March 2013
Volume 54, Issue 3
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Retina  |   March 2013
Different Intravitreal Properties of Three Triamcinolone Formulations and Their Possible Impact on Retina Practice
Author Affiliations & Notes
  • Hao Chen
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Shumao Sun
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Jie Li
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Wennan Du
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Chunhui Zhao
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Jiangping Hou
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Yu Xu
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
  • Lingyun Cheng
    From the Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; and the
    Jacobs Retina Center at Shiley Eye Center, Department of Ophthalmology, University of California, San Diego, La Jolla, California.
  • Footnotes
     Current affiliation: *Provincial Hospital, Shandong University, Jinan, Shandong, China.
  • Corresponding author: Lingyun Cheng, Institute of Ocular Pharmacology, School of Ophthalmology and Optometry, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou, Zhejiang, China, 325027; cheng@eyecenter.ucsd.edu
Investigative Ophthalmology & Visual Science March 2013, Vol.54, 2178-2185. doi:10.1167/iovs.12-11460
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      Hao Chen, Shumao Sun, Jie Li, Wennan Du, Chunhui Zhao, Jiangping Hou, Yu Xu, Lingyun Cheng; Different Intravitreal Properties of Three Triamcinolone Formulations and Their Possible Impact on Retina Practice. Invest. Ophthalmol. Vis. Sci. 2013;54(3):2178-2185. doi: 10.1167/iovs.12-11460.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: We sought to better characterize the intravitreal profile of different triamcinolone formulations.

Methods.: The study was performed in vitro and in vivo. Kenalog-40, Triesence, and Transton were characterized for ocular pharmacokinetics, particle size, crystallinity, and dissolving kinetics in vitreous following an intravitreal injection into 12 rabbit eyes. The relationship of free drug levels in the aqueous and vitreous was investigated through a dual-probe microdialysis and liquid chromatography tandem mass spectrometry.

Results.: Triesence had the most uniform particle size distribution (mean 11.51 μm) and Kenalog-40 had the largest particle sizes (mean 18.86 μm). Triesence and Kenalog-40 had 100% crystallinity, while Transton had 89% crystallinity. Triesence had a slower dissolution in vitreous than that of Kenalog-40, and Transton had the fastest dissolution, though their solubility was very similar. Following a 1.2 mg intravitreal injection in the rabbit eye, Triesence had a significantly lower ocular free drug level than Kenolog-40 (P = 0.025) and Transton (P = 0.007). Quantitative dual-probe microdialysis revealed that the aqueous free triamcinolone (Kenolog-40) was less than 1% of the vitreous free triamcinolone during the first few hours, and this percentage increased to 26.8% at 2 weeks and was 11.7% at 3 weeks following an intravitreal injection.

Conclusions.: Triesence demonstrated a significantly slower dissolution profile and lower free drug level in the vitreous than the other preserved triamcinolone, which may translate into a longer therapeutic duration and lower rate of drug-associated complications.

