IOVS Papers in Press. Published on January 29, 2015 as Manuscript iovs.14-16035
1
Cosmetic preservatives as
2
therapeutic corneal and scleral
3
tissue cross-linking (TXL) agents
4 5
Natasha Babar, MiJung Kim, Kerry Cao, Yukari Shimizu, Su-Young Kim,
6
Anna Takaoka, Stephen L. Trokel, David C. Paik
7 8 9 10
Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY, USA.
11 12
Key words: cornea, sclera, tissue cross-linking, keratoconus, progressive myopia
13 14 15
Running title: Cosmetic preservatives for corneal and scleral tissue cross-linking
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Supported in part by: Research to Prevent Blindness, the Bjorg & Stephen Ollendorf Fund, and by National Institutes of Health Grants NCRR UL1RR024156, NEI P30 EY019007, and NEI R01EY020495(dcp). Corresponding Author: David C. Paik, M.D. Department of Ophthalmology Edward S. Harkness Eye Institute Columbia University, College of Physicians and Surgeons 160 Fort Washington Ave. Room 715 New York, NY 10032 USA tel: 212-305-1622 fax: 212-342-5455 email: [email protected]
1
Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
34
Common abbreviations used throughout this study
35 36
CXL:
37
photochemically-induced corneal cross-linking, corneal collagen cross-linking, corneal
38
cross-linking, collagen cross-linking)
39
TXL: tissue cross-linking
40
UVA: Ultraviolet-A irradiation (λmax = 370 nm)
41
DSC: differential scanning calorimetry
42
FAR(s): formaldehyde releaser(s) or formaldehyde releasing agent(s)
43
BNA(s): beta-nitroalcohol(s)
44
DAU: diazolidinyl urea
45
IMU: imidazolidinyl urea
46
SMG: sodium hydroxymethylglycinate
47
DMDM: DMDM hydantoin
48
OCT: 5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane
49
HNPD: 2-hydroxymethyl-2-nitro-1,3-propanediol
50
BP: bronopol
51
HONA(s): higher order nitroalcohol(s)
UVA-riboflavin
mediated photochemical
52 53 54 55
2
cross-linking
(also
known as
56
Abstract
57
Purpose: Previously, aliphatic β-nitroalcohols have been studied as a means to
58
chemically induce tissue cross-linking (TXL) of cornea and sclera. There are a number of
59
related and possibly more potent agents known as “formaldehyde releasers” (FARs) that
60
are in commercial use as preservatives in cosmetics and other personal care products. The
61
present study was undertaken in order to screen such compounds for potential clinical
62
utility as therapeutic TXL agents.
63
Methods: A chemical registry of 62 FARs was created from a literature review and
64
included
65
carcinogenicity/mutagenicity, toxicity, hydrophobicity, and commercial availability.
66
From this registry, five (5) compounds [diazolidinyl urea (DAU), imidazolidinyl urea
67
(IMU), sodium hydroxymethylglycinate (SMG), DMDM hydantoin (DMDM), 5-Ethyl-
68
3,7-dioxa-1-azabicyclo [3.3.0] octane (OCT)] were selected for efficacy screening using
69
two independent systems, an ex vivo rabbit corneal cross-linking simulation set up and
70
incubation of cut scleral tissue pieces. Treatments were conducted at pH 7.4 or 8.5 for
71
30min. Efficacy was evaluated using thermal denaturation temperature (Tm) and cell
72
toxicity was studied using the trypan blue exclusion method.
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Results: Cross-linking effects in the five selected FARs were pH and concentration
74
dependent. Overall, the Tm shifts were in agreement with both cornea and sclera. By
75
comparison with BNAs previously reported upon, the FARs identified in this study were
76
significantly more potent but with similar or better cytotoxicity.
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Conclusions: FARs, a class of compounds well known to the cosmetic industry, may
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have utility as therapeutic TXL agents. The compounds studied thus far show promise
79
and will be further tested.
characteristics
relevant
to
TXL
80 81 82 83 84 3
such
as
molecular
weight,
85
Introduction
86
The growing clinical success of UVA-riboflavin photochemical corneal cross-
87
linking (CXL) to halt the progression of keratoconus (KC)1-8 and post-LASIK
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keratectasia8-9 suggests that increasing mechanical tissue strength in vivo can be
89
beneficial. CXL increases the stiffness of corneal tissue as shown in animal studies using
90
post-mortem mechanical strip testing.10 A majority of patients ultimately gain
91
improvements in topography and gain lines of vision.5-9 Application of CXL has been
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extended to treat corneal edema,11 corneal melting,12 and corneal infections.13
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As clinical trials involving CXL progress in the United States, suggestions have
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been made to extend its use to the sclera as a treatment for progressive myopia,14 since
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biomechanical weakening occurs during progressive globe elongation.2 Scleral cross-
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linking with UVA-riboflavin technology has been reported15 but may be difficult to carry
97
out in the posterior region of the sclera without the use of surgical means. Also, of
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concern is the potential of damaging the neural retina during UVA irradiation.15 The use
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of injectable pharmacologic agents that could cross-link the sclera as an alternative to glyceraldehyde,16
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photochemical
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glutaraldehyde,14 genipin,17 and nitroalcohols (Hoang QV, et al. IOVS 2013;54:ARVO E-
102
Abstract 5169).18
activation
is
being
explored
and
include
103
The present study serves as an extension of our previous work using
104
nitroalcohols,19-20 where we test the corneal and scleral cross-linking efficacy of several
105
related though potentially more potent chemicals from a group known as “formaldehyde
106
releasing agents (FARs).” These compounds are used as preservatives in a wide array of
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popular cosmetic and personal care products21-22 such as skin care products, body wash,
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fingernail polish and shampoo, including the former formula for Johnson & Johnson’s
109
“No More Tears” Baby Shampoo.23 FARs have also been employed in the textile industry
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as cross-linking agents to impart anti-wrinkle properties to clothing.24 Considering their
111
use in everyday items that come into direct contact with the human body, we wanted to
112
examine the efficacy and cell toxicity of FARs as tissue cross-linking agents as a first
113
step in their potential development for clinical use. To that end, in vitro and ex vivo cross-
114
linking experiments were conducted using corneal and scleral tissues as collagenous
115
tissue substrates. Effectiveness of cross-linking was evaluated using shifts in the thermal 4
116
denaturation temperature (Tm) of the collagen in corneal and scleral tissue as measured by
117
differential scanning calorimetry (DSC), where Tm is the melting temperature of a folded
118
protein.25 Past studies have employed Tm as a measure of the thermal stability of
119
biological tissue.16, 26-29 Therefore, we used differences in Tm between chemically treated
120
and control tissue to estimate extent of cross-linking and compared these values to those
121
induced by CXL (i.e. riboflavin photochemical cross-linking), which served as a positive
122
control for corneal samples. Toxicity was assessed using trypan blue exclusion staining.30
123
Our results suggest that some FARs are potent chemical agents for tissue cross-linking
124
with moderate cell toxicity thresholds.
