April 2010
Volume 51, Issue 4
Free
Physiology and Pharmacology  |   April 2010
Episcleral Implants for Topotecan Delivery to the Posterior Segment of the Eye
Author Affiliations & Notes
  • Angel M. Carcaboso
    From the Department of Pharmacology,
  • Diego A. Chiappetta
    The Group of Biomaterials and Nanotechnology for Improved Medicines (BIONIMED), the Department of Pharmaceutical Technology, and
    the National Science Research Council, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina;
  • Javier A. W. Opezzo
    From the Department of Pharmacology,
  • Christian Höcht
    From the Department of Pharmacology,
  • Adriana C. Fandiño
    the Departments of Ophthalmology and
  • J. Oscar Croxatto
    the Fundación Oftalmológica Argentina Jorge Malbrán, Buenos Aires, Argentina; and
  • Modesto C. Rubio
    From the Department of Pharmacology,
    the National Science Research Council, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina;
  • Alejandro Sosnik
    The Group of Biomaterials and Nanotechnology for Improved Medicines (BIONIMED), the Department of Pharmaceutical Technology, and
    the National Science Research Council, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina;
  • David H. Abramson
    the Department of Ophthalmic Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York.
  • Guillermo F. Bramuglia
    From the Department of Pharmacology,
  • Guillermo L. Chantada
    Hemato-Oncology Service, Hospital J.P. Garrahan, Buenos Aires, Argentina;
  • Corresponding author: Guillermo L. Chantada, Hemato-Oncology Service, Hospital J.P. Garrahan, Combate de los Pozos 1881, C1245AAL, Buenos Aires, Argentina; gchantada@yahoo.com
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2126-2134. doi:10.1167/iovs.09-4050
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Angel M. Carcaboso, Diego A. Chiappetta, Javier A. W. Opezzo, Christian Höcht, Adriana C. Fandiño, J. Oscar Croxatto, Modesto C. Rubio, Alejandro Sosnik, David H. Abramson, Guillermo F. Bramuglia, Guillermo L. Chantada; Episcleral Implants for Topotecan Delivery to the Posterior Segment of the Eye. Invest. Ophthalmol. Vis. Sci. 2010;51(4):2126-2134. doi: 10.1167/iovs.09-4050.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose.: Intravenous or periocular topotecan has been proposed as new treatment modality for patients with advanced intraocular retinoblastoma, but systemic topotecan lactone exposure induced by both approaches may cause toxicity. The purpose of this study was to develop a topotecan-loaded ocular delivery system to minimize systemic exposure and achieve selective transscleral penetration.

Methods.: Biocompatible polymer implants containing low (0.3 mg) or high (2.3 mg) topotecan load were manufactured and characterized in vitro. Adrenaline (500 μg) was coloaded to induce local vasoconstriction in vivo in 2 of 4 animal groups. Implants were inserted into the episclera of rabbits, and topotecan (lactone and total) concentrations in ocular tissues and plasma were determined over a period of 48 hours.

Results.: In vitro, implants released 30% to 50% of the loaded drug within 48 hours and 45% to 70% by day 10. In vivo, topotecan lactone was highly accumulated in locally exposed ocular tissues (ranging from 105 to 106 ng/g in sclera and choroid and 102 to103 ng/g in retina) over 48 hours with all the formulations studied. Low vitreous topotecan lactone levels (approximately 5 ng/mL) were found in animals receiving concomitant local vasoconstriction and high load implants. Topotecan lactone concentrations in plasma and in contralateral eyes were minimal or undetectable as a marker of tissue selectivity of the proposed strategy.

Conclusions.: These studies may contribute to improving the efficacy and safety of chemotherapy treatments for retinoblastoma and may support the role of the local vasculature and tissues promoting drug clearance and local accumulation during transscleral drug delivery.