Introduction
Triamcinolone acetonide (TA) is being used worldwide as a therapeutic agent for many chorioretinal diseases, such as diabetic macular edema, retinal vein occlusion, age related macular degeneration, 1 and uveitis. 2 Even with the advent of intravitreal antiangiogenesis agents, intravitreal TA remains an effective and low-cost treatment modality when used alone or combined with other treatment options. Several commercially available TA formulations are being used for intravitreal injection either at a physician's discretion or due to the availability of the product. 35 Though TA intravitreal injections generally are effective, side effects, such as cataract formation and elevated IOP are common. Most recently, the preservative-free TA formulations, Triesence and Trivaris, have been developed and are available on the market (Triesence; Alcon Pharmaceuticals, Ft. Worth, TX; and Trivaris; Allergan, Inc., Irvine, CA). The commercially available, preservative-free TAs are likely different from preserved TAs in pH value, particle size, 6 crystallinity, solubility, and dissolution kinetics in the vitreous. All of these parameters are important for better gauging treatment effect and duration, as well as understanding adverse consequences following an intravitreal injection. 7 For example, the free TA concentration in vitreous fluid and aqueous fluid may be quite different following an intravitreal injection of different TA formulations, which will affect not only the therapeutic duration, but also the possibility of side effects, such as cataract formation and IOP elevation. 
In the United States, preserved triamcinolone acetonide, such as Kenalog-40 (C24H31FO6 MW:434.50; Bristol Myers Squibb, Princeton, NJ) is the dominant TA formulation for intravitreal injection even after preservative-free TAs have become available, 8 while Transton is the counterpart of Kenalog-40 in China (C24H31FO6 MW:434.50; Kunming Jida Pharmaceuticals Co., Ltd., Yunnan, China). 4,5,912 To the best of our knowledge, the difference of ocular free TA pharmacokinetics following an intravitreal injection of Kenalog-40 or Triesence is not yet well documented. 13 In our current study, we chose Kenalog-40 and Triesence (marketed in the United States), as well as Transton (marketed in China) to compare their ocular properties to better understand their implications in daily retina practice. 
Materials and Methods
In Vitro Physicochemical Properties of the Three Different TA Formulations
Two types of commercially-available preserved TA (Kenalog-40, C24H31FO6 MW:434.50; Bristol Myers Squibb; and Transton, C24H31FO6 MW:434.50, Triamcinolone Acetonide Injection; Kunming Jida Pharmaceuticals Co., Ltd.) and one preservative-free TA (Triesence; Alcon Pharmaceuticals) were used for this study. The chemical grade triamcinolone acetonide was purchased from National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China, and used as a control. 
Osmolarity of the Supernatant from the Commercial Ampules.
The commercial ampules of each TA formulation (Triesence, Kenalog-40, Transton) were centrifuged (Eppendorf 5810R; Eppendorf, Hamburg, Germany) at 3220g for 20 minutes and 0.5 mL of the supernatant from each ampule was collected using a 30 gauge needle attached to a 1 mL syringe. The osmolarity of the supernatant was measured using an auto freezing point osmometer (FM-8; Science Development Center at Shanghai Medical University, Shanghai, China). Three ampules each were studied from Kenalog-40 and Transton. For Triesence, 2 ampules were studied. 
pH Value of the Supernatant from the Commercial Ampules.
After the centrifugation specified above, 0.6 mL of the supernatant was sampled into a pH measuring cuvette and the pH value was determined using a pH meter (SG2-ELK; Mettler-Toledo, Zurich, Switzerland). 
TA Concentration in the Supernatant from the Commercial Ampules.
After centrifugation of the TA ampule, 100 μL of the supernatant were sampled. Following filtration with a 0.45 μm filtering membrane, 50 μL of the filtered sample were mixed with 50 μL of high performance liquid chromatography (HPLC) mobile phase and 20 μL of the mixture was injected into HPLC (Agilent, Santa Clara, CA). The mobile phase consisted of methanol/water (52.5/47.5) and the flow rate was at 1 mL/min through a ZORBAX Eclipse XDB-C18 (Agilent) (4.6 × 150 mm, 5 μm) column at 30°C. TA was detected by a diode array detector at (G1315B; Agilent) 240 nm. The TA concentration was determined from a standard 7 point curve with excellent linearity (R = 0.999) between 0.5 and 20 μg/mL. 
Particle Size Analysis of the Different TA Formulations.
Two TA ampules of Triesence, Kenalog-40, and Transton were placed in an ultra-low temperature freezer overnight. The crystal powder of the three types of TA then was collected by freeze dryer (2-4 LDplus; Christ Alpha, Munich, Germany). The particle size of each TA formulation was determined using a laser particle size analyzer (Mastersizer 2000; Malvern, Worcestershire, England). 
Crystallinity of TA in the Different Formulations.
TA ampules of Triesence, Kenalog-40, and Transton were placed in a −80°C temperature freezer overnight. Then, the crystal powder of three types of TA was collected by freeze dryer (2-4 LDplus; Christ Alpha). The powder was washed by deionized water once to remove the excipients. The crystal powder was recollected by lyophilization in the same way and then analyzed using an X-ray diffractometer (X' Pert PRO; PANalytical, Eindhoven, Netherlands). The crystallinity was calculated by JADE5.0 software program used for crystal analysis (Materials Data, Inc., Livermore, CA). 
Solubility of TA from the Different Formulations.
Solubility in PBS.
The crystal powder of three types of TA was collected from the commercial ampules by lyophilization. In addition, a TA standard (purchased from National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) was used as control. The 40 mg TA powder from Triesence, Kenalog-40, Transton, and the standard TA were placed into a dialysis bag (MD25-3.5; Viskase, Darien, IL) with a 3500 molecular weight cutoff (MWCO). The dialysis bag was placed in a polyethylene bottle with 150 mL PBS (pH = 7.44). The polyethylene bottle then was placed in an orbital shaker (Thermo Fisher 481; Thermo Fisher, Marietta, GA) at 37°C with a speed of 25 revolutions per minute. At 2-, 4-, 8-, 16-, 24-, and 32-hour, and 2-, 4-, 5-, 7-, 10-, 15-, 21-, and 28-day time points, 1 mL of solution was sampled from the polyethylene bottle and 1 mL of fresh PBS was added back into the bottle. The TA concentration was determined by HPLC. 
Solubility in Vitreous.
As described above, 1.8 mg of TA powder from Triesence, Kenalog-40, Transton, and the standard TA were placed into a centrifuge tube containing 2 mL of blank rabbit vitreous. The tube was placed in the orbital shaker as described above, and centrifuged with 3220g for 10 minutes. A 20 μL supernatant then was sampled at 3-, 8-, 12-, 24-, and 36-hour, and 2-, 3-, 4-, and 5-day time points for HPLC analysis. 
In Vivo Ocular Pharmacokinetics following a Single Intravitreal Injection
TA Pharmacokinetics in Aqueous Humor following Intravitreal TA Injection.
The study was designed to characterize the free TA concentration profile in vitreous and in aqueous humor from an intravitreal injection of TA. To reduce the number of animals and minimize the variation between individual animals, we sampled the aqueous humor multiple times from the same animal at the designated time points. The aqueous humor was sampled, instead of vitreous, to avoid small TA particles being taken into samples, which would distort the free TA concentration profile to be studied. The assumption is that the free TA concentration in the vitreous and in the aqueous humor is proportional, and that their relationship can be defined in a separate dual-probe microdialysis study. 14,15 For this study, 12 pigmented rabbits were used, 4 for each type of TA. Their mean body weight was 2.65 ± 0.28 kg. Handling of animals was in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. This study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College. Only one eye was injected intravitreally with 1.2 mg of TA in 30 μL using a 27-gauge half-inch needle attached to a 1 mL syringe. For the intravitreal injection procedure, the rabbits were anesthetized by an intramuscular injection of ketamine (21 mg/kg) and xylazine (5.25 mg/kg), and topical proparacaine 0.5% also was used locally. The vial of TA was shaken well before loading the syringe, in the same manner as a 4 mg TA intravitreal injection is performed in the clinic for a human eye. After the injection at postinjection days 1 and 5, and weeks 2, 3, 4, 6, and 8, a paracentesis was performed under anesthesia, using sterile technique, through a 31-gauge needle/0.3 mL syringe to sample 50 μL of aqueous humor under the direct view of a surgical microscope (F18; Leica, Wetzlar, Germany). The samples were stored under −80°C until LC-MS/MS analysis. In addition, the vitreous TA aggregate was inspected using an indirect ophthalmoscope and noted at each aqueous humor sampling time point. At the eighth week, the rabbits were sacrificed and the whole vitreous was sampled using a snap freezing technique as described previously. 16 The whole vitreous samples were kept under −80°C until LC-MS/MS analysis. 
Microdialysis to Determine Quantitative Relationship between Free TA in Vitreous and Aqueous Humor.
For this study, three rabbits were used. The dual-probe microdialysis was performed immediately, and at weeks 2 and 3 following a 1.2 mg Kenalog-40 intravitreal injection. Only one eye of one rabbit was dual-probe microdialyzed at each time point. For the procedure, the rabbit was anesthetized by an intramuscular injection of ketamine (35 mg/kg) and xylazine (6.25 mg/kg), and an anterior chamber probe (CMA 30, 4 mm custom made, molecular weight cutoff 6000 Da; CMA Microdialysis, North Chelmsford, MA) was installed before the vitreous probe (CMA 20, 4 mm probes, molecular weight cutoff 20,000 Da; CMA Microdialysis) to avoid possible contamination of the aqueous probe by egressed vitreous fluid. In addition, aqueous probe installation causes some loss of aqueous; by installing the aqueous probe first, it allows time for the eye to recover. After the installation of the probes, bio glue (Vetbond1469SB; 3M Corp., St. Paul, MN) was applied around the probe entry at the globe surface to prevent ocular fluid leaks (Fig. 1). A minimum of 30 minutes was given to allow the eye to recover its fluid balance and IOP before the intravitreal TA injection (immediate microdialysis) or the first sample collection (weeks 2 and 3 time points). The probes were perfused at 1 μL (vitreous probe) or 2 μL (aqueous probe) per minute of 0.9% NaCl using a microsyringe pump (NE100; New Era Pump Systems, Inc., Farmingdale, NY). The vitreous and aqueous humor samples were collected every 20 minutes, and a minimum of 10 samples were collected for analysis. During the course of microdialysis, a boost of anesthesia was performed every 35 to 40 minutes using one-half the volume of the first dose. Every other boost was ketamine only starting with the first boost because xylazine stays in the system longer. 17 The same type of probe was used for determining the rate of TA recovery at 37°C using 300 ng of TA per milliliter of 0.9% NaCl. The study was performed in the same manner as the microdialysis performed in the rabbit eye. In determining the TA recovery rate from the aqueous probe, 1 and 2 μL of infusion rates were used. 
Figure 1. 
 
