Fibrin Sealant for Retinoblastoma: Where Are We?

Nancy E. Martin,1 Jonathan W. Kim,2 and David H. Abramson2

Abstract

Chemoreduction is currently the most popular treatment strategy for intraocular retinoblastoma worldwide. Despite the dramatic clinical responses obtained with multiagent systemic chemotherapy regimens, enthusi- asm for this treatment approach has been tempered by the potential toxicities of these drugs in the pediatric population. As a response to these concerns, novel approaches for the local delivery of chemotherapeutic agents to ocular structures have been investigated by both clinicians and scientists. Ocular oncologists have developed the periocular injection of carboplatin as a method for controlling intraocular tumor growth of retinoblastoma while minimizing systemic drug exposure. In parallel, the pharmaceutical industry has introduced drug-de- livery systems to the posterior segment of the globe for a variety of ocular diseases. One example of the col- laborative work by ophthalmologists and biopharmaceutical scientists is the use of fibrin sealants as a targeted drug-administration device, formulated to deliver sustained concentrations of chemotherapy at the site of ap- plication. This review integrates the recent ophthalmology and pharmaceutics literature on the potential role of fibrin sealants for periocular chemotherapy administration in the treatment of retinoblastoma.

Introduction

URVIVAL RATES FOR INTRAOCULAR RETINOBLASTOMA have im-
proved dramatically in the past century. A disease that was 95% lethal 100 years ago is now curable in more than 95% of cases.1,2 Despite these advances, clinicians continue to face challenges in improving globe salvage rates and mit- igating the long-term side effects of therapy. Over the past decade, there has been a dramatic paradigm shift away from the use of external beam radiation (EBR) and toward sys- temic chemotherapy for intraocular retinoblastoma.3 Al- though retinoblastoma is one of the few cancers cured by ra- diation alone at doses that are well tolerated by the eye, EBR has been implicated in the development of secondary nonoc- ular cancers in retinoblastoma survivors.4
Currently, the most commonly employed approach for treating retinoblastoma in developed countries is chemore- duction, a strategy involving neoadjuvant systemic chemo- therapy, followed by treatment with focal modalities, such as cryotherapy, laser treatment, or brachytherapy.5,6 In many centers, a complete six-cycle regimen with carboplatin, vin- cristine, and etoposide is given to all children with intraoc- ular disease. Unfortunately, systemic chemotherapy in the pediatric population has potential short- and long-term com-

plications. Neutropenia, fever, port infections, sepsis, ane- mia, transfusions, and associated hospitalizations have all been reported in chemotherapy-treated retinoblastoma pa- tients.7 Additionally, the association between secondary acute myelocytic leukemia and systemic chemotherapy has been suggested, especially for the podophyllotoxins.8
The most common chemotherapeutic agents employed in the treatment of retinoblastoma are carboplatin, etoposide, and vincristine.6,9 As a single agent, carboplatin has been used in both systemic and local treatment approaches. Car- boplatin is a platinum-based antineoplastic used in a variety of chemotherapy protocols for liquid and solid tumors; how- ever, its Food and Drug Administration (FDA)-approved in- dication is specifically for ovarian carcinoma. Consistent with other alkylating agents, carboplatin produces inter- strand DNA cross-linking and subsequent cell death. This mechanism is cell-cycle nonspecific. Frequent adverse effects of carboplatin following a systemic administration include thrombocytopenia, neutropenia, anemia, nausea, vomiting, and neurologic toxicities.
Carboplatin disposition has been characterized in humans after an intravenous administration with negligible protein binding, no appreciable hepatic metabolism, and mostly the renal elimination of unchanged drug.* Although the sys-

1Department of Medicine and 2Ophthalmic Oncology Service, Memorial Sloan Kettering Cancer Center, New York, NY. The authors have no proprietary interest or financial disclosure to report.
*U.S. prescribing information for Paraplatin (carboplatin aqueous solution) for injection. Princeton, NJ: Bristol Myers Squibb Company, 2004.
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temic pharmacokinetics of carboplatin have been well char- acterized and sophisticated models are available for dosing optimization,10,11 the ocular bioavailability and pharmacoki- netics of carboplatin following a systemic or periocular ad- ministration are still actively under investigation.12 A sum- mary of the published data regarding the ocular and systemic concentrations achieved after carboplatin administration through various routes is listed in Table 1. Despite advances in the pharmacokinetic modeling of drug delivery to the retina and choroid,13 the clinical application of these methods to oc- ular chemotherapy administration is still in its infancy.

