-
`
`Retina
`
`Different Intravitreal Properties of Three Triamcinolone
`Formulations and Their Possible Impact on Retina Practice
`
`Hao Chen,1 Shumao Sun,1 Jie Li,1 Wennan Du,1 Chunhui Zhao,1 Jiangping Hou,*,1 Yu Xu,1
`and Lingyun Cheng1,2
`
`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 dis
`solving 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 spec
`trometry.
`
`RESULTS. Triesence had the most uniform particle size
`distribution (mean 11.51 lm) and Kenalog 40 had the largest
`particle sizes (mean 18.86 lm). 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
`than Kenolog 40 (P ¼ 0.025) and
`ocular free drug level
`Transton (P ¼ 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. (Invest Ophthalmol Vis Sci. 2013;
`54:2178 2185) DOI:10.1167/iovs.12 11460
`
`From the 1Institute of Ocular Pharmacology, School of
`Ophthalmology and Optometry, Wenzhou Medical College, Wenz
`hou, Zhejiang, China; and the 2Jacobs Retina Center at Shiley Eye
`Center, Department of Ophthalmology, University of California, San
`Diego, La Jolla, California.
`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.
`Submitted for publication December 8, 2012; revised February
`5, 2013; accepted February 18, 2013.
`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
`Current affiliation: *Provincial Hospital, Shandong University,
`Jinan, Shandong, China.
`Institute of Ocular
`Corresponding author: Lingyun Cheng,
`Pharmacology, School of Ophthalmology and Optometry, Wenzhou
`Medical College, 270 Xueyuan Road, Wenzhou, Zhejiang, China,
`325027; cheng@eyecenter.ucsd.edu.
`
`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.3–5 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 understand
`ing 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).4,5,9–12 To the best
`of our knowledge, the difference of ocular free TA pharmaco
`kinetics 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.
`
`2178
`
`Investigative Ophthalmology & Visual Science, March 2013, Vol. 54, No. 3
`Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc.
`
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`

`IOVS, March 2013, Vol. 54, No. 3
`
`Intravitreal Properties of Different Triamcinolone
`
`2179
`
`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 lL of the
`supernatant were sampled. Following filtration with a 0.45 lm filtering
`membrane, 50 lL of the filtered sample were mixed with 50 lL of high
`performance liquid chromatography (HPLC) mobile phase and 20 lL 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 3 150 mm, 5 lm) column at 308C. 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 lg/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
`808C
`of Triesence, Kenalog 40, and Transton were placed in a
`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 378C 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 lL 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 6 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
`lL 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 lL of aqueous
`humor under the direct view of a surgical microscope (F18; Leica,
`Wetzlar, Germany). The samples were stored under 808C 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 808C 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 Micro
`dialysis) 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 lL (vitreous probe) or 2 lL
`(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
`
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`

`2180 Chen et al.
`
`IOVS, March 2013, Vol. 54, No. 3
`
`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.
`
`determining the rate of TA recovery at 378C 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 lL of infusion rates
`were used.
`
`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 6 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 3 different TA
`The physicochemical properties of
`preparations are summarized into Table 1.
`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).
`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,
`
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`