The photograph is taken from a dual-probe microdialysis. The anterior eye globe was exposed using an eye lid speculum. The vitreous probe and aqueous probe are visible, as indicated in the photograph. Probes had a 4 mm dialysis membrane. The aqueous probe membrane is seen centered in the anterior chamber; the vitreous probe membrane is in the mid cavity of the vitreous, and looks whitish and distorted through the lens.
Figure 1. 
 
The photograph is taken from a dual-probe microdialysis. The anterior eye globe was exposed using an eye lid speculum. The vitreous probe and aqueous probe are visible, as indicated in the photograph. Probes had a 4 mm dialysis membrane. The aqueous probe membrane is seen centered in the anterior chamber; the vitreous probe membrane is in the mid cavity of the vitreous, and looks whitish and distorted through the lens.
Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis of TA Concentration in the Aqueous Humor and in the Vitreous
The measurement of TA concentrations in rabbit aqueous samples was performed using LC-MS/MS as we described previously. 16,18  
Data Analysis
Data are expressed in mean ± SD. The mean dissolution rates in excised vitreous at the early time point among 3 different types of TA were compared for each pair using Student's t-test. For in vivo aqueous pharmacokinetic data the difference among 3 types of TA was evaluated using paired t-test by pairing data at each sampling time point. The pharmacokinetic parameters were extrapolated using Phoenix WinNonlin 6.2 (Pharsight, a Certara Company, St. Louis, MO) by fitting the aqueous TA concentration-time data to the extravascular input model and the noncompartmental analysis. For in vivo dual-probe microdialysis, the mean TA concentrations in vitreous or in aqueous humor were compared for each pair among 3 different times of microdialysis using nonparametric comparisons of Wilcoxon method. 
Results
Physicochemical Properties of the Different TA Preparation
The physicochemical properties of the 3 different TA preparations are summarized into Table 1
Table 1. 
 
