Multidisciplinary Ophthalmic Imaging
`
`Preclinical PET Study of Intravitreal Injections
`
`Anxo Fern´andez-Ferreiro,1–4 Andrea Luaces-Rodr´ıguez,1 Pablo Aguiar,3,5 Juan Pardo-Montero,3,6
`Miguel Gonz´alez-Barcia,2,4 Lara Garc´ıa-Varela,3 Michel Herranz,3,7 Jes´us Silva-Rodr´ıguez,3 Mar´ıa
`Gil-Mart´ınez,8 Mar´ıa A. Berm´udez,9 Alba Vieites-Prado,10 Jos´e Blanco-M´endez,1 Mar´ıa Jes´us
`Lamas,2,4 Francisco G´omez-Ulla,8,11 ´Alvaro Ruibal,3,5,12 Francisco Javier Otero-Espinar,1 and
`Francisco Gonz´alez8,11
`
`1Department of Pharmacology, Pharmacy and Pharmaceutical Technology and Industrial Pharmacy Institute, Faculty of Pharmacy,
`University of Santiago de Compostela, Santiago de Compostela, Spain
`2Pharmacy Department, Complejo Hospitalario Universitario de Santiago (SERGAS), Santiago de Compostela, Spain
`3Molecular Imaging Group, Complejo Hospitalario Universitario de Santiago (SERGAS), Health Research Institute of Santiago de
`Compostela (IDIS), Santiago de Compostela, Spain
`4Clinical Pharmacology Group, Complejo Hospitalario Universitario de Santiago (SERGAS), Health Research Institute of Santiago de
`Compostela (IDIS), Santiago de Compostela, Spain
`5Molecular Imaging Group, Department of Radiology, Faculty of Medicine, University of Santiago de Compostela, Spain
`6Medical Physics Department, Complejo Hospitalario Universitario de Santiago (SERGAS), Santiago de Compostela, Spain
`7Galician PET Radiopharmacy Unit, Galaria, Complejo Hospitalario Universitario de Santiago (SERGAS), Santiago de Compostela,
`Spain
`8Service of Ophthalmology, Complejo Hospitalario Universitario de Santiago (SERGAS), Health Research Institute of Santiago de
`Compostela (IDIS), Santiago de Compostela, Spain
`9Department of Animal Biology, Vegetal Biology and Ecology, Faculty of Biology, University of A Coru˜na, A Coru˜na, Spain
`10Clinical Neurosciences Research Laboratory, Complejo Hospitalario Universitario de Santiago (SERGAS), Health Research Institute
`of Santiago de Compostela (IDIS), Santiago de Compostela, Spain
`11Department of Surgery, University of Santiago de Compostela (CIMUS), Spain
`12Nuclear Medicine Department, Complejo Hospitalario Universitario de Santiago (SERGAS), Santiago de Compostela, Spain
`
`PURPOSE. This work aimed at describing the time course of vitreous clearance through the use
`of positron emission tomography (PET) as a noninvasive tool for pharmacokinetic studies of
`intravitreal injection.
`
`METHODS. The pharmacokinetic profile of intravitreal injections of molecules labeled with
`18Fluorine (18F) was evaluated in adult Sprague Dawley rats by using a dedicated small-animal
`PET/computed tomography scanner. Different conditions were studied: three molecules
`radiolabeled with 18F (18F-FDG, 18F-NaF, and 18F-Choline), three volumes of intravitreal
`injections (7, 4, and 2 lL), and absence or presence of eye inflammation (uveitis).
`
`RESULTS. Our results showed that there are significant pharmacokinetic differences among the
`radiolabeled molecules studied but not among the injected volumes. The presence or absence
`of uveitis was an important factor in vitreous clearance, since the elimination of the drug was
`clearly increased when this condition is present.
`
`CONCLUSIONS. Intravitreal pharmacokinetic studies based on the use of dedicated PET imaging
`can be of potential interest as noninvasive tools in ophthalmic drug development in small
`animals.
`
`intravitreal
`Keywords:
`pharmacokinetics, PET
`
`injection, radiolabeled molecules, vitreous clearance,
`
`intravitreal
`
`Correspondence: Francisco
`Gonz´alez, CIMUS, P0, D4, Universi-
`dad de Santiago de Compostela, Avd.