125 126
5
127
Materials and Methods
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Chemical registry
129
A chemical registry of formaldehyde releasers (FARs) commonly found in
130
cosmetics and other personal care products (PCPs) was compiled from a review of the
131
literature.21, 22 Information used to assemble this registry included characteristics relevant
132
to tissue cross-linking such as molecular weight, European Union maximum allowed
133
concentration
134
hydrophobicity (octanol/water partition coefficient or logP), efficacy of formaldehyde
135
release, and commercial availability of the chemicals. From the FARs identified, five
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compounds with favorable profiles were selected for cross-linking efficacy and toxicity
137
evaluation. These compounds include diazolidinyl urea (DAU), imidazolidinyl urea
138
(IMU), DMDM hydantoin (DMDM), sodium hydroxymethylglycinate (SMG), and 5-
139
Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane (OCT), which were specifically chosen
140
because of the vastness of their use in cosmetics and PCPs as well as on their ability to
141
donate formaldehyde in solution under equilibrium conditions.31-32 The cross-linking
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efficacy and toxicity of two additional FARs, bronopol (BP) and 2-hydroxymethyl-2-
143
nitro-1,3-propanediol (HNPD), which are β-nitroalcohols (BNAs) that we have
144
previously worked with, was included for comparative purposes.
(i.e.
“max
allowed”),
carcinogenicity/mutagenicity,
toxicity,
145 146
Chemicals
147
Diazolidinyl
urea
(N-Hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-
148
dioxoimidazolidin-4-yl)-N'-hydroxy-methylurea [DAU]), imidazolidinyl urea (N,N'-
149
methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea [IMU]), sodium
150
hydroxymethylglycinate (SMG), 5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane (7a-
151
Ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole
152
propanediol or bronopol (BP), hydroxypropyl methyl cellulose (HPMC, 15 centipoise),
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dextran (high molecular weight = 425-575,000 Da), sodium bicarbonate and
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ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich Corp. (St.
155
Louis,
156
imidazolidinedione [DMDM])
157
Columbia, SC). 2-hydroxymethyl-2-nitro-1-3-propanediol (HNPD) was obtained from
MO).
DMDM
hydantoin
[OCT]),
2-bromo-2-nitro-1,3-
(1,3-bis(hydroxymethyl)-5,5-dimethyl-2,4-
was obtained from Oakwood Products, Inc. (West
6
158
TCI Chemicals, Inc. (New York, NY). Riboflavin-5-phosphate was obtained from MP
159
Biomedicals (Santa Ana, CA). Dulbecco’s phosphate buffered saline (DPBS) solution
160
(MgCl2 & CaCl2 free) was obtained from Life Technologies (Carlsbad, CA). All chemical
161
solutions and buffers were prepared fresh using Millipore water (double distilled, de-
162
ionized water, 𝜌 = 18.2 MΩcm at 25 °C) on the day of cross-linking.
163 164
Chemical and riboflavin-mediated photochemical cross-linking (CXL) of the cornea
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Intact cadaveric rabbit heads with clear corneas were obtained fresh (within an
166
hour of sacrifice) in adherence with the ARVO Statement For the Use of Animals in
167
Ophthalmic and Vision Research. FAR solutions at concentrations equivalent to half the
168
maximum allowed value (1/2max) were administered in a manner designed to simulate
169
therapeutic cross-linking in patients. For all of the corneal experiments (with the notable
170
exception of CXL), the corneal epithelium was left intact. An 8mm Hessburg-Barron
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corneal reservoir was affixed to the corneal surface using the supplied syringe vacuum.
172
A single drop of proparacaine (0.5%) was applied to the corneal surface prior to reservoir
173
application. A buffer solution containing 0.1M NaHCO3 at either pH 8.5 or 7.4 was used.
174
The pH of the sample and buffer mixture was titrated to the desired pH just prior to
175
application to the eye using an appropriately concentrated HCl solution. Treatments were
176
conducted over a 30-minute period at 25°C with refreshing of the solution every five
177
minutes. The control contralateral eye was treated identically with vehicle. Immediately
178
after treatment, a central 6mm corneal button was trephined from the treated region of
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each eye, was blotted on both sides using a paper towel to remove excess solution, and
180
was analyzed using differential scanning calorimetry (DSC) [see below]. A minimum of
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two independent determinations were carried out for each condition described using a
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fresh cadaver head each time.
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As a comparison, the same ex vivo system was used to conduct photochemical
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cross-linking of rabbit cornea as previously described by Wollensak et al.,1 with some
185
changes. To that end, a central 8mm portion of the corneal epithelium was debrided using
186
a blunt-end scalpel. De-epithelialized corneal tissue was pre-soaked in 0.1% riboflavin-5-
187
phosphate solution in 1.1% HPMC for 5 mins. Thereafter, the cornea was exposed to UV
188
light (λmax = 370 nm) at an irradiance of 3 mW/cm2 with an 8mm aperture for 30 mins 7
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using the Optos XLink Corneal Collagen Cross-Linking System (Optos, Dunfermline,
190
UK). Riboflavin solution was refreshed every 3 mins for the course of the treatment. The
191
control contralateral eye was treated identically without irradiation.