Physicochemical Characteristics of Different TA Formulations
Table 1. 
 
Physicochemical Characteristics of Different TA Formulations
TA Brand Osmolarity*, mOsm/kg pH* TA*, μg/mL Particle Size, μM Crystallinity Solubility in PBS, μg/mL Solubility in Vitreous at 24 h, μg/mL
Kenalog-40 328.7 ± 1.7 5.76 ± 0.06 17.10 ± 0.68 18.86 ± 16.7 100% 12.91 ± 0.44 24.69 ± 0.82
Triesence 300.5 ± 3.5 6.90 ± 0.01 10.42 ± 0.59 11.51 ± 8.1 100% 13.42 ± 0.76 26.71 ± 3.42
Transton 309.3 ± 1.9 6.79 ± 0.1 18.28 ± 0.45 10.13 ± 7.6 88.79% 12.51 ± 0.59 23.91 ± 0.77
TA STD 12.31 ± 0.62 20.40 ± 1.13
The free TA level in the commercial vial was the lowest for Triesence. The solubility of three TA formulations was similar and the solubility of TA in vitreous was higher than that in PBS. The particle size analysis demonstrated different particle size distributions (Fig. 2). 
Figure 2
 
Particle size distribution of different formulations of triamcinolone. The x-axis is in micrometers at a log scale. The bell-shaped curve indicates the range of particle size distribution at the corresponding sizes along the x-axis, while the left y-axis indicates the percent of sample having the size shown on the corresponding x-axis. The sigmoid curve indicates the cumulative distribution of particle sizes and the cumulative percentage is shown on the right y-axis.
Figure 2
 
Particle size distribution of different formulations of triamcinolone. The x-axis is in micrometers at a log scale. The bell-shaped curve indicates the range of particle size distribution at the corresponding sizes along the x-axis, while the left y-axis indicates the percent of sample having the size shown on the corresponding x-axis. The sigmoid curve indicates the cumulative distribution of particle sizes and the cumulative percentage is shown on the right y-axis.
Triesence was the most uniformly distributed formulation with the narrowest bell-shape of distribution. Kenalog-40 showed larger median size, and wider range of distribution than Triesence and Transton. The dissolution profiles of the different TA preparations in the excised vitreous are displayed in Figure 3. Though the solubility of Triesence, Kenalog-40, and Transton was similar after a 24-hour incubation period, the dissolution profile (a kinetic process) was quite different, especially at the earlier time points of 10, 20, and 30 minutes. Transton dissolved the fastest and Triesence the slowest among the 3 TA formulations (least square mean [LSmean] 18.23 > 12.39 > 9.95 ng/mL, P < 0.05 least square means Student's t-test). 
Figure 3. 
 
Dissolution kinetics for the different formulations of triamcinolone in excised rabbit vitreous. Within the first hour of dissolution, free TA from Transton consistently was the highest and free TA from Kenolog-40 was higher than that of Triesence. The higher free TA concentrations at the earlier time points indicate a faster dissolution.
Figure 3. 
 
Dissolution kinetics for the different formulations of triamcinolone in excised rabbit vitreous. Within the first hour of dissolution, free TA from Transton consistently was the highest and free TA from Kenolog-40 was higher than that of Triesence. The higher free TA concentrations at the earlier time points indicate a faster dissolution.
In Vivo Pharmacokinetics of Different Formulations of TA in Rabbit Aqueous
The aqueous samples were analyzed using LC-MS/MS and the kinetics of each type of TA is demonstrated in Figure 4. In general, the aqueous TA concentration following a Transton intravitreal injection was significantly higher than following a Kenalog-40 injection (P = 0.0225, paired t-test) and a Triesence injection (P = 0.007, paired t-test). In addition, the TA level in the aqueous following a Triesence intravitreal injection was significantly lower than following a Kenalog-40 injection (P = 0.025). The TA in aqueous followed a first order elimination. The maximum concentration of TA in aqueous was 63.2 ng/mL for Transton, 21.1 ng/mL for Kenalog-40, and 7.2 ng/mL for Triesence. The time at which the highest TA concentration reached was postinjection 1 day for all three TAs. The area under the concentration-time curve was 815.8 ng·d/mL for Transton, 277.1 ng·d/mL for Kenalog-40, and 83.9 ng·d/mL for Triesence. 
Figure 4. 
 
The pharmacokinetic profiles of free triamcinolone in aqueous following a 1.2 mg intravitreal injection. Transton, Kenalog-40, and Triesence demonstrated a similar elimination profile, but with very different maximum concentration (Cmax) values, which was the highest for Transton and the lowest for Triesence (statistical evaluation not available due to too small sample size, n = 4). The paired t-test of means at each time point for three curves revealed statistically significant difference of free TA levels among three types of TA (Transton versus Kenalog-40, P = 0.023; Transton versus Triesence, P = 0.007; Kenalog-40 versus Triesence, P = 0.025).
Figure 4. 
 