`Barcelona 22, E-15782 Santiago de
`Compostela, Spain;
`francisco.gonzalez@usc.es.
`Francisco Javier Otero-Espinar, Fac-
`ulty of Pharmacy, University of San-
`tiago de Compostela, (USC)
`Pharmacy and Pharmaceutical Tech-
`nology Department, Praza Seminario
`de Estudos Galegos s/n E-1570 San-
`tiago de Compostela, Spain;
`francisco.otero@usc.es.
`
`AF-F and AL-R contributed equally to
`the work presented here and should
`therefore be regarded as equivalent
`authors.
`
`Submitted: March 6, 2017
`Accepted: April 26, 2017
`
`Citation: Fern´andez-Ferreiro A, Luaces-
`Rodr´ıguez A, Aguiar P, et al. Preclinical
`PET study of intravitreal injections.
`Invest Ophthalmol Vis Sci.
`2017;58:2843–2851. DOI:10.1167/
`iovs.17-21812
`
`To date, most topical and systemic drugs have not achieved
`
`adequate therapeutic levels in the vitreous, mainly owing to
`the existence of different physiological barriers.1 On one hand,
`topically instilled drugs are diluted by the tear film, thus causing
`significant drug loss in the lachrymal flow,2 and furthermore
`
`their physicochemical characteristics must be adequate to cross
`the cornea.3 On the other hand, the blood–retinal barrier
`(BRB), which comprises the retinal pigment epithelium and the
`tightly sealed walls of the retinal capillaries, complicates the
`arrival of systemic drugs to the vitreous.4 For these reasons,
`
`Copyright 2017 The Authors
`iovs.arvojournals.org j ISSN: 1552-5783
`
`2843
`
`This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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`Intravitreal Preclinical PK Study With PET/CT Imaging
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2844
`
`intravitreal administration has become an effective way to
`deliver drugs to the vitreous cavity, allowing high drug
`concentrations.5
`To achieve a sustained therapeutic drug concentration in
`the vitreous, the frequency of administration should be based
`on the half-life of the drug (t1/2). Regarding this question,
`several in vitro models have been proposed for the study of
`intravitreal pharmacokinetics, which take into account all
`aspects of the ocular anatomy and physiology.6–9 However, one
`aspect that should be taken into consideration in the in vitro
`pharmacokinetic studies is the absence of convection,10–13
`even though the principal mechanism of transport through the
`vitreous is diffusion, and convection does not play a relevant
`role in the kinetics of small molecules. Other issues such as
`protein binding, melanin binding, drug metabolism, or active
`transport are usually not taken into account in the in vitro
`studies.8,14 On the other hand, in vivo classical pharmacoki-
`netic studies of
`intravitreal
`injections are limited because
`invasive techniques are involved.15,16
`In recent years, molecular
`imaging techniques have
`become a turning point for the development and pharmaco-
`kinetic study of new drugs. These techniques involve
`noninvasive procedures in order to significantly decrease
`the number of animals used by increasing the number of
`measurements on each animal.17,18 In particular for the field
`of
`intravitreal drugs, single photon emission computed
`tomography and magnetic resonance image (MRI) have been
`the most commonly used imaging techniques, mainly to study
`pharmacokinetics14,19 and the release of drugs from implants
`and liposomes.20–22
`However, in pharmacokinetic studies performed with MRI,
`the molecules used for the labeling of the drug usually have
`very high molecular weight, which can alter the properties of
`the original drug.21
`The use of positron emission tomography (PET) has made it
`possible to label drugs with small b-emitting radioisotopes.23
`Current integrated PET/computed tomography (CT) scanners
`allow visualization of radiolabeled molecules by using a direct
`and noninvasive methodology, and the follow-up of the same
`subject over time to determine the pharmacokinetic properties
`of intravitreal injections.24–26
`Different radionuclides can be used to elaborate radiotrac-
`ers for PET scanning. The most commonly used radionuclides
`are typically isotopes with short half-lives such as 11C, 13N, 15O,
`18F, 68Ga, 82Rb, or with longer half-lives such as 124I or 89Zr. 18F
`is one of the most widely used because it is easily produced
`with a cyclotron, its positron energy of emission is 0.64 MeV, it
`is safe for patients, and it allows to obtain images with high
`resolution. Moreover, its half-life is long enough to be able to
`produce commercially manufactured fluorinated radiotracers
`at off-site locations and to be shipped to imaging services. In
`practice, 18F radionuclide is linked to different molecules to
`achieve selective transport and distribution.27
`Drug clearance in the vitreous can be influenced by various
`factors that include molecular weight, physicochemical prop-
`erties of the drug, surgical procedure, injected volumes, and
`presence of ocular inflammation.1 Also, the mechanisms of
`membrane transport and plasmatic clearance can highly
`influence the distribution and elimination of drugs after
`intravitreal administration. For this reason, fluorodeoxyglucose
`(18F-FDG), 18F-choline (18F-Choline), and 18F–sodium fluoride
`(18F-NaF) were selected in our study because of their different
`molecular weight, polarity, and transport mechanism across
`biological membranes. The aim of the present work was to
`study the effect of some of these factors on the vitreous
`clearance by using dedicated PET/CT imaging techniques for in
`vivo studies in rats.