192 193
Scleral tissue cross-linking
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The following is a modified version of the procedure that we have previously
195
described for testing tissue cross-linking (TXL) efficacy of various BNAs.18 Enucleated
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porcine globes were purchased from Visiontech, Inc. (Sunnvale, TX) and were stored at -
197
80°C until time of experimentation (1-2 months). Equatorial scleral strips approximately
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6mm x 40mm in size were obtained from multiple eyes. These strips were submerged in
199
DPBS solution containing 1mM EDTA to inactivate native collagenases and to prevent
200
tissue dehydration during sample preparation. Each strip was further cut into smaller
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4mm x 3mm pieces. The scleral pieces were individually transferred to a 24 well plate
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and were incubated in 1 ml of cross-linking solution in 0.1M NaHCO3 buffer at either pH
203
8.5 or 7.4 for 30 mins at 25°C without refreshing the solution. Four concentrations of
204
FAR solution were tested at each pH: 1) max allowed concentration 2) 1/2 max allowed
205
concentration 3) 1/10 max allowed concentration, and 4) 25 mM. Tissue samples cross-
206
linked with the BNAs BP and HNPD at concentrations of 5 mM (max allowed for BP),
207
10 mM and 25 mM were used as positive controls. Negative controls were treated
208
identically with vehicle. Post-treatment, all solutions were aspirated and samples were
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washed twice using DPBS to remove remnant cross-linking solution before being
210
analyzed by DSC. A minimum of three independent determinations were carried out for
211
each condition using scleral pieces originating from different porcine globes.
212 213
Differential Scanning Calorimetry (DSC) and cross-link analysis
214
Thermal denaturation temperature (Tm) of all samples was measured using a
215
Perkin-Elmer DSC 6000 Autosampler (Waltham, Massachusetts). Tissue samples were
216
carefully blotted in a standardized, repetitive manner to remove excess solution / DPBS
217
and transferred to pre-weighed 50ul aluminum pans.
218
hermetically sealed using a DSC pan sealing press, which is used to prevent tissue
219
dehydration due to evaporative losses. DSC scans were run using Pyris software (version 8
The pans were immediately
220
11.0) from 40°C to 75°C at a rate of 1°C/min and denaturation curves representing
221
differential heat flow over time were recorded. DSC heat flow endotherm data was
222
analyzed using the Pyris data analysis peak search function using a calculation limit of
223
±0.3°C from the apparent thermal denaturation peak.
224 225
Statistical analysis
226
Student’s t-tests were used to evaluate the significance of observed differences in
227
Tm between cross-linked and control groups. Due to the nature of the ex vivo cadaveric
228
system used for corneal cross-linking, where each cadaver provided the treated eye and
229
contralateral control, corneal samples were subjected to paired t-tests. Conversely, scleral
230
samples were subjected to non-paired t-tests assuming equal variance of data.
231
Significance of all statistical tests was based on an alpha value of 0.05 (p ≤ 0.05). All
232
ΔTm values are reported in the form of mean value followed by standard error.
233 234
FAR cytotoxicity threshold
235
FAR cell cytotoxicity assays were performed as we have previously described.33
236
Briefly, healthy human skin fibroblasts (HSFs) from ATCC (Manassas, VA) were
237
cultured in dermal cell basal media (ATCC) using a serum-free fibroblast growth kit
238
provided by the company (ascorbic acid, EGF/TGF-β1, glutamine, hydrocortisone,
239
insulin and FB growth supplements). Cells were grown in 5% CO2 and 95% ambient air
240
at 37°C until confluent. Once confluent, the cells were detached and seeded into 24 well
241
plates at a density of 5x104 cells/well and were once again allowed to reach confluence.
242
Next, cells were treated with FAR solutions over a range of concentrations (0.001mM-
243
5mM) for 24 hrs. Following cross-linking exposure, all cell media, including FAR
244
solution, was aspirated and each well was rinsed once with DPBS. Fresh media was then
245
reintroduced and the cells were allowed to recover for 48 hrs. Subsequent to cell
246
recovery, cell toxicity was assessed using a modified version of the trypan blue staining
247
protocol. To that end, all culture media was aspirated and each well was rinsed with
248
DPBS. Next, 0.4% trypan blue solution (Gibco, Grand Island, NY, USA) was added to
249
each well for 3 minutes at 25°C. The staining solution was then aspirated and cells were
9
250
washed twice with DPBS. Finally, extent of trypan blue staining and morphology of cells
251
was visualized using an inverted microscope (Fisher Scientific Cat#12-560-45).
252 253
Results
254
Identification of FARs
255
From a broad review of the literature, we were able to identify a total of 62
256
formaldehyde-releasing agents that can potentially be used for corneal and scleral tissue
257
cross-linking. These include FARs commonly found in cosmetics and personal care
258
products as well as those that are used in the textile industry. Table 1 depicts the
259
structures, chemical formulae, toxicity, and other pertinent information of the seven
260
FARs that were chosen for evaluation in this study. This is an excerpt from the larger
261
chemical registry that was compiled. Neither of the chemicals that were tested in this
262
study are known carcinogens (various MSDS). They range in size up to < 400Da with
263
IMU being the largest at 388Da and SMG the smallest at 104Da [MW = 127 – 23 (Na) =
264
104Da]. In most but not all cases, mutagenicity data is available, and these chemicals
265
have been found to be non-mutagenic using Ames, micronucleus and other standard
266
assays. Furthermore, they exhibit low organismal toxicity as indicated by high (>1,000
267
mg/kg,) rat oral LD50 values. The exception is BP which has a relatively low LD50 Oral,
268
rat = 180mg/kg (Table 1).
