eberhart², Ian Pitha², Donald J Zack^{1

2}, Peter A Campochiaro², Justin Hanes^{8

9

10

11

12

13

14}, Laura M Ensign^{15

16

17

18

19

20

21

22}

Affiliations

¹ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
² Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
⁴ The Daniel K. Inouye College of Pharmacy, University of Hawaii Hilo, Hilo, HI, USA.
⁵ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
⁶ Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
⁷ The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
⁸ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
⁹ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁰ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹¹ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹² The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹³ Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁴ Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁵ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁶ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁷ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁸ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁹ The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
²⁰ Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
²¹ Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
²² Department of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.

PMID: 32895514
PMCID: PMC7655548
DOI: 10.1038/s41551-020-00606-8

Gelling hypotonic polymer solution for extended topical drug delivery to the eye

Yoo Chun Kim et al. Nat Biomed Eng. 2020 Nov.

. 2020 Nov;4(11):1053-1062.

doi: 10.1038/s41551-020-00606-8. Epub 2020 Sep 7.

Authors

Affiliations

¹ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
² Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
⁴ The Daniel K. Inouye College of Pharmacy, University of Hawaii Hilo, Hilo, HI, USA.
⁵ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
⁶ Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
⁷ The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
⁸ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
⁹ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁰ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹¹ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹² The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. hanes@jhmi.edu.
¹³ Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁴ Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hanes@jhmi.edu.
¹⁵ Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁶ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁷ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁸ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
¹⁹ The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. lensign@jhmi.edu.
²⁰ Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
²¹ Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.
²² Department of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lensign@jhmi.edu.

PMID: 32895514
PMCID: PMC7655548
DOI: 10.1038/s41551-020-00606-8

Abstract

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

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Conflict of interest statement

Competing interests

Y.C.K., A.D., L.E., and J.H. are inventors on patents/patent applications related to this technology.

Figures

**Fig 1 |. Hypotonicity drives water absorption that leads to uniform ocular surface coating and gelation.**
a, Representative optical coherence tomography (OCT) images from rat eyes taken immediately after topical application (Left) of 12% Hypo, 12% Iso, and 18% Iso eye drops and after manually blinking the eyelids (Right). Images are false-colored to differentiate the eye structures (blue) and the topical formulations (red), n = 5 – 6 independent eyes and three images were obtained per eye. White arrows point to the eye drop layer. Scale bar = 100 μm and applies to all images. b, Ensemble averaged mean square displacement (<MSD>) of 200 nm nanoparticles in various liquids and gels at 37°C *in vitro* and after application *in vivo* (particles visualized *ex vivo*). Each data point represents the ensemble average of 50 – 100 nanoparticles tracked in an individual sample, n = 4 – 7 independent animals. Data represented as the mean ± SEM. Statistical analyses conducted by one-way ANOVA with multiple comparisons with respect to the 12% Hypo *in vivo* group. The *in vivo* data groups with boxes around them correspond to those shown in c. c, Representative schematics showing the *ex vivo* tracked trajectories (~20 s) of nanoparticles in the 12% Hypo (Left) formulation compared with 12% Iso (Middle) and 18% Iso (Right) after ocular application *in vivo*.

Fig 2 |. Hypotonic gelling formulation (12% hypo) provides increased intraocular absorption of water-soluble BT (0.15% w/v) compared with the conventional gelling formulation (18% iso) or the commercially available formulation (Alphagan P).
**a-c**, BT concentrations in the rat cornea (a), conjunctiva (b) and aqueous humor (c) measured at 1, 4, and 8 h after the last dose (twice-daily dosing for 5 d, n = 5–9 independent animals). d,e, Change in IOP (ΔIOP) in normotensive rabbits measured for 10 h after a single dose comparing 12% hypo(BT) with 18% iso(BT) or Alphagan P (d) and the 12% hypo vehicle alone (Vehicle) (e) (n = 5–6 independent animals). Data are mean ± s.e.m. Dotted lines mark the starting average IOP (ΔIOP = 0). f, BT concentrations in rabbit serum for up to 3 h after a single dose (n = 4 independent animals). Data are mean ± s.e.m. Statistical analyses conducted by one-way ANOVA with multiple comparisons.