The pharmacokinetic profiles of free triamcinolone in aqueous following a 1.2 mg intravitreal injection. Transton, Kenalog-40, and Triesence demonstrated a similar elimination profile, but with very different maximum concentration (Cmax) values, which was the highest for Transton and the lowest for Triesence (statistical evaluation not available due to too small sample size, n = 4). The paired t-test of means at each time point for three curves revealed statistically significant difference of free TA levels among three types of TA (Transton versus Kenalog-40, P = 0.023; Transton versus Triesence, P = 0.007; Kenalog-40 versus Triesence, P = 0.025).
During clinical observation, indirect ophthalmoscopy revealed that, in general, a smaller drug depot size was noted for Transton and Kenalog-40 (Fig. 5) when compared to Triesence at the fourth week or later post injection. On day 56 post injection, all rabbits were sacrificed, and the total mean vitreous TA and mean plasma TA concentrations are summarized in Table 2
Figure 5. 
 
Fundus photographs of the triamcinolone vitreous drug depot at the fourth week following the initial intravitreal injection. All drug depots settled in the inferior vitreous cavity. The photographs were acquired using a fundus camera (NF505; Nikon, Tokyo, Japan) with a 20 diopter fundus lens (Double aspheric; Volk Optical, Inc., Mentor, OH) prepositioned on the native camera lens. In this way, a wider field of fundus image is acquired, which allows for a display of the inferior drug depot at the peripheral fundus. An additional 20 diopter lens caused the reflection rings seen at the center of the images. The Triesence drug depots appeared larger, thicker, and whitish due to the light reflected back from the depot. In contrast, the Kenalog-40 and Transton drug depots looked smaller and yellowish, because the thinner depot allowed the red reflection from the fundus to penetrate and mix with the white reflection from the depot itself.
Figure 5. 
 
Fundus photographs of the triamcinolone vitreous drug depot at the fourth week following the initial intravitreal injection. All drug depots settled in the inferior vitreous cavity. The photographs were acquired using a fundus camera (NF505; Nikon, Tokyo, Japan) with a 20 diopter fundus lens (Double aspheric; Volk Optical, Inc., Mentor, OH) prepositioned on the native camera lens. In this way, a wider field of fundus image is acquired, which allows for a display of the inferior drug depot at the peripheral fundus. An additional 20 diopter lens caused the reflection rings seen at the center of the images. The Triesence drug depots appeared larger, thicker, and whitish due to the light reflected back from the depot. In contrast, the Kenalog-40 and Transton drug depots looked smaller and yellowish, because the thinner depot allowed the red reflection from the fundus to penetrate and mix with the white reflection from the depot itself.
Table 2. 
 
TA Remaining in Vitreous and Plasma Concentration at the End of Study
Table 2. 
 
TA Remaining in Vitreous and Plasma Concentration at the End of Study
TA Type TA Amount in Whole Vitreous, ng TA Concentration in Plasma, ng/mL N of Eye Drug Aggregate Visible
Transton 1091.3 ± 2175.9 0.98 ± 1.6 4 1 of 4
Kenalog-40 1884.4 ± 3031.7 0.58 ± 0.4 4 1 of 4
Triesence 3264.75 ± 1733.5 1.36 ± 1.2 3* 3 of 3
The Simultaneous Kinetics of Free Kenalog-40 in the Aqueous and Vitreous In Vivo, following an Intravitreal Injection of 1.2 mg Suspension
The vitreous probe TA recovery rate was 23.4 ± 2.7% at 37°C and under a perfusion rate of 1 μL per minute. In contrast, the aqueous probe TA recovery rate was 11.3 ± 1% at 37°C under a perfusion rate of 1 μL/min. With the perfusion rate of 2 μL/min, the recovery rate was only 6.6 ± 1.1%. The vitreous probe recovery rate was significantly higher than that of aqueous probe (P < 0.0001, t-test). The aqueous probe recovery rate was significantly higher at 1 μL/min perfusion than that at 2 μL/min perfusion (P < 0.0001). Immediately following an intravitreal injection of triamcinolone, the free drug gradually increased in dialysate of vitreous and aqueous, and the level of free TA reached a near constant around 150 minutes post injection (Fig. 6, blue lines). It is clear that the changes in TA levels in vitreous and aqueous are proportional. For the microdialysis performed at two and three weeks post injection, the free TA in the aqueous and vitreous showed a near constant level during microdialysis. The mean TA concentrations were 0.21 ± 0.12, 6.03 ± 1.54, and 2.38 ± 0.96 ng/mL in the aqueous humor, and 137.72 ± 27.33, 46.57 ± 6.89, and 42.28 ± 12.6 ng/mL in the vitreous humor immediately after, and at 2 and 3 weeks post injection, respectively. Taking the probes' recovery rates, and the difference of the recovery rates between the vitreous probe and the aqueous humor probe, the calculated TA concentration in the aqueous humor at 2 weeks post injection was 26.8% of that in the vitreous and at 3 weeks post injection it was 11.7% that of free TA in the vitreous. The TA in the aqueous humor was less than 1% of the free TA in the vitreous within the first few hours following a 1.2 mg intravitreal injection. 
Figure 6. 
 
Free TA concentrations in the vitreous (solid lines) and in the corresponding aqueous humor (dotted lines) from the microdialysis. The free TA in the vitreous and the aqueous humor demonstrated a synchronized increase or proportional change (blue lines) immediately following the intravitreal injection. The free TA levels in the aqueous humor at 2 weeks post injection was higher than at 3 weeks post injection; however, the free TA levels in the vitreous and aqueous humor, at the time points, were constant and analogous to one another.
Figure 6. 
 