`
`MATERIALS AND METHODS
`
`Our work was designed as an experimental study in rats
`scanned in a dedicated PET/CT system after intravitreal
`injections of different radiolabeled molecules, different vol-
`umes, and absence/presence of
`inflammatory eye disease
`(uveitis).
`
`Animals
`
`This study was carried out on male adult Sprague Dawley rats
`with an average weight of 300 g, supplied by the animal facility
`of the University of Santiago de Compostela (Santiago de
`Compostela, Spain). During the experiments, the animals were
`kept in individual cages with free access to food and water in a
`room under controlled temperature (228C 6 18C) and humidity
`(60% 6 5%) and with day–night cycles regulated by artificial
`light (12/12 hours). The animals were treated as indicated in
`the ARVO Statement for the Use of Animals in Ophthalmic and
`Vision Research and according to the guidelines for laboratory
`animals.28,29 Experiments were approved by the Galician
`Network Committee for Ethical Research and followed the
`Spanish and European Union (EU) rules (86/609/CEE, 2003/
`65/CE, 2010/63/EU, RD 1201/2005, and RD53/2013).
`
`Intravitreal Injection Procedure
`
`Intravitreal injection was performed according to the proce-
`dure described previously by Chiu et al.30 Firstly, the animals
`were placed in a gas chamber containing 2% isoflurane in
`oxygen. When unconscious, the animals were removed from
`the chamber but kept under anesthesia with a mask (1.5%
`isoflurane in oxygen). The procedure was initiated by applying
`one drop of topical anesthesia (Colircusi Anestesico Doble:
`tetracaine 1 mg/mL and oxybuprocaine 4 mg/mL) on the eye
`followed by mydriatic eye drops (phenylephrine 100 mg/mL
`[Colircusi Fenilefrina] and tropicamide 10 mg/mL [Colircusi
`Tropicamide]) to visualize the eye fundus. Thereafter, radiola-
`beled molecules were injected into the vitreous through the
`pars plana by using a Hamilton syringe with a 34-G needle. The
`injection procedure was performed with a surgical microscope
`(Takagi OM-5 220-2; Takagi, Tokyo, Japan). Pictures of the
`procedure were taken by means of a digital camera (Nikon D-
`200; Nikon, Tokyo, Japan) attached to the microscope. Eyes
`with lens damage, or with significant bleeding when the
`intravitreal injection was made, were discarded from the study.
`
`Experiments
`
`The experiments were carried out by using intravitreal
`injections with three radiolabeled molecules and three
`different injection volumes, in healthy eyes and in eyes with
`lipopolysaccharide (LPS)–induced uveitis.
`
`Effect of the Type of Injected Radiolabeled
`Molecules
`
`Three different molecules were labeled with 18F to evaluate
`the intravitreal pharmacokinetics. The radiolabeled molecules
`to be injected were 18F-NaF, 18F-FDG, and 18F-Choline, with
`molecular weights of 41, 182, and 122 g/mol, respectively
`(Fig. 1).