The systemic treatment of advanced intraocular retinoblastoma with vitreous seeding represents a challenge because drug penetration to the avascularized vitreous is limited by the presence of the blood-retinal barrier. 1,2 The standard of care for these patients in most centers around the world includes tumor chemoreduction with intravenously administered carboplatin, etoposide, and vincristine followed by consolidation with local therapy or external beam radiotherapy. 3 However, the discouraging results in terms of eye preservation in this subgroup motivated the clinical testing of novel treatment modalities, such as carboplatin given periocularly, which is currently part of the ongoing Children Oncology Group trial for eyes with vitreous seeding. 4  
It is postulated that periocularly administered anti-retinoblastoma drugs may achieve better ocular penetration while minimizing systemic drug exposure in the treatment of these tumors. 58 Carboplatin was the first drug to be used by the periocular route in retinoblastoma, but its use is being reconsidered because of local toxicity, including optic nerve atrophy, 7,9 and its limited ability to be curative. 10 However, there are few drugs other than carboplatin that have been characterized for periocular use in this disease, and identifying other candidates is a primary challenge for clinicians. Topotecan (TPT), a camptothecin derivative, has been studied as an alternative for the treatment of retinoblastoma. After the report of single cases from other groups, we reported encouraging activity in a series of children with extraocular disease treated under compassionate basis with intravenously administered TPT. 11,12 Subsequent studies in tumor-bearing animal models showed that the association of TPT and carboplatin results in a very active drug combination, with potentially synergistic activity. 13 However, hematopoietic toxicity limits the clinical use of these two agents concomitantly by the intravenous (IV) route. 14 One way of potentially taking advantage of this combination and reducing its toxicity would be to give one agent by the IV route and the remaining one by the periocular route, therefore reducing the overall systemic exposure in patients while achieving active drug levels in the eye. The response rate to intravenously administered TPT associated with periocular carboplatin in previously untreated patients is being investigated in a phase III study sponsored by St Jude Children's Research Hospital (Memphis, TN). In the phase III study, some patients receive periocular carboplatin concomitantly (clinicaltrials.gov identifier: NCT00186888). TPT is also being tested intravenously as second-line treatment for relapsed or resistant retinoblastoma. Because of the potentially severe local toxicity associated with periocular carboplatin, we studied periocular TPT in phase I trials 8 to consider its clinical use alone or in association with IV carboplatin for the treatment of retinoblastoma. 
It is well known that local drug delivery to the vitreous is hampered by the innate local ocular barriers that protect the eye from potentially toxic xenobiotics. 15 Confirming this point, in a study of the ocular pharmacokinetics of periocular TPT in a non–tumor-bearing animal model, we observed profuse ocular barrier activity after the periocular administration of TPT that limited penetration to the vitreous. 16 Specifically, we found that a significant proportion of the periocularly administered TPT reached the vitreous through the systemic circulation, probably because of rapid orbital clearance of the drug. In a phase I clinical trial of periocular TPT in children with relapsed or resistant intraocular retinoblastoma, we observed a low toxicity profile for this new treatment modality and were able to characterize TPT plasma concentrations in these patients. 8 We found that the systemic drug exposure was significant, though lower than the exposure induced in the same patients after IV administration of similar dosages. Based on our preclinical and clinical results for periocular TPT we inferred that, though promising, this strategy may remain relatively inefficient in the clinical setting as a way of delivering TPT by the transscleral route. Therefore, we aimed to design a method for a more selective periocular administration of TPT that would allow for the safe use of this drug alone or in association with carboplatin for the treatment of retinoblastoma with vitreous seeding. Because this concern is also applicable to other drugs, it has motivated researchers to propose several strategies to maximize drug penetration into the eye and to achieve a prolonged exposure, 15 which could be especially important for S-phase specific drugs such as TPT. 17 Recently, intense research has been focused on the design of a biocompatible polymer-based drug delivery system (DDS) to release drugs in the orbital space, primarily for the delivery of drugs for the treatment of other (nontumor) ocular conditions. 18 Based on our previous results, we hypothesized that a DDS implanted locally could saturate the adjacent eye barriers and achieve selective drug delivery to the vitreous of the treated eye while minimizing systemic exposure. Thus, in this study we developed and characterized, in vitro and in vivo, a TPT-loaded “double-faced” polymeric DDS composed of a drug-free face designed to isolate the drug reservoir from the orbital conjunctival tissues and a drug-loaded face to concentrate TPT in the adjacent scleral surface when inserted episclerally. We specifically studied the ability of the DDS, in combination with a pharmacologic strategy to constrain local blood vessels, to achieve high and long-term local drug accumulation in ocular tissues, which could enhance the effect of TPT in retinoblastoma. 
Materials and Methods
Commercial TPT (Hycamtin, containing 4 mg TPT, 48 mg mannitol, and 20 mg tartaric acid) and TPT standard were donated by Glaxo-SmithKline (Buenos Aires, Argentina). At physiological pH, the active form of TPT, a closed lactone ring (LTPT; stable at acidic pH), is reversibly hydrolyzed to an inactive carboxylate form. 19  
Poly(ε-caprolactone) polymer (PCL; molecular weight 14,000 Da; Tm 60°C) and adrenaline were purchased from Sigma-Aldrich (Buenos Aires, Argentina). The remaining reagents and chromatography solvents were of analytical grade. 
Implant Manufacture and Characterization
TPT implants were manufactured by a simple, fast, and reproducible melt-molding-compression method using a stainless steel mold (7-mm diameter). 20 A diagram of the procedure is shown in Figure 1. Depending on the proportions of the matrix polymer powder and active principles, different implants were manufactured. For the initial experiments, we developed implants with a low load (LL) of TPT. We subsequently explored the effect of including a vasoconstrictor (adrenaline) in the PCL matrix and increasing drug load, developing implants named low load with vasoconstrictor (LLv), high load (HL), and high load with vasoconstrictor (HLv; Table 1). When included in the formulation, adrenaline and PCL powders were blended. Blank drug-free implants containing identical excipient amounts were also produced, as were pure PCL implants. 
Figure 1.
 