Free TA concentrations in the vitreous (solid lines) and in the corresponding aqueous humor (dotted lines) from the microdialysis. The free TA in the vitreous and the aqueous humor demonstrated a synchronized increase or proportional change (blue lines) immediately following the intravitreal injection. The free TA levels in the aqueous humor at 2 weeks post injection was higher than at 3 weeks post injection; however, the free TA levels in the vitreous and aqueous humor, at the time points, were constant and analogous to one another.
Among the three time points, the free TA levels in the vitreous were significantly higher within the first few hours following the intravitreal injection (P = 0.0127 vs. 2 weeks post injection and 0.0101 vs. 3 weeks post injection; nonparametric comparisons for each pair using the Wilcoxon method), and the free TA levels in the vitreous at 2 and 3 weeks post injection were similar (P = 0.218). Free TA levels in the aqueous humor demonstrated a significant difference among the three time points, with the highest level at 2 weeks post injection, the second highest at 3 weeks post injection, and the lowest within the first few hours following the intravitreal injection (2-week vs. 3-week, P = 0.0004; 1-hour vs. 2- or 3-week, P < 0.0001). 
Discussion
Triamcinolone is a crystal drug with limited solubility, causing it to form a drug depot following an intravitreal injection, and leading it to provide slow release and a long lasting therapeutic effect. It is important to note that only dissolved free triamcinolone has a therapeutic effect, and that the amount of free drug in ocular fluid can vary greatly due to the different formulation parameters and associated dissolution kinetics. To the best of our knowledge, our study is the most comprehensive study to date on the ocular pharmacokinetics following an intravitreal injection of different formulations of triamcinolone, preserved and preservative-free. 
It has been known that particle size, crystallinity, and dispersion profile all may affect the dissolution kinetics of a given drug. In our ex vivo vitreous dissolution study, we noted that the solubilities of Triesence, Kenalog-40, and Transton were comparable (26.71, 24.69, and 23.91 μg/mL, respectively), but the dissolution kinetics in the vitreous at 37°C were different. The preservative-free Triesence dissolved much more slowly than Kenalog-40 and Transton, which dissolved the fastest. These dissolution features have important clinical implications for the pharmacotherapeutics of TAs following an intravitreal injection. In vivo, the vitreous fluid or aqueous fluid of the eye is turning over constantly at a rate of 1 to 2 μL per minute. 19,20 We hypothesized that the steady state free drug level of a slow dissolving triamcinolone will be significantly lower in the vitreous and aqueous of a living eye than that of a fast dissolving triamcinolone formulation. This hypothesis was supported by our in vivo pharmacokinetic study and clinical observation, which revealed a faster dissipation of the vitreous drug depot, and higher free TA levels in aqueous and vitreous humor for Transton and Kenalog-40 when compared to Triesence. These results suggested that intravitreal Triesence may provide a longer therapeutic duration and less TA-related complications, such as cataract and IOP elevation, when compared to an equivalent intravitreal injection of Kenalog-40 or Transton, because these complications are dose-dependent, or more specifically, free TA level–dependent. Evidence to support this point are as follows. An elevated IOP is more frequent in an intravitreal TA injection than it is in a subtenon TA injection and the level of free TA is much higher in intravitreally injected eyes. 18,2124 In addition, a randomized clinical trial has demonstrated significantly more IOP elevations and cataract surgeries performed after a 4 mg TA injection than were observed following a 1 mg intravitreal injection. 25  
We believe that the drug level in the aqueous humor is proportional to the drug level in the vitreous for a given drug. Our dual-probe microdialysis study within the first few hours following an intravitreal injection clearly demonstrated synchronized changes of free TA levels between the vitreous and aqueous humor. In our study, the TA recovery rate for the vitreous probe was higher than that for the aqueous probe, possibly due to the different diffusion membrane areas (the membrane diameter is 0.5 mm for the vitreous probe, while only 0.24 mm for aqueous probe). Both probes had a 4 mm length membrane, so the membrane surface area for the vitreous probe is approximately 6.68 mm2, but only 4.45 mm2 for the aqueous probe. Taking the different recovery rates into consideration, the free TA concentration in the vitreous at two and 3 weeks following a 1.2 mg (equivalent to 4 mg for the human eye) TA intravitreal injection would be 199 ± 29 and 181 ± 54 ng/mL, respectively; the corresponding free TA in the aqueous would be 53 ± 14 ng/mL at week 2 and 21 ± 8 ng/mL at week 3, which constitutes 27% and 12% of the level of TA in the vitreous. In contrast, the free TA was very high (589 ± 117 ng/mL) in the vitreous and very low (2 ± 1 ng/mL) in the aqueous during the first few hours following an intravitreal TA injection. The free TA in either aqueous or vitreous during the first few hours may not be comparable with the drug levels at two and three weeks post injection because the vitreous status (extent of liquefaction caused by intravitreal injection) may be quite different. It has been shown that the viscosity of a medium negatively affects the diffusion of TA. 26,27  
The difference of the free TA levels among 3 TA formulations may come from the strong conglomeration of Triesence in the ocular fluid as seen in balanced saline solution enriched with bicarbonate, dextrose, and glutathione (BSS Plus). 6 The conglomerate will reduce the surface area and decrease the dissolution rate, though Triesence is formulated at a smaller particle size than Kenalog-40. Triesence and Kenalog-40 have a 100% crystallinity, which should not cause the different vitreous dissolution rates observed. In contrast, Transton has a lower crystallinity, which may be the key factor for the higher dissolution rate seen in our study. It should be noted that the preservative-free TA prepared by a compounding pharmacy may be quite different from Triesence. Moshfeghi et al. demonstrated that preservative-free TA prepared by the compounding pharmacy had much less conglomerate than Triesence. 6 Indeed, Kim et al. showed that the preservative-free TA prepared by the compounding pharmacy had similar intravitreal pharmacokinetics to Kenalog-40 by sampling the whole rabbit vitreous following a 4 mg intravitreal injection. 28 However, in a most recent study, Zacharias et al. reported that Kenalog-40 was lasting longer in a rabbit eye model than Triesence after a single 4 mg intravitreal injection. 13 In that study, 4 mg TA was injected into a liquefied rabbit vitreous and a series of fundus images were taken to quantify the vitreous TA aggregate. They measured only two dimensions of the TA aggregate image and concluded that after week 12, Kenalog-40 had a significantly bigger pixels area than the Triesence. However, final TA concentration in retina and choroid measured by HPLC/MS at the end of the study (19 weeks) was not significantly different between Kenalog-40 and Triesence. 
In summary, our study demonstrated that an intravitreal injection of Triesence had significantly less free TA in ocular fluid than an equivalent dose of intravitreal Kenalog-40 or Transton. Low concentrations of free TA may decrease the rate of steroid-related complications, such as elevated IOP and cataract. In addition, a low dissolution rate of Triesence will translate into a longer ocular residence of the drug depot and a possible longer therapeutic duration. We acknowledge the limitation of our study, which did not perform pharmacodynamics to compare Triesence with the other two preserved TA formulations. The free TA level in aqueous is associated with the level of free TA in the vitreous and the proportion may depend on the individual drug and vitreous status. A more liquefied vitreous may render higher aqueous humor–free drug. Our study was conducted using the rabbit eye as a model, which has a denser vitreous and larger lens than human eyes. The proportionality of free drug in the aqueous and vitreous should not simply be generalized into other species. Nonetheless, the finding of a lower dissolution rate of Triesence in our study is valid because these three types of triamcinolone were tested simultaneously in the same species. 
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Footnotes
 Supported by National Natural Science Foundation of China Grant No. 31271022, Wenzhou Science and Technology Projects Y20100077, Zhejiang Provincial Grant for High Level Healthcare Talents, and International Research Project of MOST, China, 2012DFB30020. The authors alone are responsible for the content and writing of the paper.
Footnotes
 Disclosure: H. Chen, None; S. Sun, None; J. Li, None; W. Du, None; C. Zhao, None; J. Hou, None; Y. Xu, None; L. Cheng, None
Figure 1. 
 