`The radioisotope 18F was obtained from the nuclear
`reaction 18O (proton, neutron) carried out in our PET Trace
`800 cyclotron, according to the method described by Saha.31
`The radiosynthesis of 18F-Na was made with a carbonate-type
`anion-exchange resin column, in such a way that the 18F is
`retained into the column and it is recovered as 18F–sodium
`
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`Intravitreal Preclinical PK Study With PET/CT Imaging
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2845
`
`FIGURE 1. Chemical structure of (A) 18F-Choline, (B) 18F-FDG, and (C)
`18F-NaF.
`
`fluoride by elution with potassium carbonate solution. 18F-FDG
`and 18F-Choline were produced on a TRACERlab MX synthe-
`sizer (GE Healthcare, Waukesah, WI, USA) by using cassettes
`and reagent kits from ABX (Advanced Biochemical Com-
`pounds, Radeberg, Germany). The nucleophilic substitution
`standard method was used in the case of 18F-FDG and for the
`reaction of 18F-fluoromethyl triflate with dimethylethanol-
`amine on a Sep-Pak column used in the case of 18F-Choline.32,33
`All procedures to obtain radiolabeled molecules were
`performed under good-manufacturing-practice conditions fol-
`lowing the specific standards of European Pharmacopoeia.34
`The purity and stability quality control requirements were
`undertaken via high-pressure liquid chromatography/ion chro-
`matography (930 Compact IC Flex con; Metrohm AG, Herisau,
`Switzerland) and thin layer chromatography. Osmolality
`(mOsm/kg) and pH were determined with a vapor pressure
`osmometer (VAPRO 5520; ELITECH Group, Paris, France) and a
`pH meter (WTW inoLab; WTW, Weilheim, Germany).
`
`Effect of the Injected Volumes
`
`the injected volume on the intravitreal
`The effect of
`pharmacokinetics of
`the abovementioned molecules was
`evaluated by using three different volumes: 2, 4, and 7 lL.
`
`Effect of the Presence of Inflammation
`
`Intravitreal pharmacokinetics was assessed in a uveitis animal
`model previously used by our group33 and then compared to
`the intravitreal pharmacokinetics in healthy eyes. To induce
`uveitis, rats were inoculated into the right posterior paw with 1
`mg/kg Escherichia coli LPS diluted in 0.1 mL phosphate-
`buffered saline by using a BD Micro-Fine syringe (BD, Oxford,
`UK) with 30-G needles. The presence of uveitis was assessed
`by direct inspection of the eye, using the surgical microscope.
`The animals were kept under such conditions for 24 hours. To
`reduce the number of animals, the influence of volume and
`presence or absence of uveitis were examined only for 18F-NaF
`(monoexponential kinetics) and 18F-FDG (biexponential kinet-
`ics). Four animals (eight eyes) were used in each condition
`studied.
`
`Data Acquisition and Analysis
`
`PET Data Acquisition. After the intravitreal injections of 1
`MBq in each eye for all experimental conditions, dynamic PET
`acquisition was carried out to generate eight images of 15
`minutes’ duration for the first 1.5 hours. Afterwards, single PET
`images were obtained at 4 and 6 hours after drug administra-
`tion. PET and CT images were acquired by using an Albira PET/
`CT Preclinical Imaging System (Bruker Biospin, Woodbridge,
`CT, USA). Animals were kept under anesthesia with a mask
`(1.5% isoflurane in oxygen). Respiration frequency and body
`temperature were monitored during the anesthesia period. The
`PET subsystem comprises three rings of eight compact
`modules based on monolithic crystals coupled to multianode
`photomultiplier tubes, forming an octagon with an axial field
`of view (FOV) of 40 mm per ring and a transaxial FOV of 80
`mm in diameter. The CT system comprises a commercially
`available microfocus x-ray tube and a CsI scintillator 2D
`pixelated flat panel x-ray detector. Scatter and random
`coincidences were corrected by using the protocols imple-
`mented in the scanner. Attenuation correction was not
`performed. Images were reconstructed by using the maximum
`likelihood expectation maximization algorithm. Twelve itera-
`tions were performed with a reconstructed image pixel size of
`0.4 3 0.4 3 0.4 mm3.