Challenges and approaches to ocular drug delivery in retinoblastoma
A critical aspect of drug efficacy is to ensure the drug reaches its intended site of action, for an appropriate dura- tion of time, to exert its intended effect. Several factors pose serious challenges in the case of retinoblastoma to this issue in drug product development. Due to the presence of the blood-retina barrier, intraocular drug concentrations are less than plasma concentrations after a systemic administra- tion.14–17 Several drug-transport mechanisms located in the retinal pigment epithelium (RPE), including P-glycoprotein as well as organic anion and cation transporters, serve as ef- flux pumps precluding optimal ocular drug delivery.16,17 Twenty-nine (29) such transporters have been shown to be expressed in the blood-retina barrier and are thought to be important for maintaining its barrier function.17 One ap- proach to bypass these recognized obstacles is direct intrav- itreal injection. Despite recent clinical advances in ocular therapeutics through the intravitreal injection (e.g., Kenalog, Macugen, Avastin, Lucentis, etc.), direct injection is strongly discouraged in the presence of retinoblastoma due to con- cerns of secondary tumor seeding or spread beyond the globe.18 Thus, alternative approaches have been investigated to facilitate intraocular drug delivery through periocular or subconjunctival transscleral delivery modalities.

Transscleral drug administration is governed by scleral permeability and barrier functions of the RPE only recently appreciated.14,19–24 Conventional principles that govern the likelihood of a drug crossing a biologic membrane do not apply to the sclera. For example, scleral permeability is not influenced by the lipophilicity of the drug and the sclera per- mits the passage of large molecules, including proteins. These features of the sclera are in notable contrast to the cornea, conjunctiva, and other biologic membranes. Despite the relative ease with which molecules can cross the sclera, the RPE acts as a tight barrier and is the rate-limiting mem- brane for drug permeability. Given these insights into trans- scleral and RPE permeability, techniques including Coulomb-controlled iontophoresis and direct subtenon or subconjunctival injections have been explored to optimize carboplatin delivery to the posterior segment.14,19,25,26
Carboplatin has been shown to cross the sclera5,18,27,28; however, the agent is found to be rapidly cleared from the vitreous.5 This has led to increased interest in controlled re- lease formulations of trans-scleral carboplatin. One promis- ing approach is impregnating fibrin sealant products with carboplatin for increased drug delivery and/or sustained in- traocular concentrations.5,19,28

Attributes of fibrin sealant and its drug-delivery potential
Fibrin sealant is an FDA-approved surgical adhesive in- dicated as an adjunct to hemostasis and is currently being employed as a biodegradable, semisolid drug delivery de- vice for transscleral drug delivery. It is available as a kit con- taining four components, human sealer protein concentrate (human fibrinogen and factor XIII), bovine fibrinolysis in- hibitor solution, human thrombin, and calcium chloride so- lution. After multiple heating and reconstitution steps, sealer protein and thrombin solutions are each loaded into a sy- ringe and then inserted into a Duploject System. The Du- ploject System is a two-syringe clip with a common plunger that forces equal volumes of the two solutions through a join-

Route of

TABLE 1. REPORTED OCULAR AND SYSTEMIC CARBOPLATIN CONCENTRATIONS AT 1–2 H
POSTDOSE IN MULTIPLE SPECIES FOLLOWING VARIOUS ROUTES OF ADMINISTRATION
Reported carboplatin concentrations (mcg/mL)