The photograph is taken from a dual-probe microdialysis. The anterior eye globe was exposed using an eye lid speculum. The vitreous probe and aqueous probe are visible, as indicated in the photograph. Probes had a 4 mm dialysis membrane. The aqueous probe membrane is seen centered in the anterior chamber; the vitreous probe membrane is in the mid cavity of the vitreous, and looks whitish and distorted through the lens.
Figure 1. 
 
The photograph is taken from a dual-probe microdialysis. The anterior eye globe was exposed using an eye lid speculum. The vitreous probe and aqueous probe are visible, as indicated in the photograph. Probes had a 4 mm dialysis membrane. The aqueous probe membrane is seen centered in the anterior chamber; the vitreous probe membrane is in the mid cavity of the vitreous, and looks whitish and distorted through the lens.
Figure 2
 
Particle size distribution of different formulations of triamcinolone. The x-axis is in micrometers at a log scale. The bell-shaped curve indicates the range of particle size distribution at the corresponding sizes along the x-axis, while the left y-axis indicates the percent of sample having the size shown on the corresponding x-axis. The sigmoid curve indicates the cumulative distribution of particle sizes and the cumulative percentage is shown on the right y-axis.
Figure 2
 
Particle size distribution of different formulations of triamcinolone. The x-axis is in micrometers at a log scale. The bell-shaped curve indicates the range of particle size distribution at the corresponding sizes along the x-axis, while the left y-axis indicates the percent of sample having the size shown on the corresponding x-axis. The sigmoid curve indicates the cumulative distribution of particle sizes and the cumulative percentage is shown on the right y-axis.
Figure 3. 
 
Dissolution kinetics for the different formulations of triamcinolone in excised rabbit vitreous. Within the first hour of dissolution, free TA from Transton consistently was the highest and free TA from Kenolog-40 was higher than that of Triesence. The higher free TA concentrations at the earlier time points indicate a faster dissolution.
Figure 3. 
 
Dissolution kinetics for the different formulations of triamcinolone in excised rabbit vitreous. Within the first hour of dissolution, free TA from Transton consistently was the highest and free TA from Kenolog-40 was higher than that of Triesence. The higher free TA concentrations at the earlier time points indicate a faster dissolution.
Figure 4. 
 