Diagram of the melt-molding compression technique developed to produce one-side coated TPT-loaded PCL implants. Briefly, to produce a PCL coating (drug-free layer of pure polymer), grounded PCL is introduced into the bottom of a stainless steel mold composed of a static platform and a matrix (a cross-section is represented). A homogeneous mixture of PCL and TPT (with hydrophilic excipients) is poured onto the pure PCL layer, and the mold plunger is mounted (left). The filled mold is exposed to a 1.7 kg/m2 pressure and heated (70°C, 1 hour) to melt the polymer and entrap the drug and excipients (middle). The system is cooled (4°C, 0.5 hour) to allow for the solidification of the implants. Implant with dimensions (right). P, pressure.
Figure 1.
 
Diagram of the melt-molding compression technique developed to produce one-side coated TPT-loaded PCL implants. Briefly, to produce a PCL coating (drug-free layer of pure polymer), grounded PCL is introduced into the bottom of a stainless steel mold composed of a static platform and a matrix (a cross-section is represented). A homogeneous mixture of PCL and TPT (with hydrophilic excipients) is poured onto the pure PCL layer, and the mold plunger is mounted (left). The filled mold is exposed to a 1.7 kg/m2 pressure and heated (70°C, 1 hour) to melt the polymer and entrap the drug and excipients (middle). The system is cooled (4°C, 0.5 hour) to allow for the solidification of the implants. Implant with dimensions (right). P, pressure.
Table 1.
 
Characteristics of the TPT-Loaded PCL Implants
Table 1.
 