Fig 3 |. Hypotonic gelling formulation (12% Hypo) solubilizes and provides improved intraocular delivery of two insoluble drugs, brinzolamide (BRZ, 1% w/v) and cyclosporine A (CsA, 0.05% w/v), compared to the conventional gelling formulation (18% Iso) or the commercially available formulations (Azopt®, Restasis®).
Brinzolamide concentrations in the rat **(a)** cornea, **(b)** conjunctiva, and **(c)** aqueous measured at 1, 4 and 8 h after the last dose (twice-daily dosing for 5 days, n = 5 – 10 independent animals). Change in intraocular pressure (ΔIOP) in normotensive rabbits measured for 8 h after a single dose, comparing **(d)** 12% Hypo(BRZ) to 18% Iso(BRZ) or to Azopt (n = 5 – 6 independent animals). Data shown as mean ± SEM. Dotted lines mark the starting average IOP (ΔIOP = 0). CsA concentrations in the rat **(e)** cornea and **(f)** conjunctiva measured at 1, 4, and 8 h after the last dose (twice-daily dosing for 5 days, n = 6 – 9 independent animals). Statistical analyses conducted by one-way ANOVA with multiple comparisons.

Fig. 4 |. Once-daily dosing of the hypotonic gelling formulation (12% Hypo) containing water-soluble acriflavine hydrochloride (ACF, 0.5% w/v) and sunitinib malate (SM, 0.4% w/v) in mice suppresses laser-induced choroidal neovascularization (CNV).
Laser-induced rupture of Bruch’s membrane was performed in both eyes, and each mouse received the drug-containing drop in one eye and the 12% Hypo vehicle (Vehicle) in the contralateral eye immediately after laser and once per day thereafter (total 7 days). Both **(a)** 12% Hypo(ACF) (n = 6 independent eyes, one image was obtained per CNV spot) and **(b)** 12% Hypo(SM) (n = 12 independent eyes, one image was obtained per CNV spot) provided a significant reduction in the mean area of CNV. Data shown as mean ± SEM. Statistical analyses conducted by a two-tailed Mann-Whitney test. Right panels show fluorescent images of CNV spots with areas representative of the means for each group. Scale bar = 200 μm and applies to all images.

**Fig 5 |. Hypotonic gelling formulation (12% Hypo) provides therapeutically relevant delivery of sunitinib malate (SM, 0.4% w/v) to the posterior segment with once-daily dosing in rabbits and pigs.**
a, Dutch belted rabbits were dosed unilaterally with 12% Hypo(SM) once daily for 14 days. Ocular tissues were collected 6 h after the last dose (n = 6 independent animals). Combined levels of sunitinib and N-desethyl sunitinib are shown with a dotted line indicating the K_i for VEGFR/PDGFR. b, Juvenile Yorkshire pigs received 12% Hypo(SM) once daily for 5 days unilaterally. Ocular tissues were collected 1 h after the last dose (n = 4 independent eyes). Combined levels of sunitinib and N-desethyl sunitinib are shown with a dotted line indicating the K_i for VEGFR/PDGFR. Drug concentrations achieved in the retina and choroid/retinal pigmented epithelium (Ch/RPE) exceed the inhibitory concentration. c, Laser-induced rupture of Bruch’s membrane was performed in Juvenile Yorkshire pigs and each pig received either 12% Hypo(SM), SM dissolved in saline (SM in Saline), or 12% Hypo vehicle (Vehicle) bilaterally. Topical eye drops were given daily for a total of 15 days. Right panels show fluorescent images of CNV spots with areas representative of the means for each group. Scale bar = 100 μm and applies to all images. The 12% Hypo(SM) (n = 40 CNV spots and one image was obtained per CNV spot) provided a significant reduction in mean area of CNV compared to 12% Hypo vehicle (Vehicle) (n = 21 CNV spots and one image was obtained per CNV spot) or SM in Saline (n = 19 CNV spots and one image was obtained per CNV spot). Statistical analyses conducted by linear mixed model. Data shown as mean ± SEM.

**Fig 6 |. Hypotonic gelling formulation (12% Hypo) is indistinguishable from no treatment (Untreated) with twice-daily dosing for 5 weeks in rabbits.**
12% Hypo was compared to an isotonic formulation (12% Iso), isotonic balanced salt solution (BSS) and no treatment (Untreated) with bilateral administration of twice-daily dosing for 5 weeks in rabbits. Ocular surface damage, as assessed by **(a)** lissamine green staining and scoring and **(b)** counting the number of blinks in a 3 min period 3 h after the first dose on a given day, as well as **(C)** H&E staining of cornea tissues collected at 5 weeks (n = 3 independent eyes and one image was obtained per group), was indistinguishable across all groups.