administration Species Dose Aqueous Vitreous Plasma

Intravenous Rabbit39 18.7 mg/kg NR ~332 ~6223 (1 h)
NR ~685 ~3452 (2 h)
Rabbit43 25 mg/kg NR ~0.1–0.3 NR
Primate42 560 mg/m2 ~6 ~0.3 ~30
Human11 560 mg/m2 ~5 ~4 NR
Periocular in BSS Rabbit5 ~11.25 mg ~18 ~1.8 ~1.6
(~0.8)a
Subconjunctival Rabbit39 5 mg NR ~4560 (1 h) ~182 (1 h)
NR ~3305 (2 h) ~327 (2 h)
Iontophoresis Rabbit39 5 mg NR ~1575 (1 h) ~261 (1 h)
Peribulbar Primate42 10 mg NR
~2 ~1655 (2 h)
~2.4 ~178 (2 h)
~0.9
Periocular through Rabbit5 ~11.25 mg ~57 ~12 ~7.4
fibrin sealant (~4)a
NR, not reported.
aConcentration reported in the contralateral, untreated eye.

ing piece and mixes them in the needle. Once mixed, the thrombin cleaves the fibrinogen into fibrin monomers, which rapidly polymerize to form a clot at the injection site.† Sev- eral investigators have incorporated carboplatin into the above described reconstitution process with varying con- centrations to prepare a final carboplatin in fibrin sealant for- mulation.5,18,27
While evaluating carboplatin in balanced salt solution (BSS) versus fibrin sealant preparations, Simpson and col- leagues5 demonstrated that formulation has profound influ- ence on the ability to sustain intraocular therapeutic con- centrations of carboplatin, mainly due to the property of fibrin sealant slowly releasing the drug from its matrix. Par- due and colleagues18 characterize four key features of fibrin sealant that make it an ideal depot formulation for the trans- scleral delivery of carboplatin. These features include: (1) the clot is formed on the scleral surface focusing drug delivery at a particular interface; (2) fibrin sealant is a semisolid medium for drug delivery, allowing the incorporation of the anhydrous form of a drug in concentrations in excess of its solubility limit, thereby providing more drug in a smaller volume of vehicle over an extended time frame; (3) im- munogenicity and foreign body reactions are minimized with the use of human protein in the fibrin sealant; and (4) fibrin sealant is a natural controlled-release delivery system, since it is degraded through fibrinolysis over time.
The use of fibrin sealant products as drug-delivery devices is not unique to transscleral chemotherapy.29 Several recent studies investigated the use of fibrin sealant for a variety of targeted delivery approaches, including local drug delivery in head and neck cancers.30 intrapleural chemotherapy de-

livery in mesothelioma,31 perivascular delivery of losartan for arterial injury,32 and antibiotic delivery in burn patients.33 Further, the use of fibrin sealant products has been investi- gated for transscleral delivery of antibiotics, corticosteroids, methotrexate, and growth factors.34–36

Recent investigations of carboplatin in fibrin sealant for retinoblastoma
Several investigators have recently published in vitro/vivo characterizations of carboplatin in fibrin sealant for con- trolled release transscleral delivery (Table 2).5,18,27 The first report of transscleral diffusion of carboplatin, using fibrin sealant, was published in 2002 by Simpson and colleagues5 Both in vitro characterization of the permeability of carbo- platin in fibrin sealant or BSS formulations in human eye bank sclera (n = 10) as well as in vivo comparisons in Dutch Belted rabbits were performed (n = 44).
The in vitro portion of the experiment utilized a perfusion apparatus with carboplatin placed in either vehicle on the epis- cleral side and constant perfusion with BSS simulating sink con- ditions on the choroidal side. Serial perfusate was collected and carboplatin concentrations measured with atomic absorption spectroscopy. The concentration-time profiles for carboplatin in fibrin sealant and BSS demonstrate that fibrin sealant deliv- ery decreases the peak concentration received on the choroidal side of the sclera but provides a more consistent concentration throughout the sampling time frame. The investigators report that the in vitro portion of the experiment demonstrates that carboplatin in fibrin sealant releases from the formulation and permeates across the sclera more slowly than in BSS.