`PET Data Analysis. After reconstruction, quantitative
`measurements were obtained by using the Amide’s Medical
`Image Data Examiner.35 Different regions of interest (ROIs)
`were manually drawn containing the signal on each eye. The
`ROIs were then replicated on the different temporal image
`frames to obtain the decrease curve of the radioisotope over
`time, conveniently corrected for radioactive decay.
`Statistical Analysis. The curves of percentage of radio-
`tracer in the eye versus time were fitted to the mono- and
`bicompartimental pharmacokinetic model by using nonlinear
`least squares regression analysis. The area under the percent-
`age of radiotracer time curve AUC360
`from zero to infinity was
`0
`calculated by log-trapezoidal rule. The statistical analysis of
`experiments was performed by using a 1-way analysis of
`variance (ANOVA) and Tukey’s multiple comparisons test. The
`nonlinear fitting and the statistical analysis were made by using
`the GraphPad Prism 6.0 software (2014; GraphPad Software,
`Inc., San Diego, CA, USA).
`
`RESULTS
`
`All radiolabeled molecules were clearly detected in the vitreous
`cavity at the initial time of the study and it was possible to
`observe how the signal decreased over time. Figure 2 shows
`the coronal views of the fused PET/CT images from the initial
`frame (10 minutes after the injection) to the last frame (360
`minutes after the injection).
`
`FIGURE 2. Fused image PET/CT showing the signal evolution in the rat eyes throughout time (minutes).
`
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`Intravitreal Preclinical PK Study With PET/CT Imaging
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2846
`
`and 18F-Choline tracers appeared to fit a two-compartment
`model with a biphasic clearance from the vitreous. The
`obtained average intravitreal half-lives for these radiolabeled
`molecules were13.99 minutes for 18F-FDG and 35.18 minutes
`for 18F-Choline for the initial rapid elimination phase (a), and
`214.2 minutes and 1351 minutes, respectively, for the slow
`elimination phase (b). Table 1 shows the pharmacokinetic
`parameters obtained by fitting the data to a bicompartmental
`model. On the other hand, the clearance curve from 18F-Na
`showed a one-compartment pharmacokinetic model, and the
`average intravitreal half-life was 113.2 minutes. Table 2 shows
`the pharmacokinetic parameters obtained by fitting the data to
`a one-compartment pharmacokinetic model.
`When comparing the area under the curve between 0 and
`360 minutes (AUC360
`) among three radiolabeled molecules, it
`0
`was observed that 18F-Choline remains significantly longer in
`the eye than 18F-FDG and 18F-NaF (Fig. 3B).
`The radiolabeled molecules leave the eye and reach the
`systemic circulation, following different kinetic curves. Fur-
`thermore, the distribution at system level is also significantly
`different. Figure 4 shows that 18F-NaF is captured by bone
`structures, while 18F-FDG and 18F-Choline are captured by
`internal organs.
`The radiolabeled molecules used for the intravitreal
`injection had radiochemical purity for 18F-FDG higher than
`95% with a specific activity of approximately 1000 MBq/mL.
`The 18F-Choline had radiochemical purity higher than 95%
`with a specific activity of approximately 500 MBq/mL. All
`radiotracers showed percentages of fluorine bound to the
`radiotracer that were higher than 95% at 8 hours post
`synthesis. The osmolality of all radiolabeled solutions was
`approximately 280 6 10 mOsm/kg with a pH »7.4.
`
`Effect of the Injected Volumes
`
`Figure 5 shows no differences between the different volumes
`of intravitreal injections (2, 4, and 7 lL) for 18F-Na and 18F-FDG
`radioisotopes, which follow the same kinetics as previously
`described in Figure 3A. Tables 1 and 2 show that no statistically
`significant differences were found between pharmacokinetic
`parameters in relation to the injected volumes of both 18F-FDG
`(Table 1) and 18F-Na (Table 2). Finally, it should be noted that a
`transient vascular collapse in the retinal vessels was observed
`after administration of 7 lL, but not for 2 and 4 lL.
`
`FIGURE 3.
`Influence of the drug type on its intravitreal release (mean
`6 SD, n ¼ 8). (A) Intravitreal pharmacokinetic profile of 18F-FDG, 18F-
`injection of 4 lL.
`NaF, and 18F-Choline after
`(B)
`intravitreal
`Representation of AUC360
`(% min) for all radiotracers. *1-way ANOVA
`0
`analysis and Tukey multiple comparison test show significant
`differences among the three different compounds (a < 0.01).