Characteristics of the TPT-Loaded PCL Implants
Coating Matrix
Formulation (TPT load, mg) PCL (mg) PCL (mg) Hycamtin* (mg) TPT Powder† (mg) Adrenaline (mg) Added Exciplents‡ (mg) Final Weight§ (mg)
LL (0.3) 20 95 5 0 0 0 117 ± 7
LLv (0.3) 20 95 5 0 0.5 0 122 ± 4
HL (2.3) 50 45 5 2 0 0 93 ± 8
HLv (2.3) 50 45 5 2 0.5 0 98 ± 4
Blank (0) 50 45 0 0 0.5 5 94 ± 5
Scanning electron microscopy (SEM; JSM-35C; JEOL, Tokyo, Japan) was performed to characterize the surface and matrix structure of the implants before and after in vitro incubations. 
In Vitro Release Assay
Individual LLv and HLv implants (n = 3 each) were placed in 100-mL glass flasks containing phosphate-buffered solution (PBS; 50 mL, pH 7.4) and were incubated at 37°C. After 1 hour, the complete content of the flasks was collected and replaced with fresh PBS solution. This sampling process was repeated daily for 10 days. The total TPT concentration was measured in every collected sample by HPLC with fluorometric detection, as previously described. 16  
To characterize the long-term stability of the PCL matrix, the swelling process (absorption of fluids) and the weight loss of the implants, blank implants and pure PCL implants (n = 3 each) were incubated in the same conditions as for the in vitro release assay. At 1, 7, 21, and 28 days, the implants were collected and carefully wiped to remove superficial liquids, and the wet weight was recorded. After a drying period of 24 hours at 37°C, the dry weight was recorded. 
In Vivo Studies and Sampling Procedures
New Zealand albino rabbits (1.8–2.2 kg) used for this study were handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Four groups of rabbits (LL, LLv, HL, HLv) were used to study ocular and plasma TPT concentrations after they were exposed to TPT implants. A mixture of ketamine (37.5 mg/kg) and xylazine (5 mg/kg) was used to anesthetize the animals, and the DDS was implanted as shown in Figure 2
Figure 2.
 
Surgical insertion of the DDS. (a) The implant is introduced through the conjunctival incision in the superior temporal episcleral zone of the right eye, with the uncoated side facing the sclera. (b) The incision is sutured with 5-0 sutures. (c) Final appearance of the implanted eye.
Figure 2.
 