The pharmacokinetic profiles of free triamcinolone in aqueous following a 1.2 mg intravitreal injection. Transton, Kenalog-40, and Triesence demonstrated a similar elimination profile, but with very different maximum concentration (Cmax) values, which was the highest for Transton and the lowest for Triesence (statistical evaluation not available due to too small sample size, n = 4). The paired t-test of means at each time point for three curves revealed statistically significant difference of free TA levels among three types of TA (Transton versus Kenalog-40, P = 0.023; Transton versus Triesence, P = 0.007; Kenalog-40 versus Triesence, P = 0.025).
Figure 4. 
 
The pharmacokinetic profiles of free triamcinolone in aqueous following a 1.2 mg intravitreal injection. Transton, Kenalog-40, and Triesence demonstrated a similar elimination profile, but with very different maximum concentration (Cmax) values, which was the highest for Transton and the lowest for Triesence (statistical evaluation not available due to too small sample size, n = 4). The paired t-test of means at each time point for three curves revealed statistically significant difference of free TA levels among three types of TA (Transton versus Kenalog-40, P = 0.023; Transton versus Triesence, P = 0.007; Kenalog-40 versus Triesence, P = 0.025).
Figure 5. 
 
Fundus photographs of the triamcinolone vitreous drug depot at the fourth week following the initial intravitreal injection. All drug depots settled in the inferior vitreous cavity. The photographs were acquired using a fundus camera (NF505; Nikon, Tokyo, Japan) with a 20 diopter fundus lens (Double aspheric; Volk Optical, Inc., Mentor, OH) prepositioned on the native camera lens. In this way, a wider field of fundus image is acquired, which allows for a display of the inferior drug depot at the peripheral fundus. An additional 20 diopter lens caused the reflection rings seen at the center of the images. The Triesence drug depots appeared larger, thicker, and whitish due to the light reflected back from the depot. In contrast, the Kenalog-40 and Transton drug depots looked smaller and yellowish, because the thinner depot allowed the red reflection from the fundus to penetrate and mix with the white reflection from the depot itself.
Figure 5. 
 
Fundus photographs of the triamcinolone vitreous drug depot at the fourth week following the initial intravitreal injection. All drug depots settled in the inferior vitreous cavity. The photographs were acquired using a fundus camera (NF505; Nikon, Tokyo, Japan) with a 20 diopter fundus lens (Double aspheric; Volk Optical, Inc., Mentor, OH) prepositioned on the native camera lens. In this way, a wider field of fundus image is acquired, which allows for a display of the inferior drug depot at the peripheral fundus. An additional 20 diopter lens caused the reflection rings seen at the center of the images. The Triesence drug depots appeared larger, thicker, and whitish due to the light reflected back from the depot. In contrast, the Kenalog-40 and Transton drug depots looked smaller and yellowish, because the thinner depot allowed the red reflection from the fundus to penetrate and mix with the white reflection from the depot itself.
Figure 6. 
 
Free TA concentrations in the vitreous (solid lines) and in the corresponding aqueous humor (dotted lines) from the microdialysis. The free TA in the vitreous and the aqueous humor demonstrated a synchronized increase or proportional change (blue lines) immediately following the intravitreal injection. The free TA levels in the aqueous humor at 2 weeks post injection was higher than at 3 weeks post injection; however, the free TA levels in the vitreous and aqueous humor, at the time points, were constant and analogous to one another.
Figure 6. 
 
Free TA concentrations in the vitreous (solid lines) and in the corresponding aqueous humor (dotted lines) from the microdialysis. The free TA in the vitreous and the aqueous humor demonstrated a synchronized increase or proportional change (blue lines) immediately following the intravitreal injection. The free TA levels in the aqueous humor at 2 weeks post injection was higher than at 3 weeks post injection; however, the free TA levels in the vitreous and aqueous humor, at the time points, were constant and analogous to one another.
Table 1. 
 
Physicochemical Characteristics of Different TA Formulations
Table 1. 
 
Physicochemical Characteristics of Different TA Formulations
TA Brand Osmolarity*, mOsm/kg pH* TA*, μg/mL Particle Size, μM Crystallinity Solubility in PBS, μg/mL Solubility in Vitreous at 24 h, μg/mL
Kenalog-40 328.7 ± 1.7 5.76 ± 0.06 17.10 ± 0.68 18.86 ± 16.7 100% 12.91 ± 0.44 24.69 ± 0.82
Triesence 300.5 ± 3.5 6.90 ± 0.01 10.42 ± 0.59 11.51 ± 8.1 100% 13.42 ± 0.76 26.71 ± 3.42
Transton 309.3 ± 1.9 6.79 ± 0.1 18.28 ± 0.45 10.13 ± 7.6 88.79% 12.51 ± 0.59 23.91 ± 0.77
TA STD 12.31 ± 0.62 20.40 ± 1.13
Table 2. 
 
TA Remaining in Vitreous and Plasma Concentration at the End of Study
Table 2. 
 
TA Remaining in Vitreous and Plasma Concentration at the End of Study
TA Type TA Amount in Whole Vitreous, ng TA Concentration in Plasma, ng/mL N of Eye Drug Aggregate Visible
Transton 1091.3 ± 2175.9 0.98 ± 1.6 4 1 of 4
Kenalog-40 1884.4 ± 3031.7 0.58 ± 0.4 4 1 of 4
Triesence 3264.75 ± 1733.5 1.36 ± 1.2 3* 3 of 3
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