`
`Effect of the Type of Radiolabeled Molecules
`
`Effect of the Presence of Inflammation
`
`The values measured from the ROI, containing each eye
`throughout time, were obtained for the three radiolabeled
`molecules, giving rise to significantly different kinetic curves
`(Fig. 3A). On the one hand, the clearance curves from 18F-FDG
`
`Figure 6A shows that inflammation slightly, but with statistical
`significance, increased the vitreous clearance of 18F-FDG. This
`effect was quantified by comparing the AUC360
`of radiolabeled
`0
`molecules in uveitis and under normal conditions. Figure 6B
`
`TABLE 1. Pharmacokinetic Parameters Obtained by Fitting the Data to a Bicompartmental Model for 18F-FDG and 18F-Choline
`
`18F-FDG
`
`4 lL*
`
`18F-Choline
`
`Pharmacokinetic Parameters
`a, min1
`t1/2a, min
`B, min1
`t1/2b, min
`AUC360
`, % min
`0
`R2
`
`2 lL*
`
`Normal†
`
`Uveitis†
`
`7 lL*
`
`7 lL
`
`0.0336
`20.65
`0.00285
`243.0
`70.13 6 5.31
`0.9958
`
`0.03341
`20.75
`0.002421
`286.4
`88.15 6 7.86
`0.9958
`
`0.0416
`16.66
`0.00218
`317.8
`70.01 6 5.70
`0.9938
`
`0.0495
`13.99
`0.00324
`214.2
`82.05 6 15.67
`0.9963
`
`0.01970
`35.18
`0.00051
`1351
`201.3 6 18.83
`0.9971
`
`* No statistical differences for AUC360
`(% min) were observed between different injection volumes (a not significant [n.s.]).
`0
`(% min) between normal and uveitis eyes for a < 0.01.
`† Statistical differences for AUC360
`0
`
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2847
`
`TABLE 2. Pharmacokinetic Parameters Obtained by Fitting the Data to Monocompartmental Model With 18F-NaF
`
`18F-NaF
`
`4 lL*
`
`Pharmacokinetic Parameters
`k, min1
`t1/2, min
`AUC360
`, % min
`0
`R2
`
`2 lL*
`
`Normal†
`
`Uveitis†
`
`0.00669
`103.6
`140.15 6 14.93
`0.9982
`
`0.00656
`105.7
`135.23 6 14.09
`0.9982
`
`0.00805
`86.11
`123.69 6 21.09
`0.9952
`
`7 lL*
`
`0.00612
`113.2
`137.03 6 5.72
`0.9956
`
`* No statistical differences were observed for AUC360
`0
`† No statistical differences were observed for AUC360
`0
`
`(% min) between different injection volumes (a n.s.).
`(% min) between normal and uveitis eyes (a n.s.).
`
`shows that eyes with uveitis had smaller AUC360
`than normal
`0
`eyes. In addition, statistically significant differences were found
`between the pharmacokinetic parameters in uveitis and
`normal conditions for the case of 18F-FDG (Table 1). It must
`be mentioned that animals receiving an LPS injection
`developed a fibrinous reaction in the anterior chamber of the
`eye, which produced a pupillary membrane and an irregular
`pupil after drug-induced mydriasis, caused by the adhesion of
`the iris to the lens (Fig. 7). The uveitis model was successfully
`achieved in the same way as obtained in our previous studies.36
`
`The use of small animals, such as Sprague Dawley rats, has
`many advantages because of their small size, the availability of
`research animal facilities, and multiple disease models suitable
`for them.44,45 However, since they have a small vitreous
`volume, classic pharmacokinetic studies become difficult, with
`in vivo imaging being an ideal technique, as no invasive
`modalities are required to obtain experimental results.46,47 To
`the best of our knowledge, our work is the first study of
`intravitreal pharmacokinetics with PET/CT in rats. Previous
`intravitreal pharmacokinetic studies have required larger
`
`DISCUSSION
`
`Intravitreal injections are increasingly used in a multitude of
`retinal ophthalmic conditions such as age-related macular
`degeneration,37 diabetic macular edema,38 macular holes,39
`branch and central retinal vein occlusion,40 and endophthal-
`mitis.41 The development of new intravitreal drugs or systems
`that modify their release involves wide preclinical develop-
`ment42 in which pharmacokinetic studies play a key role.43
`
`FIGURE 4. Representation of the systemic distribution of radiotracers
`at different times after intravitreal administration. (A) Coronal views
`after injection of 18F-FDG. (B) Sagittal views after injection of 18F-NaF.