Surgical insertion of the DDS. (a) The implant is introduced through the conjunctival incision in the superior temporal episcleral zone of the right eye, with the uncoated side facing the sclera. (b) The incision is sutured with 5-0 sutures. (c) Final appearance of the implanted eye.
To assess the suitability of LL implants (DDS containing 0.3 mg TPT) to achieve selective TPT ocular delivery and low systemic exposure, a first cohort of animals received LL implants (n = 4). After evaluating the results showing low vitreous levels of TPT, a second cohort (n = 5) received LLv implants. In these two groups, a terminal 100-μL vitreous sample was aspirated with a 25-gauge needle in the anesthetized animals 5 hours after the insertion of the DDS, and blood samples were obtained through the ear vein at 1, 2, and 4 hours. Total TPT and LTPT were determined in vitreous and plasma, as previously described. 16 To study longer term (24-hour) exposure in ocular tissues, three more rabbits were similarly exposed to LLv implants, and their plasma, vitreous, and ocular tissues were analyzed for total TPT and LTPT 24 hours later. These rabbits were anesthetized, and the implants removed; 30 minutes later, the rabbits were euthanatized with an IV or an intracardiac bolus injection of sodium thiopental (100 mg in 5 mL saline). Eyes were enucleated, and the right eyes were divided in two equal halves. One half was exposed (i.e., in contact with the DDS), and the other half was nonexposed. Left eyes were excised in the same way, and one of the halves was processed. All the excised tissues were washed in ice-cold PBS, and the sclera, choroid, and retina were immediately dissected. The excess PBS in the tissues was absorbed with a sponge, and tissues were weighed. To quantitatively extract TPT from the tissues, the tissues were exposed to two freeze-thaw cycles in 100 μL distilled water, and finally 400 μL cold methanol were added. Supernatants were analyzed for total TPT and LTPT. 
To further characterize the transscleral penetration process of TPT and to achieve high and long-term (24- to 48-hour) TPT concentrations in the vitreous, animal groups HL and HLv (n = 6 and n = 8 rabbits, respectively) receiving high-load implants (DDS containing 2.3 mg TPT; Table 1) were included in the experiments. To evaluate the transscleral release kinetics of the drug after implanting the DDS in these two groups, two methods were used. First, to characterize the early vitreous penetration profile of the drug released from the DDS, a microdialysis-based multiple-sampling schedule was performed. Second, a terminal vitreous sample was aspirated 24 or 48 hours later. During the microdialysis sampling period, TPT concentrations were assessed by means of the principle by which drugs in the targeted tissue cross the capillary-like semipermeable microdialysis probe continuously perfused with a drug-free physiological fluid, until equilibrium was reached. 21 For these experiments, 2 hours after the surgical insertion of the DDS, in a location 3 mm away from the limbus and 120° away from the implant site, the surface of the sclera was exposed by making an incision in the conjunctiva, and the microdialysis probe (Fig. 3) was inserted into the vitreous space through an incision made with a 25-gauge needle. A second probe was inserted into the contralateral eye in the same location. Both probes were fixed to the sclera with two vicryl 7-0 sutures (Fig. 3). Microdialysis membranes were then perfused with PBS (pH 7.4) at a flow rate of 0.5 μL/min using an infusion pump (KDS230; KD Scientific, Holliston, MA) and were left for 30 to 40 minutes to equilibrate with the vitreous before sample collection started. Dialysates were collected every 40 minutes over a period of 160 minutes, covering the 160- to 320-minute time range after the DDS implantation, and total TPT was analyzed. At the end of the sample collection, a concentrated TPT solution (500 ng/mL) was perfused through the probe to calculate the probe recovery by the retrodialysis method. 22 The recovery value obtained for the probes was 30% ± 9% and 28% ± 6% (mean ± SD for the HL and HLv groups, respectively), which allowed calibration of the probes to calculate the actual intravitreal TPT concentrations. At the end of the microdialysis study, probes were withdrawn, a 5-0 suture was used to close the sclera, and animals were allowed to recover. Either 24 or 48 hours later, a vitreous sample (100 μL) was aspirated with a 25-gauge needle and was analyzed for total TPT and LTPT. During the experiment, plasma samples were obtained through a jugular catheter at 0.25, 0.5, 1, 2, 4, 6, and 24 (or 48) hours after the insertion of the DDS. Plasma exposure to the drug was defined as the area under the concentration-time curve (AUC), calculated as previously published. 16 At the end of the 24- or 48-hour experiment, DDS was removed. Thirty minutes later animals were euthanatized, eyes were enucleated, and tissues were processed as detailed for total TPT and LTPT content. 
Figure 3.
 
Diagram of the ocular microdialysis concentric probe designed and manufactured in the laboratory. A 10-mm-long microdialysis membrane (outer diameter [OD] 200 μm, 10,000 molecular weight cutoff; Asahi Medical Co., Japan), an inlet plastic tube (inner diameter, 500 μm), and an outlet silica tube (OD 145 μm) were used to build the probes. Epoxy adhesive (gray dots) was used to glue the components and to seal the end of the dialysis membrane. The probe was mounted in a nitrocellulose semirigid support, which allowed fixing the canula to the sclera with two sutures of 7-0 silk.
Figure 3.
 