TABLE 2. AVAILABLE STUDIES EVALUATING CARBOPLATIN IN FIBRIN SEALANT FOR RETINOBLASTOMA
Study Design highlights Outcome

Simpson5 In vitro perfusion of CPT across CPT releases from fibrin sealant
human sclera in fibrin sealant formulation and permeates across
versus BSS sclera more slowly than in BSS.
In vivo measurement in rabbits of CPT delivered through fibrin sealant
ocular tissue concentrations of CPT provides higher concentrations in
after subconjunctival injection of the exposed sclera at 48 h versus
CPT in fibrin sealant versus BSS (n = 44) BSS. CPT in fibrin sealant provides
less exposure to the untreated eye.
Pardue18 In vivo retinal function assessments Transient reduction in the dark-
with ERG and histopathology of adapted b-wave amplitudes was
enucleated eyes in rabbits (n = 14) noted in 2 of 5 rabbits on Day 2.
after subconjunctival injection of Histopathologic evaluation revealed
CPT in fibrin sealant or BSS and normal structure in all eyes treated
controls with CPT at the doses used
in the study.
Van Quill27 In vivo randomized, controlled Complete or near complete
efficacy trial in transgenic mice intraocular tumor regression in 10 of
using placebo, low-dose, or high- 11 eyes (91%) was achieved with low-
dose CPT in fibrin sealant (n = 33) dose CPT without histologic
evidence of toxicity. High-dose CPT
with a similar reduction in mean
tumor burden, yet severe toxicity
noted
BSS, balanced salt solution; CPT, carboplatin; ERG, electroretinogram.

†US Package Insert for Tisseel VH (two-component fibrin sealant vapor heated). Baxter Healthcare Corporation (Deerfield, IL), February 2000.

The in vivo portion of the study by Simpson and col- leagues5 examined choroid and vitreous tissue concentra- tions of carboplatin after a subconjunctival injection of car- boplatin in fibrin sealant or BSS in rabbits. Injection volumes were 300 μL with carboplatin concentrations of 37.5 and 10 mg/mL in fibrin sealant and BSS, respectively. Ocular tissue concentrations were measured after enucleation at 1.5 h, 48 h, and 2 weeks after drug administration. Since injection con- centrations were different in each vehicle, ocular tissue con- centrations were normalized to an initial injection concen- tration of 10 mg/mL to make comparisons between the two groups. The investigators concluded that carboplatin deliv- ered through fibrin sealant provided higher concentrations at the scleral surface at 48 h versus a BSS formulation. In ad- dition, the data demonstrated that fibrin sealant provides less exposure to the untreated eye.
Simpson and colleagues5 demonstrated that normalized carboplatin concentrations in the choroid and vitreous are comparable at 48 h and 2 weeks in both fibrin sealant and BSS treatment groups. An ideal sustained release formula- tion should provide therapeutic concentrations over the pro- posed dosing interval. As such, the goal would be to create a formulation that provides higher drug concentrations at later time points in the dosing interval than the immediate release preparation—in this case, the BSS formulation. Car- boplatin choroid and vitreous concentrations achieved through fibrin sealant are consistently higher than when for- mulated in BSS simply due to the delivery of a higher dose to a smaller surface area of sclera. This is the unique feature of fibrin sealant allowing for a reservoir of drug in excess of its solubility limit delivered in a focal area. However, the comparable normalized concentrations at 48 h and 2 weeks in these tissues suggest that there is room for improvement in the carboplatin in fibrin sealant formulation. Collectively, these data suggest that fibrin sealant may provide focal de- livery of carboplatin across the sclera. Additional formula- tion permutations with in vitro dissolution profiling would be beneficial next steps in formulation development. Identi- fying the specific formulation attributes that provide sus- tained therapeutic carboplatin concentrations over the dos- ing interval is essential for the development of a truly sustained release delivery mechanism.
Pardue and colleagues18 subsequently reported retinal function assessments in rabbits by using electroretinographic (ERG) and histopathologic examinations of enucleated eyes after subconjunctival injections of carboplatin in fibrin sealant (n = 5), carboplatin in BSS (n = 5), fibrin sealant alone (n = 2), and BSS alone (n = 2). All carboplatin treated rabbits received 300-μL doses containing approximately 25 mg/mL of carboplatin in fibrin sealant or 12 mg/mL of car- boplatin in BSS. Serial ERGs in light- and dark-adapted con- ditions were performed at baseline and 2 days and at 1, 2, and 3 weeks postdrug administration. Histopathologic as- sessments were conducted at 3 weeks. Pardue and colleagues reported transient reductions in the dark-adapted b-wave amplitudes noted 2 days post-treatment for carboplatin in fi- brin-sealant-treated eyes in 2 of 5 rabbits. Histopathologic evaluation under light microscopy revealed normal structure in all eyes treated with carboplatin at the doses used in the study. The investigators concluded that despite the slight at- tenuation noted on ERG, no effects on retinal structure or function were appreciated at the end of the study. They con-