`(C) Coronal views after injection of 18F-Choline.
`
`FIGURE 5.
`Influence of the injection volume on vitreal release (mean
`6 SD, n ¼ 8). Intravitreal pharmacokinetic profile of 18F-FDG (A) and
`18F-NaF (B) after intravitreal injection of 2, 4, and 7 lL.
`
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2848
`
`FIGURE 7. Anterior segment of two eyes 24 hours after pad injection
`of LPS, showing signs of uveitis. Left: Fibrinous reaction producing a
`pupillary membrane. Right: Irregular pupil after drug-induced mydri-
`asis caused by the adhesion of the iris to the lens.
`
`are 1.47, 1.57, and 1.20 angstroms, respectively); therefore,
`changing oxygen or hydrogen for fluoride does not entail
`substantial modifications in the molecular structure by steric
`impediments. Furthermore, in terms of Taft Es parameters,56
`fluoride and hydroxyl substituents have very similar character-
`istics (þ0.78 vs. þ0.69); therefore, their substitution does not
`compromise either the structural activity of the compound or
`its interaction with receptors. The electronegativity of fluoride
`and hydrogen atoms is different
`(4.0 vs. 2.1), hence
`interchanging them can substantially affect the physicochem-
`ical properties of the molecule (pKa, hydrogen bond capacity,
`or lipophilicity). On the contrary, fluoride and oxygen have
`similar values (4.0 vs. 3.5), so no major changes should be
`expected when interchanged.55 Owing to the relatively short
`half-life of 18F, the fluorinated radiotracers have limited use in
`studies of pharmacokinetics or biodistribution of drugs with
`long half-lives in the vitreous cavity. For these long-term
`studies, using other radiotracers with long half-lifes such as 124I
`(Kuntner et al.57 and Dangl et al.58) or 89Zr (Van Loon et al.59)
`is more adequate.
`Fluorinated radiotracers, as the ones used in this work, have
`the advantage of their low positron emission energy (the
`lowest of all the radiolabels used in PET). Furthermore, the
`greater sensitivity of modern PET technology allows the use of
`low radioactivity levels, so the dose received and absorbed by
`the animal is significantly below the dose limit.60 Additionally,
`during the disintegration of 18F, no c rays or a and b particles
`are emitted, reducing the dose received by animals and
`increasing safety.61 On the other hand, cytotoxicity and acute
`irritation of fluorinated radiotracers have been described as
`safe in previous reports.23 In our study, no alterations in the
`eye of the animals were observed after the administration of
`the fluorinated radiotracers.
`Our findings showed significant differences between the
`different radiolabeled molecules we used. The reason for these
`differences could rest on the mechanism used for crossing the
`BRB. In the rat retina there are transporters for glucose and
`cationic amino acids, which probably are used by 18F-FDG and
`18F-Choline to leave the vitreous cavity.62 The biexponential
`kinetics we observed is also common for intravitreal drugs
`such as bevacizumab and ranibizumab.50,63 Furthermore,
`it
`must be mentioned that hyaluronic acid, which is part of the
`vitreous humor, has a highly negative charge at physiological
`pH levels. Because of this, it could interact with positively
`charged molecules, such as choline, by generating polyelec-
`trolyte complexes with low solubility.64 This is probably the
`reason why choline is released at a slower rate than glucose.
`On the other hand, our findings showed that 18F-NaF is
`eliminated from the vitreous,
`following monoexponential
`kinetics, which could be explained by assuming passive
`diffusion through the BRB because this compound diffuses
`freely across membranes.65 It would be similar to the release
`kinetics of other intravitreal drugs, such as aflibercept.66,67
`
`Influence of inflammation on vitreal release (mean 6 SD, n
`FIGURE 6.