Diagram of the ocular microdialysis concentric probe designed and manufactured in the laboratory. A 10-mm-long microdialysis membrane (outer diameter [OD] 200 μm, 10,000 molecular weight cutoff; Asahi Medical Co., Japan), an inlet plastic tube (inner diameter, 500 μm), and an outlet silica tube (OD 145 μm) were used to build the probes. Epoxy adhesive (gray dots) was used to glue the components and to seal the end of the dialysis membrane. The probe was mounted in a nitrocellulose semirigid support, which allowed fixing the canula to the sclera with two sutures of 7-0 silk.
In Vivo Release Assay
To study the ability of the DDS to release the drug in vivo, LLv implants were recovered from animals of the LLv group over a period of 7 days. To determine the amount of TPT remaining (not released) in the DDS, the polymer was dissolved in methylene chloride (2 mL), and TPT was extracted with distilled water (4 × 1 mL) and analyzed by HPLC. The difference between the initial TPT load and the unreleased TPT was the amount of drug released in vivo. 
Histopathology Evaluation
The effect of HLv and blank implants on the exposed and contralateral eyes was assessed in vivo in 10 rabbit eyes. Briefly, the surgical procedure used in the studies was followed to expose n = 3 eyes to HLv implants and n = 3 eyes to blank implants. Control eyes contralateral to the implanted eyes were also studied (n = 4). After surgery, the animals were recovered and housed normally until they were killed 48 hours later, and eyes were enucleated. Histology evaluation was performed as previously described. 16  
Results
Solid products with convex surfaces, 7 mm in diameter and 2.5 to 3 mm in thickness, were obtained (see photograph in Fig. 1). Implants were characterized by an intense yellow color on the scleral side from TPT and a white, glossy appearance in the orbital side coated with pure PCL. According to SEM, the scleral surfaces of the implants were porous, and the orbital surfaces were smooth (Figs. 4a, 4b). Hydrophilic excipients and drugs formed needle-looking crystal structures into the original implant matrix, which disappeared after 10 days of the in vitro release study (Figs. 4c–e). 
Figure 4.
 
Scanning electron micrographs of LLv implants. (a) Scleral side of an intact LLv implant. (b) Orbital side of the same implant. (c) Image of a fractured implant, showing the porous scleral side (top) and the smooth orbital side (bottom). (d) Detail of the inner implant matrix showing fine needle-like crystals formed by the hydrophilic molecules (TPT + excipients) loaded into the implant. (e) Detail of the matrix after 10 days of incubation in PBS at 37°C; absence of crystals was observed. Original magnification: (a, b) 56×; (c) 24×; (d, e) 2000×. Scale bar: (a, b) 100 μm; (c) 1 mm; (d, e) 10 μm.
Figure 4.
 
Scanning electron micrographs of LLv implants. (a) Scleral side of an intact LLv implant. (b) Orbital side of the same implant. (c) Image of a fractured implant, showing the porous scleral side (top) and the smooth orbital side (bottom). (d) Detail of the inner implant matrix showing fine needle-like crystals formed by the hydrophilic molecules (TPT + excipients) loaded into the implant. (e) Detail of the matrix after 10 days of incubation in PBS at 37°C; absence of crystals was observed. Original magnification: (a, b) 56×; (c) 24×; (d, e) 2000×. Scale bar: (a, b) 100 μm; (c) 1 mm; (d, e) 10 μm.
Implant weights and drug loads are shown in Table 1. The drug could be completely recovered as LTPT by solvent extraction after the implant was dissolved in methylene chloride, confirming the stability of the drug through the manufacturing process (data not shown). 
In Vitro and In Vivo Release from TPT DDS
In vitro release profiles of LLv and HLv implants are presented in Figure 5. The release rate was biphasic (faster during the first 2 days and slower thereafter). During the first 2 days, 150 μg TPT was released from LLv implants and 700 μg was released from HLv implants (Figs. 5a, 5b), accounting for 50% and 30% of the total drug load, respectively. Afterward, a slower release rate between days 2 and 10 was observed, reaching 70% and 45% of the total loaded dose, respectively. The adrenaline release profile was similar (Fig. 5c). Although the instability of adrenaline at pH 7.4 precludes obtaining accurate data, the detection of adrenaline traces during the whole in vitro experiment confirms its entrapment and a sustained release trend. The shape and size of the implants was fully conserved during the 10 days of the in vitro study (Fig. 5d) and after the in vivo studies (Figs. 5e, 5f). Disappearance of the yellow color from the scleral side of the implants throughout the in vitro and in vivo studies further evidenced drug release (Figs. 5d–f). In vivo release data from eight implants are displayed in Figure 5a, overlapping the in vitro release curve, as a sign of the in vitro/in vivo release correlation. The release in vivo was also confirmed by the imprinting of the released TPT (yellow area) on the surface of the sclera (Fig. 5g). In vitro water absorption by blank implants was 1.9% ± 0.2% at day 1 and 4.5% ± 0.6% at day 7. These values coincided with the weight loss, as displayed in Figure 5h (1.8% of original weight at day 1 and 4.5% at day 7). Pure PCL implants showed <2% weight loss after 28 days of incubation. 
Figure 5.
 