269 270
Efficacy of corneal cross-linking
271
The ability of five FARs (DAU, IMU, SMG, DMDM, OCT) to cross-link intact
272
cadaveric rabbit cornea, a substrate for collagenous tissue, was assessed using an ex vivo
273
tissue cross-linking (TXL) simulation set up. Cross-linking effects were measured using
274
differential scanning calorimetry (DSC), an assay method based on changes in thermal
275
denaturation temperature (Tm). Our results indicate that two out of the seven FARs
276
studied, DAU and SMG, are effective collagen cross-linking agents for the cornea with
277
the epithelium left intact (in the ex vivo simulation setup) at half maximum allowed
278
concentrations. DAU was effective at pH 8.5 and SMG was effective at both pH 8.5 and
279
7.4. This was evidenced by shifts in the thermal denaturation temperature of corneal
280
tissue (Fig.1). SMG at pH 8.5 showed the greatest upwards shift in Tm (ΔTm = 3.573 ± 10
281
0.578°C, p < 0.05) followed by DAU at pH 8.5 (ΔTm = 3.210 ± 0.742°C, p < 0.05). SMG
282
also showed effective cross-linking at pH 7.4 (ΔTm = 2.281 ± 0.697°C, p < 0.05). Some
283
inconsistencies in the shifts in Tm induced by SMG at pH 7.4, however, were noted and a
284
sample size of n = 8 was required to reach statistical significance. A negative shift in Tm
285
on the order of ~0.5°C was observed for DAU at pH 7.4 and for IMU at both pH 8.5 and
286
7.4 (Fig.1) but the shift was only significant for IMU at pH 8.5 (ΔTm = -0.69 ± 0.697°C,
287
p < 0.05). The lack of effect under these conditions may reflect issues related to epithelial
288
permeability since both DAU and IMU are significantly larger than SMG. Furthermore,
289
an increase in Tm was observed for DMDM at both pH 8.5 and 7.4 (ΔTm = 2.04 ±
290
0.225°C and 2.13 ± 0.273°C, respectively) and for OCT at pH 8.5 (ΔTm = 1.10 ±
291
0.246°C). However, these observed increases in Tm were not statistically significant using
292
paired controls, which included the contralateral eye for each sample. Rabbit cornea
293
cross-linked using UVA-riboflavin (CXL) showed an increase in Tm comparable to
294
values previously reported. In the present study, the ΔTm following CXL = 1.73 ±
295
0.487°C. We previously reported a ΔTi = 1.9°C,19 which was similar to that reported by
296
Spoerl et al. at 2.5°C.34 Ti values indicate the onset of thermal shrinkage (Ts) temperature
297
and change in Ti (ΔTi) is another parameter popularly used to measure extent of tissue
298
cross-linking. As previously reported, the CXL effect is relatively mild from a “thermal
299
transition shifting” standpoint if one considers the potential magnitude of shifts in Tm that
300
may be induced using chemical agents. Lastly, it is worth noting that corneal tissue
301
remained clear to visual inspection using either chemical or photochemical cross-linking
302
treatment.
303 304
Efficacy of scleral cross-linking
305
The results for scleral tissue cross-linking are generally comparable to the results
306
for corneal samples, although different methods of application were used (i.e. refreshing
307
solution every five mins for corneal experiments and not refreshing for scleral
308
experiments). In this case, two additional FARs, BP and HNPD, were also tested. We
309
found that SMG, DAU, and DMDM induced statistically significant cross-linking effects
310
at pH 8.5 and 7.4 (with the exception of SMG at pH 7.4) at concentrations as low as half
311
max allowed. In addition, both a concentration and pH dependent effect was observed for 11
312
the FARs (Fig. 2). A notable exception to the concentration dependent effect is seen in
313
the thermal denaturation data for SMG at pH 7.4 and 39.06 mM, where little change in
314
Tm is observed (ΔTm= 0.007 ± 0.222°C, p = 0.493) although a dramatic upward shift is
315
seen for the same concentration using a pH of 8.5 (ΔTm= 9.073 ± 0.450°C, p < 0.05). The
316
reason for this difference is unclear since in general, upwards shifts in Tm occur for most
317
FARs, albeit consistently greater for pH 8.5 over 7.4. It should be noted that SMG is
318
highly basic in un-buffered solution, requiring the addition of significant amounts of acid
319
in order to achieve the targeted pH of 7.4. Thus, we speculate that the procedure for
320
titrating the buffered SMG solution to pH 7.4 may have impacted the efficacy of TXL in
321
this case. This phenomenon might also explain the inconsistencies in TXL efficacy
322
experienced when intact cornea was cross-linked using SMG at pH 7.4, as mentioned
323
above.
324
We also tested FARs at a concentration of 25 mM in order to directly compare the
325
cross-linking “potency” of each FAR in comparison to the others. We chose a relatively
326
high concentration for this comparison because we wanted to elicit a noticeable cross-
327
linking effect using the BNAs HNPD and BP within 30 mins. DAU showed the greatest
328
shifts in thermal denaturation temperature at 25 mM for both pH 8.5 and 7.4 (ΔTm= 7.713
329
± 0.226°C and 4.347 ± 0.538°C, respectively, p 2,000 mg/kg63
LD50 Oral - rat 2,100 mg/kg, LD50 Dermal - rabbit >2,000 mg/kg54 LD50 Oral – rat – 3,720 mg/kg; LD50 Oral – rat - >2,000 mg/kg50 LD50 Oral - rat >3,600 mg/kg; LD50 Dermal rabbit - 1,948 mg/kg53
Bronopol [BP; CAS No: 52-51-7; MW: 199.99 g/mol; Formula: C3H6BrNO4] 2-hydroxymethyl-2-nitro1,3-propanediol [HNPD; CAS No: 126-11-4; MW: 151.12 g/mol; Formula: C4H9NO5]
1.150±0.63161
-0.115±0.770
62
0.121 (5 mM)
-
Non-mutagenic§51
Non-mutagenic||
52
LD50 Oral - rat 180 mg/kg51
LD50 Oral - rat1,917 mg/kg; LD50 Oral - mouse – 10,550 mg/kg62
* non-mutagenic: Ames, Micronucleus Assay † non-mutagenic: Ames - 100% Sodium hydroxymethylglycinate, Mouse Micronucleus; Rat Hepatocyte/DNA Repair Assay; In vivo – In vitro Rat Hepatocyte UDS Assay ‡ non-mutagenic: Ames – Salmonella - 55% DMDM - 0.001-5 ul/plate; Salmonella / Mammalian-Microsome Preincubation Mutagenicity Assay - Salmonella-2.0ul/plate; mutagenic: L5178 TK+/- Mouse Lymphoma Assay; 0.01-1.0ug/ml; L5178 TK+/- Mouse Lymphoma Assay; 0.006-0.2 ul/ml; Chromosome Aberrations Assay; Chinese Hamster Ovary Cells, 0.3 ul/ml § non-mutagenic: Ames - Salmonella – with and without metabolic activation - dose not specified || non-mutagenic: Ames - Salmonella with and without metabolic activation, 1000 ug/plate; Chromosomal Aberration
Table 2: FAR toxicity thresholds for human skin fibroblasts Concentration (mM)
DAU
IMU
SMG
DMDM
OCT
HNPD
BP
5
Dead
Dead
Dead
Dead
Dead
Dead
Dead
1
Dead
Dead
Dead
Dead
Dead
Dead
Dead
0.1
Alive
Alive
Alive
Alive
Alive
Alive
Dead
0.01
Alive
Alive
Alive
Alive
Alive
Alive
Dead
0.001
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Control
Alive
Alive
Alive
Alive
Alive
Alive
Alive
*Human skin fibroblasts (Passage 2) were exposed to FARs for 24 hrs followed by a 48 hr recovery in fresh cell media.