trasted their findings with previously reported retinal tox- icity after intravitreal carboplatin injections and suggested that the drug delivery modality may explain the decreased toxicity.
More recently, Van Quill et al.27 reported the results of a randomized, controlled trial in mice to determine the effi- cacy of subconjunctival carboplatin in fibrin sealant in the treatment of transgenic murine retinoblastoma. Three groups of 11 LHβ-Tag transgenic mice were treated with a single 30-μL injection of fibrin sealant with low dose carbo- platin (0.66 mg), high dose carboplatin (1.23 mg), or placebo. Mice were sacrificed on Day 22 of treatment, and the eyes were evaluated for histopathology. The primary outcome measure was mean tumor burden per eye in each group. The best therapeutic results were seen in eyes treated with low- dose carboplatin in fibrin sealant, where complete or near complete intraocular tumor regression was achieved in 10 of 11 eyes (91%) without associated histologic evidence of tox- icity. The high dose group had a similar reduction in mean tumor burden; however, 50% of eyes in this group demon- strated histopathologic evidence of severe toxicity.
Interestingly, Gorodetsky et al.37 reported detailed in vitro experiments confirming the integrity of carboplatin drug product in the fibrin milieu. The investigators noted that car- boplatin actually increased the rate of clot formation (i.e., thrombin-induced fibrin clotting time was decreased) in a concentration-dependent manner. This characterization of the carboplatin-fibrin interaction is critical for our under- standing of optimizing formulation development, in that it quantitatively describes the release characteristics of carbo- platin from the fibrin matrix delivery system.
The above studies pave the foundation for the future in- vestigation of transscleral delivery of carboplatin in fibrin sealant to intraocular lesions in retinoblastoma. The current literature suggests that periocular carboplatin in aqueous so- lution and fibrin sealant permeates across the sclera intact and can modulate tumor growth and treat vitreous seeds. Sustained vitreous concentrations of carboplatin have been demonstrated in vivo by impregnating fibrin sealant with the drug and placing the depot formulation on the sclera. Fibrin sealant has been shown to concentrate periocular carboplatin delivery in a focal area on the sclera, thereby minimizing both periocular and systemic exposure.

Future directions for optimizing carboplatin in fibrin sealant
Fibrin sealant, a semisolid medium for drug delivery, al- lows incorporation of the anhydrous form of carboplatin in concentrations in excess of its aqueous solubility limit of 14 mg/mL. The formulation provides a larger amount of drug in a smaller volume and permits sustained drug delivery by using natural fibrinolysis from the fibrin matrix of the car- boplatin reservoir. Given these unique features, the approach of combining a fibrin sealant with a chemotherapeutic agent appears promising for targeted, sustained drug delivery to the posterior segment of the globe. However, there are sev- eral important issues that remain unanswered and warrant further investigation. The most suitable formulation of car- boplatin in fibrin sealant remains unknown. That is, the de- tails of when and how to add the carboplatin to the fibrin sealant during the mixing process has not been fully opti-