Drug release profile from TPT-loaded implants. (a) In vitro cumulative release from LLv implants (closed circles; mean ± SD of n = 3 values) and in vivo release values calculated as the difference between the initial TPT load and the unreleased TPT in recovered implants (open circles; individual values). (b) In vitro cumulative release from HLv implants (mean ± SD of n = 3 values). (c) Adrenaline in vitro cumulative release from LLv implants (mean ± SD of n = 3 values). (d) LLv implants scleral side before (left) and after (right) 10 days of in vitro release. (e) Scleral side of LLv implants as preimplanted (left) or recovered from rabbits at 8 hours, 3 days, and 6 days (left to right). (f) Orbital sides of the implants in (d). (g) Enucleated treated (left) and contralateral eyes devoid of conjunctival and muscular tissues of an albino rabbit 8 hours after the insertion of the DDS. (h) Weight loss (percentage of initial weight) of Blank implants and pure PCL implants after in vitro incubation (mean ± SD of three values).
Figure 5.
 
Drug release profile from TPT-loaded implants. (a) In vitro cumulative release from LLv implants (closed circles; mean ± SD of n = 3 values) and in vivo release values calculated as the difference between the initial TPT load and the unreleased TPT in recovered implants (open circles; individual values). (b) In vitro cumulative release from HLv implants (mean ± SD of n = 3 values). (c) Adrenaline in vitro cumulative release from LLv implants (mean ± SD of n = 3 values). (d) LLv implants scleral side before (left) and after (right) 10 days of in vitro release. (e) Scleral side of LLv implants as preimplanted (left) or recovered from rabbits at 8 hours, 3 days, and 6 days (left to right). (f) Orbital sides of the implants in (d). (g) Enucleated treated (left) and contralateral eyes devoid of conjunctival and muscular tissues of an albino rabbit 8 hours after the insertion of the DDS. (h) Weight loss (percentage of initial weight) of Blank implants and pure PCL implants after in vitro incubation (mean ± SD of three values).
In Vivo Experiments
Figure 6a displays TPT vitreous levels 5 hours after the insertion of low-loaded implants (LL and LLv groups). A statistically significant increase in TPT vitreous levels was found in the treated eye compared with the contralateral eye in both groups (P < 0.001; t-test). TPT vitreous levels into the treated eye were significantly higher for the LLv group compared with the LL group, whereas the penetration to the contralateral eye was low and similar in both groups. Plasma TPT levels were below 4 ng/mL at 1, 2, and 4 hours and below 1 ng/mL at 24 hours. LTPT in vitreous and plasma was below the detection limit (0.5 ng/mL) at all time points. 
Figure 6.
 
TPT vitreous and plasma concentrations in vivo. (a) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous humor aspirated from rabbits implanted in the right eye with LL (n = 4) and LLv implants (n = 5; *P < 0.01 compared with the ipsilateral eye in the LL group; t-test). (b) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous during the 160- to 320-minute period after the insertion of an HL implant in the right eye. Both eyes were sampled by microdialysis (HL group; n = 6). Midpoints of the sample collection time intervals are represented. (c) Total TPT levels (mean ± SD) in right and left vitreous during the 160- to 320-minute period after the insertion of an HLv implant in the right eye. Both eyes were sampled by microdialysis (HLv group; n = 8; *P < 0.05 compared with the left eye; Mann-Whitney rank sum test). (d) Plasma levels of total TPT in the HL and HLv groups. Time is represented in logarithmic scale to discern the early values (mean ± SD of 4–8 values; *P < 0.01 compared with the plasma level value for the HLv group at the same time point; t-test). (e) Total TPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; *P < 0.05 compared with HL and LLv groups; one-way ANOVA with Holm-Sidak multiple comparison; TPT levels in left vitreous were below the limit of detection). (f) LTPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; LTPT levels in left vitreous were below the limit of detection). N.D., not determined.
Figure 6.
 