1
Cosmetic preservatives as
2
therapeutic corneal and scleral
3
tissue cross-linking (TXL) agents
4 5
Natasha Babar, MiJung Kim, Kerry Cao, Yukari Shimizu, Su-Young Kim,
6
Anna Takaoka, Stephen L. Trokel, David C. Paik
7 8 9 10
Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY, USA.
11 12
Key words: cornea, sclera, tissue cross-linking, keratoconus, progressive myopia
13 14 15
Running title: Cosmetic preservatives for corneal and scleral tissue cross-linking
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Supported in part by: Research to Prevent Blindness, the Bjorg & Stephen Ollendorf Fund, and by National Institutes of Health Grants NCRR UL1RR024156, NEI P30 EY019007, and NEI R01EY020495(dcp). Corresponding Author: David C. Paik, M.D. Department of Ophthalmology Edward S. Harkness Eye Institute Columbia University, College of Physicians and Surgeons 160 Fort Washington Ave. Room 715 New York, NY 10032 USA tel: 212-305-1622 fax: 212-342-5455 email: [email protected]
1
Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
34
Common abbreviations used throughout this study
35 36
CXL:
37
photochemically-induced corneal cross-linking, corneal collagen cross-linking, corneal
38
cross-linking, collagen cross-linking)
39
TXL: tissue cross-linking
40
UVA: Ultraviolet-A irradiation (λmax = 370 nm)
41
DSC: differential scanning calorimetry
42
FAR(s): formaldehyde releaser(s) or formaldehyde releasing agent(s)
43
BNA(s): beta-nitroalcohol(s)
44
DAU: diazolidinyl urea
45
IMU: imidazolidinyl urea
46
SMG: sodium hydroxymethylglycinate
47
DMDM: DMDM hydantoin
48
OCT: 5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane
49
HNPD: 2-hydroxymethyl-2-nitro-1,3-propanediol
50
BP: bronopol
51
HONA(s): higher order nitroalcohol(s)
UVA-riboflavin
mediated photochemical
52 53 54 55
2
cross-linking
(also
known as
56
Abstract
57
Purpose: Previously, aliphatic β-nitroalcohols have been studied as a means to
58
chemically induce tissue cross-linking (TXL) of cornea and sclera. There are a number of
59
related and possibly more potent agents known as “formaldehyde releasers” (FARs) that
60
are in commercial use as preservatives in cosmetics and other personal care products. The
61
present study was undertaken in order to screen such compounds for potential clinical
62
utility as therapeutic TXL agents.
63
Methods: A chemical registry of 62 FARs was created from a literature review and
64
included
65
carcinogenicity/mutagenicity, toxicity, hydrophobicity, and commercial availability.
66
From this registry, five (5) compounds [diazolidinyl urea (DAU), imidazolidinyl urea
67
(IMU), sodium hydroxymethylglycinate (SMG), DMDM hydantoin (DMDM), 5-Ethyl-
68
3,7-dioxa-1-azabicyclo [3.3.0] octane (OCT)] were selected for efficacy screening using
69
two independent systems, an ex vivo rabbit corneal cross-linking simulation set up and
70
incubation of cut scleral tissue pieces. Treatments were conducted at pH 7.4 or 8.5 for
71
30min. Efficacy was evaluated using thermal denaturation temperature (Tm) and cell
72
toxicity was studied using the trypan blue exclusion method.
73
Results: Cross-linking effects in the five selected FARs were pH and concentration
74
dependent. Overall, the Tm shifts were in agreement with both cornea and sclera. By
75
comparison with BNAs previously reported upon, the FARs identified in this study were
76
significantly more potent but with similar or better cytotoxicity.
77
Conclusions: FARs, a class of compounds well known to the cosmetic industry, may
78
have utility as therapeutic TXL agents. The compounds studied thus far show promise
79
and will be further tested.
characteristics
relevant
to
TXL
80 81 82 83 84 3
such
as
molecular
weight,
85
Introduction
86
The growing clinical success of UVA-riboflavin photochemical corneal cross-
87
linking (CXL) to halt the progression of keratoconus (KC)1-8 and post-LASIK
88
keratectasia8-9 suggests that increasing mechanical tissue strength in vivo can be
89
beneficial. CXL increases the stiffness of corneal tissue as shown in animal studies using
90
post-mortem mechanical strip testing.10 A majority of patients ultimately gain
91
improvements in topography and gain lines of vision.5-9 Application of CXL has been
92
extended to treat corneal edema,11 corneal melting,12 and corneal infections.13
93
As clinical trials involving CXL progress in the United States, suggestions have
94
been made to extend its use to the sclera as a treatment for progressive myopia,14 since
95
biomechanical weakening occurs during progressive globe elongation.2 Scleral cross-
96
linking with UVA-riboflavin technology has been reported15 but may be difficult to carry
97
out in the posterior region of the sclera without the use of surgical means. Also, of
98
concern is the potential of damaging the neural retina during UVA irradiation.15 The use
99
of injectable pharmacologic agents that could cross-link the sclera as an alternative to glyceraldehyde,16
100
photochemical
101
glutaraldehyde,14 genipin,17 and nitroalcohols (Hoang QV, et al. IOVS 2013;54:ARVO E-
102
Abstract 5169).18
activation
is
being
explored
and
include
103
The present study serves as an extension of our previous work using
104
nitroalcohols,19-20 where we test the corneal and scleral cross-linking efficacy of several
105
related though potentially more potent chemicals from a group known as “formaldehyde
106
releasing agents (FARs).” These compounds are used as preservatives in a wide array of
107
popular cosmetic and personal care products21-22 such as skin care products, body wash,
108
fingernail polish and shampoo, including the former formula for Johnson & Johnson’s
109
“No More Tears” Baby Shampoo.23 FARs have also been employed in the textile industry
110
as cross-linking agents to impart anti-wrinkle properties to clothing.24 Considering their
111
use in everyday items that come into direct contact with the human body, we wanted to
112
examine the efficacy and cell toxicity of FARs as tissue cross-linking agents as a first
113
step in their potential development for clinical use. To that end, in vitro and ex vivo cross-
114
linking experiments were conducted using corneal and scleral tissues as collagenous
115
tissue substrates. Effectiveness of cross-linking was evaluated using shifts in the thermal 4
116
denaturation temperature (Tm) of the collagen in corneal and scleral tissue as measured by
117
differential scanning calorimetry (DSC), where Tm is the melting temperature of a folded
118
protein.25 Past studies have employed Tm as a measure of the thermal stability of
119
biological tissue.16, 26-29 Therefore, we used differences in Tm between chemically treated
120
and control tissue to estimate extent of cross-linking and compared these values to those
121
induced by CXL (i.e. riboflavin photochemical cross-linking), which served as a positive
122
control for corneal samples. Toxicity was assessed using trypan blue exclusion staining.30
123
Our results suggest that some FARs are potent chemical agents for tissue cross-linking
124
with moderate cell toxicity thresholds.