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Comment in

A topical gel for extended ocular drug release.
Seah I, Loh XJ, Su X. Seah I, et al. Nat Biomed Eng. 2020 Nov;4(11):1024-1025. doi: 10.1038/s41551-020-00645-1. Nat Biomed Eng. 2020. PMID: 33159191 No abstract available.

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References

1. Urtti A, Pipkin JD, Rork G & Repta AJ Controlled Drug Delivery Devices for Experimental Ocular Studies with Timolol .1. Invitro Release Studies. Int J Pharm 61, 235–240, doi:Doi 10.1016/0378-5173(90)90214-O (1990). - DOI
1. Hermann MM, Papaconstantinou D, Muether PS, Georgopoulos G & Diestelhorst M Adherence with brimonidine in patients with glaucoma aware and not aware of electronic monitoring. Acta Ophthalmol 89, E300–E305, doi:10.1111/j.1755-3768.2010.02050.x (2011). - DOI - PubMed
1. Agrawal AK, Das M & Jain S In situ gel systems as ‘smart’ carriers for sustained ocular drug delivery. Expert Opin Drug Del 9, 383–402, doi:10.1517/17425247.2012.665367 (2012). - DOI - PubMed
1. Dumortier G, Grossiord JL, Agnely F & Chaumeil JC A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm Res 23, 2709–2728, doi:10.1007/s11095-006-9104-4 (2006). - DOI - PubMed
1. Rowe RC, Sheskey PJ, Owen S. n. C. & American Pharmacists Association. Handbook of pharmaceutical excipients / edited by Rowe Raymond C., Sheskey Paul J., Quinn Marian E.. 6th edn, (APhA/Pharmaceutical Press;, 2009).

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[1] Urtti A, Pipkin JD, Rork G & Repta AJ Controlled Drug Delivery Devices for Experimental Ocular Studies with Timolol .1. Invitro Release Studies. Int J Pharm 61, 235–240, doi:Doi 10.1016/0378-5173(90)90214-O (1990). - DOI

[2] Urtti A, Pipkin JD, Rork G & Repta AJ Controlled Drug Delivery Devices for Experimental Ocular Studies with Timolol .1. Invitro Release Studies. Int J Pharm 61, 235–240, doi:Doi 10.1016/0378-5173(90)90214-O (1990). - DOI

[3] Hermann MM, Papaconstantinou D, Muether PS, Georgopoulos G & Diestelhorst M Adherence with brimonidine in patients with glaucoma aware and not aware of electronic monitoring. Acta Ophthalmol 89, E300–E305, doi:10.1111/j.1755-3768.2010.02050.x (2011). - DOI - PubMed

[4] Hermann MM, Papaconstantinou D, Muether PS, Georgopoulos G & Diestelhorst M Adherence with brimonidine in patients with glaucoma aware and not aware of electronic monitoring. Acta Ophthalmol 89, E300–E305, doi:10.1111/j.1755-3768.2010.02050.x (2011). - DOI - PubMed

[5] Agrawal AK, Das M & Jain S In situ gel systems as ‘smart’ carriers for sustained ocular drug delivery. Expert Opin Drug Del 9, 383–402, doi:10.1517/17425247.2012.665367 (2012). - DOI - PubMed

[6] Agrawal AK, Das M & Jain S In situ gel systems as ‘smart’ carriers for sustained ocular drug delivery. Expert Opin Drug Del 9, 383–402, doi:10.1517/17425247.2012.665367 (2012). - DOI - PubMed

[7] Dumortier G, Grossiord JL, Agnely F & Chaumeil JC A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm Res 23, 2709–2728, doi:10.1007/s11095-006-9104-4 (2006). - DOI - PubMed

[8] Dumortier G, Grossiord JL, Agnely F & Chaumeil JC A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm Res 23, 2709–2728, doi:10.1007/s11095-006-9104-4 (2006). - DOI - PubMed

[9] Rowe RC, Sheskey PJ, Owen S. n. C. & American Pharmacists Association. Handbook of pharmaceutical excipients / edited by Rowe Raymond C., Sheskey Paul J., Quinn Marian E.. 6th edn, (APhA/Pharmaceutical Press;, 2009).

[10] Rowe RC, Sheskey PJ, Owen S. n. C. & American Pharmacists Association. Handbook of pharmaceutical excipients / edited by Rowe Raymond C., Sheskey Paul J., Quinn Marian E.. 6th edn, (APhA/Pharmaceutical Press;, 2009).

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Gelling hypotonic polymer solution for extended topical drug delivery to the eye

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Gelling hypotonic polymer solution for extended topical drug delivery to the eye

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