`IOVS, March 2013, Vol. 54, No. 3
`
`Intravitreal Properties of Different Triamcinolone
`
`2181
`
`TABLE 1. Physicochemical Characteristics of Different TA Formulations
`
`Osmolarity*,
`mOsm/kg
`
`328.7 6 1.7
`300.5 6 3.5
`309.3 6 1.9
`
`TA Brand
`
`Kenalog 40
`Triesence
`Transton
`TA STD
`
`pH*
`
`TA*,
`lg/mL
`
`Particle
`Size, lM
`
`Crystallinity
`
`5.76 6 0.06
`6.90 6 0.01
`6.79 6 0.1
`
`17.10 6 0.68
`10.42 6 0.59
`18.28 6 0.45
`
`18.86 6 16.7
`11.51 6 8.1
`10.13 6 7.6
`
`100%
`100%
`88.79%
`
`STD, standard.
`* Indicates the parameters was derived from measurement of the supernatant of the TA preparation.
`
`Solubility in
`PBS, lg/mL
`
`12.91 6 0.44
`13.42 6 0.76
`12.51 6 0.59
`12.31 6 0.62
`
`Solubility in
`Vitreous at
`24 h, lg/mL
`
`24.69 6 0.82
`26.71 6 3.42
`23.91 6 0.77
`20.40 6 1.13
`
`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).
`
`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 (P¼ 0.0225, paired t test) and a Triesence
`injection (P ¼ 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 ¼
`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.
`
`indirect ophthalmoscopy re
`During clinical observation,
`vealed 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 summa
`rized in Table 2.
`
`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 6 2.7% at 378C
`and under a perfusion rate of 1 lL per minute. In contrast, the
`aqueous probe TA recovery rate was 11.3 6 1% at 378C under
`a perfusion rate of 1 lL/min. With the perfusion rate of 2 lL/
`min, the recovery rate was only 6.6 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 lL/min perfusion
`than that at 2 lL/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
`
`Kenalog-40
`
`Size Distribution
`
`Transton
`
`10
`9
`
`~ 1
`e.., 6
`
`.2 4
`~ 3
`
`60 es
`
`100
`90
`80
`70
`
`50
`40
`30
`20
`10
`
`JOO
`90
`80
`70
`60
`50
`,10
`30
`20
`10
`0
`
`e4
`"
`E 3
`"
`~ 2
`
`tt
`
`O 0. 01
`
`0. l
`
`Triesence
`
`ll
`lO
`9
`~ 8
`
`" 6
`
`t 1
`5 5
`0 4
`> 3
`2
`
`10
`Size (µm)
`
`100
`
`1000
`
`0. 01
`
`0. l
`
`10
`Size (µm)
`
`100
`
`1000
`
`lOO
`90
`80
`70
`60
`50
`40
`30
`20
`lO
`0
`
`O 0. Ol
`
`0. l
`
`10
`Size (µm)
`
`100
`
`1000
`
`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.
`
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`2182 Chen et al.
`
`IOVS, March 2013, Vol. 54, No. 3
`
`8 5 - - - - - - - - - - - - - - - - - -~
`80
`
`LSmean 18.23(Transton)
`vs
`12.39(Kenalog)
`vs
`9.95(Triesence)
`
`-A- Transton
`_._ Kenatog-40
`-.- Triesence
`
`,-_ 75
`
`E 10
`el)
`-3 ss
`g 60
`
`·= 30 e 2s c 8 20
`
`C:: 15
`0
`U 10
`
`10
`
`20
`
`30
`
`40
`50
`1400 1410 1420 1430 1440 1450
`Time (minutes)
`
`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.
`
`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 6 0.12, 6.03 6 1.54, and 2.38 6
`0.96 ng/mL in the aqueous humor, and 137.72 6 27.33, 46.57
`6 6.89, and 42.28 6 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 concentra
`tion 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
`
`60
`55
`
`humor was less than 1% of the free TA in the vitreous within
`the first few hours following a 1.2 mg intravitreal injection.
`Among the three time points, the free TA levels in the
`vitreous were significantly higher within the first few hours
`following the intravitreal injection (P¼ 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 ¼ 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 ¼ 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 comprehen
`sive 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 lg/mL, respective
`ly), but the dissolution kinetics in the vitreous at 378C were
`different. The preservative free Triesence dissolved much more
`slowly than Kenalog 40 and Transton, which dissolved the
`
`TA_type
`- Transton
`- Kenalog-40
`- Triesence
`
`,,,.._
`
`'-./
`
`50
`45
`:e 40
`Cl
`C 35
`C 30
`0 Jg
`25
`C
`Q)
`0
`20
`C
`0
`(.) 15
`<(I
`I- 10
`5
`0
`-5
`0
`
`:
`
`:
`
`-.
`
`10
`
`40
`30
`20
`Time Point (days)
`
`50
`
`60
`
`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;
`0.025).
`Transton versus Triesence, P
`0.007; Kenalog 40 versus Triesence, P
`
`Downloaded from iovs.arvojournals.org on 01/07/2022
`
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`
`

`

`IOVS, March 2013, Vol. 54, No. 3
`
`Intravitreal Properties of Different Triamcinolone
`
`2183
`
`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.
`
`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 lL
`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.18,21–24 In
`addition, a randomized clinical trial has demonstrated signifi
`cantly 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 syn
`chronized 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
`
`TABLE 2. TA Remaining in Vitreous and Plasma Concentration at the End of Study
`
`TA Type
`
`Transton
`Kenalog 40
`Triesence
`
`TA Amount in
`Whole Vitreous, ng
`
`TA Concentration
`in Plasma, ng/mL
`
`N of Eye
`
`Drug Aggregate Visible
`
`1091.3 6 2175.9
`1884.4 6 3031.7
`3264.75 6 1733.5
`
`0.98 6 1.6
`0.58 6 0.4
`1.36 6 1.2
`
`4
`4
`3*
`
`1 of 4
`1 of 4
`3 of 3
`
`* One of 4 rabbits died immediately before the 6 week time point.
`
`Downloaded from iovs.arvojournals.org on 01/07/2022
`
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`
`

`

`2184 Chen et al.
`
`IOVS, March 2013, Vol. 54, No. 3
`
`~------------------------~ ID
`- Vitreous-14iour
`- Vitreous-2-wks
`- Vitreous-3-wks
`· Aqueous-14iour
`· · · · Aqueous-2-wks
`· Aqueous-3-wks
`
`400
`3 00
`200
`
`100
`70
`50
`
`3 0
`20
`
`:::::.
`~ 10
`-S
`C
`0
`1
`Q) ..,
`..,
`
`C
`0
`
`<(
`I -
`
`7
`5
`3
`2
`
`0 .7
`0 .5
`
`0 .3
`0.2
`
`0 .1
`0.07
`0 .05
`
`0 ,0 3
`0.02
`
`0.01
`
`0
`
`50
`
`200
`150
`100
`Time point ( minutes)
`
`250
`
`300
`
`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.
`
`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 6 29 and
`181 6 54 ng/mL, respectively; the corresponding free TA in
`the aqueous would be 53 6 14 ng/mL at week 2 and 21 6 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 6 117 ng/mL) in the vitreous and very low (2 6 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 free TA levels among 3 TA
`The difference of
`formulations may come from the strong conglomeration o