125 126
5
127
Materials and Methods
128
Chemical registry
129
A chemical registry of formaldehyde releasers (FARs) commonly found in
130
cosmetics and other personal care products (PCPs) was compiled from a review of the
131
literature.21, 22 Information used to assemble this registry included characteristics relevant
132
to tissue cross-linking such as molecular weight, European Union maximum allowed
133
concentration
134
hydrophobicity (octanol/water partition coefficient or logP), efficacy of formaldehyde
135
release, and commercial availability of the chemicals. From the FARs identified, five
136
compounds with favorable profiles were selected for cross-linking efficacy and toxicity
137
evaluation. These compounds include diazolidinyl urea (DAU), imidazolidinyl urea
138
(IMU), DMDM hydantoin (DMDM), sodium hydroxymethylglycinate (SMG), and 5-
139
Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane (OCT), which were specifically chosen
140
because of the vastness of their use in cosmetics and PCPs as well as on their ability to
141
donate formaldehyde in solution under equilibrium conditions.31-32 The cross-linking
142
efficacy and toxicity of two additional FARs, bronopol (BP) and 2-hydroxymethyl-2-
143
nitro-1,3-propanediol (HNPD), which are β-nitroalcohols (BNAs) that we have
144
previously worked with, was included for comparative purposes.
(i.e.
“max
allowed”),
carcinogenicity/mutagenicity,
toxicity,
145 146
Chemicals
147
Diazolidinyl
urea
(N-Hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-
148
dioxoimidazolidin-4-yl)-N'-hydroxy-methylurea [DAU]), imidazolidinyl urea (N,N'-
149
methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea [IMU]), sodium
150
hydroxymethylglycinate (SMG), 5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane (7a-
151
Ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole
152
propanediol or bronopol (BP), hydroxypropyl methyl cellulose (HPMC, 15 centipoise),
153
dextran (high molecular weight = 425-575,000 Da), sodium bicarbonate and
154
ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich Corp. (St.
155
Louis,
156
imidazolidinedione [DMDM])
157
Columbia, SC). 2-hydroxymethyl-2-nitro-1-3-propanediol (HNPD) was obtained from
MO).
DMDM
hydantoin
[OCT]),
2-bromo-2-nitro-1,3-
(1,3-bis(hydroxymethyl)-5,5-dimethyl-2,4-
was obtained from Oakwood Products, Inc. (West
6
158
TCI Chemicals, Inc. (New York, NY). Riboflavin-5-phosphate was obtained from MP
159
Biomedicals (Santa Ana, CA). Dulbecco’s phosphate buffered saline (DPBS) solution
160
(MgCl2 & CaCl2 free) was obtained from Life Technologies (Carlsbad, CA). All chemical
161
solutions and buffers were prepared fresh using Millipore water (double distilled, de-
162
ionized water, 𝜌 = 18.2 MΩcm at 25 °C) on the day of cross-linking.
163 164
Chemical and riboflavin-mediated photochemical cross-linking (CXL) of the cornea
165
Intact cadaveric rabbit heads with clear corneas were obtained fresh (within an
166
hour of sacrifice) in adherence with the ARVO Statement For the Use of Animals in
167
Ophthalmic and Vision Research. FAR solutions at concentrations equivalent to half the
168
maximum allowed value (1/2max) were administered in a manner designed to simulate
169
therapeutic cross-linking in patients. For all of the corneal experiments (with the notable
170
exception of CXL), the corneal epithelium was left intact. An 8mm Hessburg-Barron
171
corneal reservoir was affixed to the corneal surface using the supplied syringe vacuum.
172
A single drop of proparacaine (0.5%) was applied to the corneal surface prior to reservoir
173
application. A buffer solution containing 0.1M NaHCO3 at either pH 8.5 or 7.4 was used.
174
The pH of the sample and buffer mixture was titrated to the desired pH just prior to
175
application to the eye using an appropriately concentrated HCl solution. Treatments were
176
conducted over a 30-minute period at 25°C with refreshing of the solution every five
177
minutes. The control contralateral eye was treated identically with vehicle. Immediately
178
after treatment, a central 6mm corneal button was trephined from the treated region of
179
each eye, was blotted on both sides using a paper towel to remove excess solution, and
180
was analyzed using differential scanning calorimetry (DSC) [see below]. A minimum of
181
two independent determinations were carried out for each condition described using a
182
fresh cadaver head each time.
183
As a comparison, the same ex vivo system was used to conduct photochemical
184
cross-linking of rabbit cornea as previously described by Wollensak et al.,1 with some
185
changes. To that end, a central 8mm portion of the corneal epithelium was debrided using
186
a blunt-end scalpel. De-epithelialized corneal tissue was pre-soaked in 0.1% riboflavin-5-
187
phosphate solution in 1.1% HPMC for 5 mins. Thereafter, the cornea was exposed to UV
188
light (λmax = 370 nm) at an irradiance of 3 mW/cm2 with an 8mm aperture for 30 mins 7
189
using the Optos XLink Corneal Collagen Cross-Linking System (Optos, Dunfermline,
190
UK). Riboflavin solution was refreshed every 3 mins for the course of the treatment. The
191
control contralateral eye was treated identically without irradiation.