mized. In addition, the ideal dose of carboplatin in fibrin sealant, both for formulation stability as well as therapeutic utility, has not been confirmed. These dose and formulation details are essential for optimal carboplatin delivery to mit- igate tumor burden with minimal toxicity.
Dose selection is often controversial, especially for agents with limited exposure-response data, such as antineoplastics (where doses are often based on maximum tolerated doses, and there is limited or no therapeutic index between efficacy and toxicity profiles). Further, there are sparse data regard- ing optimal drug exposure for localized drug delivery. Some key principles governing dose selection in this case include: providing sufficient and sustained concentrations of drug in- traocularly to suppress or regress tumor growth; limiting drug exposure systemically and to unaffected ocular tissue; and working within volume constraints for periocular de- livery especially in children (maximum volume of aqueous solution reported for periocular delivery is 2 mL).
In vitro pharmacodynamic and efficacy data in models of retinoblastoma provide a modest framework for targeting optimal concentrations at the site of tumor. Perhaps the most useful dose-selection guidance available is the collective ex- perience of specialists in the field of retinoblastoma and the limited published case reports describing the efficacy and toxicity of carboplatin administered periocularly in vivo. Hayden and colleagues describe studies using repeated car- boplatin doses in the range of 30–300 μg every 72 h in mice and 5-mg single subconjunctival injections in rabbits.38,39 With clinical experience in retinoblastoma patients, the pe- riocular carboplatin dose reported is approximately 20 mg.9,40,41 The collective information suggests that the doses studied to date are highly constrained by the volume that can be administered periocularly and by the aqueous solu- bility of carboplatin. Despite evidence of tumor regression, case reports indicate local toxicities in humans at the 20-mg dose, including ocular motility changes and ischemic optic neuropathy.40,41 Whether these changes are related to car- boplatin or to the periocular technique is controversial. Schmack and colleagues suggest that the toxicity associated with periocular administration of carboplatin may be mod- ifiable by using refined focal techniques of drug adminis- tration, such as application through fibrin sealant.40 The use of fibrin sealant as both a targeted drug administration de- vice and as a drug reservoir for sustained concentrations at the site of application appears promising in this regard.
Other features to explore include the potential for dose- dependent drug release from the fibrin sealant as well as po- tential differences in drug release with using various fibrin sealant products. Maintaining therapeutic intraocular con- centrations of carboplatin is both a function of the rate of drug release from the fibrin sealant governed by in vivo fibrinolysis and drug clearance from the vitreous. To date, there is limited understanding of how well our in vitro and animal models of drug delivery mirror this critical aspect of drug release from a fibrin matrix in human eyes with retinoblastoma. In addition, the relative safety and efficacy of transscleral administration of carboplatin in fibrin sealant in patients afflicted with retinoblastoma, compared to cur- rently available treatment modalities has not been examined. Although superior tumor control with fibrin sealant is sug- gested by the transgenic retinoblastoma studies, it is not cer- tain that fibrin sealant delivery will provide greater effec-

tiveness in human cases with subretinal and vitreous seed- ing. This is due to the limitations of the transgenic mouse model, which does not fully mimic the human clinical sce- nario for tumor seeding. Finally, whether carboplatin or an- other antineoplastic agent would be the best agent for this approach is unknown.

Fibrin sealant for retinoblastoma: Where are we?
The two drivers for the application of fibrin sealant for retinoblastoma are: (1) there is no consistently successful therapy to treat retinoblastoma with extensive subretinal or vitreous seeding, (2) the toxicity of systemic chemotherapy might be avoided with the local delivery of effective phar- macologic agents in appropriate therapeutic concentrations. Periocular carboplatin has transient efficacy in the setting of subretinal and vitreous seeds, but it is rarely curative. Un- fortunately, its repeated use is often limited by local side ef- fects.

Conclusions
Delivering carboplatin in fibrin sealant has the potential to provide sustained therapeutic concentrations over a longer duration in the target tissue than current clinical ap- proaches. The safety and efficacy of an optimized formula- tion of carboplatin in fibrin sealant in patients has not been proven. At present, fibrin sealant offers the potential for bet- ter control and, possibly, cures for some patients with ad- vanced retinoblastoma. Further investigation is warranted to characterize the efficacy and toxicity profile of this novel drug delivery modality in pediatric patients with retinoblas- toma.

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Received: September 21, 2007
Accepted: June 9, 2008

Reprint Requests: David H. Abramson Ophthalmic Oncology Service
Memorial Sloan Kettering Cancer Center
1275 York Avenue New York, NY 10021 NSC 241240