`¼ 8). (A) Intravitreal pharmacokinetic profile of 18F-FDG and 18F-NaF
`after a 7-lL intravitreal
`injection in normal eyes and in eyes with
`uveitis. (B) Representation of AUC360
`(% min) for 18F-FDG and 18F-NaF
`0
`in these conditions. *Statistical significant differences between normal
`and uveitis eyes for a < 0.01.
`
`numbers of animals and more complex techniques to
`determine vitreous drug levels at different time points.48–50
`In our study serial measurements were obtained at multiple
`time points after the intravitreal injection in the same animal.
`The advantage of preclinical PET/CT images in this field is very
`important because the technique is noninvasive, and it yields
`images in 3D and real time.51 PET/CT is also becoming a
`relevant procedure for ophthalmic research, as it has been
`used for diagnosis of intraocular tumors,52 neurophysiological
`studies,53,54 or pharmacokinetic studies with topical ophthal-
`mic formulations.23 Although PET is a very sensitive technique,
`it presents some limitations related to low spatial resolution. As
`an example, the delineation of the vitreous area is troublesome
`and challenging owing to the small size of the eyeball, and
`therefore our measurements cannot be restricted exclusively to
`the vitreous area.
`More than 10% of currently used drugs contain fluorine
`atoms that can be labeled with 18F. Moreover, the substitution
`of oxygen atoms or hydroxyl groups by fluorine is relatively
`easy with no critical changes in the properties of
`the
`molecule.55 Fluoride and oxygen have a very similar radius,
`whereas that of hydrogen is slightly smaller (van der Waals radii
`
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`Intravitreal Preclinical PK Study With PET/CT Imaging
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`IOVS j June 2017 j Vol. 58 j No. 7 j 2849
`
`Our findings showed that the injected volume had no
`significant influence on vitreous drug clearance. Different
`studies have been carried out in human eyes68 and in murine
`models69,70 using a wide range of
`intravitreal
`injection
`volumes (2–20 lL), but they did not include an evaluation
`of their impact on the vitreous drug clearance. On the other
`hand, it has been pointed out that an increase of intraocular
`pressure could result in an increase of hydraulic flow, derived
`from the excess of volume introduced.10 This increase in
`intraocular pressure could be the cause of the transitory
`collapse we observed with the administration of 7 lL.
`However, this process seems not to have an effect on the
`vitreous clearance of low-molecular-weight drugs,71,72 such
`as the ones we used, where all radiolabeled molecules had
`molecular weights below 500 Da. Finally,
`it has to be
`mentioned that the vitreous volume of a rat is smaller than
`that of humans (approximately 50 lL in rats versus 4.5 mL in
`humans)73 (Vezina M, et al. IOVS 2011;52:ARVO E-Abstract
`3219). This difference must be kept in mind if our results are
`to be translated to humans.
`Our results showed an increase in the intravitreal clearance
`of the 18F-FDG radiotracer in eyes with inflammation (uveitis)
`when compared to healthy eyes. On the contrary, no
`significant differences were observed for 18F-NaF. Studies using
`MRI techniques have shown that inflammation in rabbit eyes,
`induced by LPS, can increase the permeability of BRB.74,75 On
`the other hand, additional studies have demonstrated that in
`inflammatory conditions, as in tumors, a high FDG uptake and
`a high GLUT-1 expression level is observed.76 Of note, 18F-NaF
`is not affected by changes produced by the inflammatory
`process probably because it
`is freely diffusible across
`membranes.65 However,
`the increase in permeability and
`GLUT transporter under inflammatory conditions can increase
`the clearance of 18F-FDG from the vitreous. Since the
`magnitude of the clearance changes we found were small, it
`would be necessary to carry out additional studies to properly
`establish the influence of inflammation on the BRB permeabil-
`ity. It is possible that the severity of the inflammatory process
`determines the increase of BRB permeability and hence, the
`intravitreal clearance rate.
`in
`inhaled anesthesia,
`Finally, although the effect of
`particular isoflurane, on drug permeability has been exten-
`sively studied in the blood–brain barrier (BBB), no studies have
`shown any type of modification in the status of the BRB.77
`Inhaled isoflurane in rats decreases the transfer of small
`hydrophilic molecules across the BBB, either by reducing the
`perfused capillary surface area or by a direct