192 193
Scleral tissue cross-linking
194
The following is a modified version of the procedure that we have previously
195
described for testing tissue cross-linking (TXL) efficacy of various BNAs.18 Enucleated
196
porcine globes were purchased from Visiontech, Inc. (Sunnvale, TX) and were stored at -
197
80°C until time of experimentation (1-2 months). Equatorial scleral strips approximately
198
6mm x 40mm in size were obtained from multiple eyes. These strips were submerged in
199
DPBS solution containing 1mM EDTA to inactivate native collagenases and to prevent
200
tissue dehydration during sample preparation. Each strip was further cut into smaller
201
4mm x 3mm pieces. The scleral pieces were individually transferred to a 24 well plate
202
and were incubated in 1 ml of cross-linking solution in 0.1M NaHCO3 buffer at either pH
203
8.5 or 7.4 for 30 mins at 25°C without refreshing the solution. Four concentrations of
204
FAR solution were tested at each pH: 1) max allowed concentration 2) 1/2 max allowed
205
concentration 3) 1/10 max allowed concentration, and 4) 25 mM. Tissue samples cross-
206
linked with the BNAs BP and HNPD at concentrations of 5 mM (max allowed for BP),
207
10 mM and 25 mM were used as positive controls. Negative controls were treated
208
identically with vehicle. Post-treatment, all solutions were aspirated and samples were
209
washed twice using DPBS to remove remnant cross-linking solution before being
210
analyzed by DSC. A minimum of three independent determinations were carried out for
211
each condition using scleral pieces originating from different porcine globes.
212 213
Differential Scanning Calorimetry (DSC) and cross-link analysis
214
Thermal denaturation temperature (Tm) of all samples was measured using a
215
Perkin-Elmer DSC 6000 Autosampler (Waltham, Massachusetts). Tissue samples were
216
carefully blotted in a standardized, repetitive manner to remove excess solution / DPBS
217
and transferred to pre-weighed 50ul aluminum pans.
218
hermetically sealed using a DSC pan sealing press, which is used to prevent tissue
219
dehydration due to evaporative losses. DSC scans were run using Pyris software (version 8
The pans were immediately
220
11.0) from 40°C to 75°C at a rate of 1°C/min and denaturation curves representing
221
differential heat flow over time were recorded. DSC heat flow endotherm data was
222
analyzed using the Pyris data analysis peak search function using a calculation limit of
223
±0.3°C from the apparent thermal denaturation peak.
224 225
Statistical analysis
226
Student’s t-tests were used to evaluate the significance of observed differences in
227
Tm between cross-linked and control groups. Due to the nature of the ex vivo cadaveric
228
system used for corneal cross-linking, where each cadaver provided the treated eye and
229
contralateral control, corneal samples were subjected to paired t-tests. Conversely, scleral
230
samples were subjected to non-paired t-tests assuming equal variance of data.
231
Significance of all statistical tests was based on an alpha value of 0.05 (p ≤ 0.05). All
232
ΔTm values are reported in the form of mean value followed by standard error.
233 234
FAR cytotoxicity threshold
235
FAR cell cytotoxicity assays were performed as we have previously described.33
236
Briefly, healthy human skin fibroblasts (HSFs) from ATCC (Manassas, VA) were
237
cultured in dermal cell basal media (ATCC) using a serum-free fibroblast growth kit
238
provided by the company (ascorbic acid, EGF/TGF-β1, glutamine, hydrocortisone,
239
insulin and FB growth supplements). Cells were grown in 5% CO2 and 95% ambient air
240
at 37°C until confluent. Once confluent, the cells were detached and seeded into 24 well
241
plates at a density of 5x104 cells/well and were once again allowed to reach confluence.
242
Next, cells were treated with FAR solutions over a range of concentrations (0.001mM-
243
5mM) for 24 hrs. Following cross-linking exposure, all cell media, including FAR
244
solution, was aspirated and each well was rinsed once with DPBS. Fresh media was then
245
reintroduced and the cells were allowed to recover for 48 hrs. Subsequent to cell
246
recovery, cell toxicity was assessed using a modified version of the trypan blue staining
247
protocol. To that end, all culture media was aspirated and each well was rinsed with
248
DPBS. Next, 0.4% trypan blue solution (Gibco, Grand Island, NY, USA) was added to
249
each well for 3 minutes at 25°C. The staining solution was then aspirated and cells were
9
250
washed twice with DPBS. Finally, extent of trypan blue staining and morphology of cells
251
was visualized using an inverted microscope (Fisher Scientific Cat#12-560-45).
252 253
Results
254
Identification of FARs
255
From a broad review of the literature, we were able to identify a total of 62
256
formaldehyde-releasing agents that can potentially be used for corneal and scleral tissue
257
cross-linking. These include FARs commonly found in cosmetics and personal care
258
products as well as those that are used in the textile industry. Table 1 depicts the
259
structures, chemical formulae, toxicity, and other pertinent information of the seven
260
FARs that were chosen for evaluation in this study. This is an excerpt from the larger
261
chemical registry that was compiled. Neither of the chemicals that were tested in this
262
study are known carcinogens (various MSDS). They range in size up to < 400Da with
263
IMU being the largest at 388Da and SMG the smallest at 104Da [MW = 127 – 23 (Na) =
264
104Da]. In most but not all cases, mutagenicity data is available, and these chemicals
265
have been found to be non-mutagenic using Ames, micronucleus and other standard
266
assays. Furthermore, they exhibit low organismal toxicity as indicated by high (>1,000
267
mg/kg,) rat oral LD50 values. The exception is BP which has a relatively low LD50 Oral,
268
rat = 180mg/kg (Table 1).
269 270
Efficacy of corneal cross-linking
271
The ability of five FARs (DAU, IMU, SMG, DMDM, OCT) to cross-link intact
272
cadaveric rabbit cornea, a substrate for collagenous tissue, was assessed using an ex vivo
273
tissue cross-linking (TXL) simulation set up. Cross-linking effects were measured using
274
differential scanning calorimetry (DSC), an assay method based on changes in thermal
275
denaturation temperature (Tm). Our results indicate that two out of the seven FARs
276
studied, DAU and SMG, are effective collagen cross-linking agents for the cornea with
277
the epithelium left intact (in the ex vivo simulation setup) at half maximum allowed
278
concentrations. DAU was effective at pH 8.5 and SMG was effective at both pH 8.5 and
279
7.4. This was evidenced by shifts in the thermal denaturation temperature of corneal
280
tissue (Fig.1). SMG at pH 8.5 showed the greatest upwards shift in Tm (ΔTm = 3.573 ± 10
281
0.578°C, p < 0.05) followed by DAU at pH 8.5 (ΔTm = 3.210 ± 0.742°C, p < 0.05). SMG
282
also showed effective cross-linking at pH 7.4 (ΔTm = 2.281 ± 0.697°C, p < 0.05). Some
283
inconsistencies in the shifts in Tm induced by SMG at pH 7.4, however, were noted and a
284
sample size of n = 8 was required to reach statistical significance. A negative shift in Tm
285
on the order of ~0.5°C was observed for DAU at pH 7.4 and for IMU at both pH 8.5 and
286
7.4 (Fig.1) but the shift was only significant for IMU at pH 8.5 (ΔTm = -0.69 ± 0.697°C,
287
p < 0.05). The lack of effect under these conditions may reflect issues related to epithelial
288
permeability since both DAU and IMU are significantly larger than SMG. Furthermore,
289
an increase in Tm was observed for DMDM at both pH 8.5 and 7.4 (ΔTm = 2.04 ±
290
0.225°C and 2.13 ± 0.273°C, respectively) and for OCT at pH 8.5 (ΔTm = 1.10 ±
291
0.246°C). However, these observed increases in Tm were not statistically significant using
292
paired controls, which included the contralateral eye for each sample. Rabbit cornea
293
cross-linked using UVA-riboflavin (CXL) showed an increase in Tm comparable to
294
values previously reported. In the present study, the ΔTm following CXL = 1.73 ±
295
0.487°C. We previously reported a ΔTi = 1.9°C,19 which was similar to that reported by
296
Spoerl et al. at 2.5°C.34 Ti values indicate the onset of thermal shrinkage (Ts) temperature
297
and change in Ti (ΔTi) is another parameter popularly used to measure extent of tissue
298
cross-linking. As previously reported, the CXL effect is relatively mild from a “thermal
299
transition shifting” standpoint if one considers the potential magnitude of shifts in Tm that
300
may be induced using chemical agents. Lastly, it is worth noting that corneal tissue
301
remained clear to visual inspection using either chemical or photochemical cross-linking
302
treatment.
303 304
Efficacy of scleral cross-linking
305
The results for scleral tissue cross-linking are generally comparable to the results
306
for corneal samples, although different methods of application were used (i.e. refreshing
307
solution every five mins for corneal experiments and not refreshing for scleral
308
experiments). In this case, two additional FARs, BP and HNPD, were also tested. We
309
found that SMG, DAU, and DMDM induced statistically significant cross-linking effects
310
at pH 8.5 and 7.4 (with the exception of SMG at pH 7.4) at concentrations as low as half
311
max allowed. In addition, both a concentration and pH dependent effect was observed for 11
312
the FARs (Fig. 2). A notable exception to the concentration dependent effect is seen in
313
the thermal denaturation data for SMG at pH 7.4 and 39.06 mM, where little change in
314
Tm is observed (ΔTm= 0.007 ± 0.222°C, p = 0.493) although a dramatic upward shift is
315
seen for the same concentration using a pH of 8.5 (ΔTm= 9.073 ± 0.450°C, p < 0.05). The
316
reason for this difference is unclear since in general, upwards shifts in Tm occur for most
317
FARs, albeit consistently greater for pH 8.5 over 7.4. It should be noted that SMG is
318
highly basic in un-buffered solution, requiring the addition of significant amounts of acid
319
in order to achieve the targeted pH of 7.4. Thus, we speculate that the procedure for
320
titrating the buffered SMG solution to pH 7.4 may have impacted the efficacy of TXL in
321
this case. This phenomenon might also explain the inconsistencies in TXL efficacy
322
experienced when intact cornea was cross-linked using SMG at pH 7.4, as mentioned
323
above.
324
We also tested FARs at a concentration of 25 mM in order to directly compare the
325
cross-linking “potency” of each FAR in comparison to the others. We chose a relatively
326
high concentration for this comparison because we wanted to elicit a noticeable cross-
327
linking effect using the BNAs HNPD and BP within 30 mins. DAU showed the greatest
328
shifts in thermal denaturation temperature at 25 mM for both pH 8.5 and 7.4 (ΔTm= 7.713
329
± 0.226°C and 4.347 ± 0.538°C, respectively, p 2,000 mg/kg63
LD50 Oral - rat 2,100 mg/kg, LD50 Dermal - rabbit >2,000 mg/kg54 LD50 Oral – rat – 3,720 mg/kg; LD50 Oral – rat - >2,000 mg/kg50 LD50 Oral - rat >3,600 mg/kg; LD50 Dermal rabbit - 1,948 mg/kg53
Bronopol [BP; CAS No: 52-51-7; MW: 199.99 g/mol; Formula: C3H6BrNO4] 2-hydroxymethyl-2-nitro1,3-propanediol [HNPD; CAS No: 126-11-4; MW: 151.12 g/mol; Formula: C4H9NO5]
1.150±0.63161
-0.115±0.770
62
0.121 (5 mM)
-
Non-mutagenic§51
Non-mutagenic||
52
LD50 Oral - rat 180 mg/kg51
LD50 Oral - rat1,917 mg/kg; LD50 Oral - mouse – 10,550 mg/kg62
* non-mutagenic: Ames, Micronucleus Assay † non-mutagenic: Ames - 100% Sodium hydroxymethylglycinate, Mouse Micronucleus; Rat Hepatocyte/DNA Repair Assay; In vivo – In vitro Rat Hepatocyte UDS Assay ‡ non-mutagenic: Ames – Salmonella - 55% DMDM - 0.001-5 ul/plate; Salmonella / Mammalian-Microsome Preincubation Mutagenicity Assay - Salmonella-2.0ul/plate; mutagenic: L5178 TK+/- Mouse Lymphoma Assay; 0.01-1.0ug/ml; L5178 TK+/- Mouse Lymphoma Assay; 0.006-0.2 ul/ml; Chromosome Aberrations Assay; Chinese Hamster Ovary Cells, 0.3 ul/ml § non-mutagenic: Ames - Salmonella – with and without metabolic activation - dose not specified || non-mutagenic: Ames - Salmonella with and without metabolic activation, 1000 ug/plate; Chromosomal Aberration
Table 2: FAR toxicity thresholds for human skin fibroblasts Concentration (mM)
DAU
IMU
SMG
DMDM
OCT
HNPD
BP
5
Dead
Dead
Dead
Dead
Dead
Dead
Dead
1
Dead
Dead
Dead
Dead
Dead
Dead
Dead
0.1
Alive
Alive
Alive
Alive
Alive
Alive
Dead
0.01
Alive
Alive
Alive
Alive
Alive
Alive
Dead
0.001
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Control
Alive
Alive
Alive
Alive
Alive
Alive
Alive
*Human skin fibroblasts (Passage 2) were exposed to FARs for 24 hrs followed by a 48 hr recovery in fresh cell media.