TPT vitreous and plasma concentrations in vivo. (a) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous humor aspirated from rabbits implanted in the right eye with LL (n = 4) and LLv implants (n = 5; *P < 0.01 compared with the ipsilateral eye in the LL group; t-test). (b) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous during the 160- to 320-minute period after the insertion of an HL implant in the right eye. Both eyes were sampled by microdialysis (HL group; n = 6). Midpoints of the sample collection time intervals are represented. (c) Total TPT levels (mean ± SD) in right and left vitreous during the 160- to 320-minute period after the insertion of an HLv implant in the right eye. Both eyes were sampled by microdialysis (HLv group; n = 8; *P < 0.05 compared with the left eye; Mann-Whitney rank sum test). (d) Plasma levels of total TPT in the HL and HLv groups. Time is represented in logarithmic scale to discern the early values (mean ± SD of 4–8 values; *P < 0.01 compared with the plasma level value for the HLv group at the same time point; t-test). (e) Total TPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; *P < 0.05 compared with HL and LLv groups; one-way ANOVA with Holm-Sidak multiple comparison; TPT levels in left vitreous were below the limit of detection). (f) LTPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; LTPT levels in left vitreous were below the limit of detection). N.D., not determined.
Figure 6b shows total TPT vitreous levels in the HL group, as assessed by microdialysis sampling during the 160- to 320-minute time period after the insertion of the DDS. No differences between the implanted and the control eye were found at any of the time points in this study. In animals receiving HLv implants, however, a significantly higher penetration to the treated eye was found compared with the contralateral eye in the 220- to 300-minute time interval (Fig. 6c). When compared with the HL group, vitreous TPT levels in the HLv rabbits were higher at 260 and 300 minutes (P < 0.05, t-test). Microdialysis sampling did not allow for analyzing LTPT because lactone was converted to carboxylate during the 40-minute sample collection in pH 7.4 buffer. Therefore, additional experiments were performed in rabbits with HLv implants sampled by vitreous aspiration at 5 hours; 12% ± 3% LTPT was found in the vitreous total TPT amount (n = 3 rabbits). These experiments also allowed confirmation of comparable total TPT vitreous concentration values at 5 hours, as sampled by vitreous aspiration (13 ± 6 ng/mL) and microdialysis (9 ± 5 ng/mL). 
TPT plasma exposure was assessed in both HL and HLv groups for 48 hours (Fig. 6d). LTPT AUC could not be calculated because LTPT was not detectable in most of the plasma samples. In the few samples with detectable amounts of LTPT, concentrations were lower than 5 ng/mL. Total TPT plasma levels were similar in both groups, with the exception of the early 30-minute plasma sample, significantly higher in the HL group than in the HLv group. Total TPT plasma exposures (AUC0–48 h) were 112 ± 17 ng/h/mL and 179 ± 40 ng/h/mL for the HL and HLv groups, respectively (mean ± SE), with no statistically significant differences among them. 
At 24 hours, vitreous total TPT and LTPT levels in the implanted eye were higher in HLv rabbits than in HL and LLv rabbits (Figs. 6e, 6f); at 48 hours, total TPT and LTPT vitreous levels were still detectable in the implanted eyes of the HLv group. TPT was not detectable in the contralateral eye vitreous at 24 and 48 hours in any of the groups. 
Figure 7 shows total TPT and LTPT accumulation in the right eye half exposed to LLv, HL, and HLv implants for 24 or 48 hours compared with the nonexposed half of the right eye and with the left eye. Higher accumulations were observed in right-exposed tissues in all groups and at all time points compared with the right nonexposed and the left tissues. The statistical power to detect differences in TPT accumulation among tissues was limited by the variability of the data and the number of collected eyes (n = 3) for each condition studied. However, the trends (HLv>HL>LLv and right-exposed>right-nonexposed>left) were consistently repeated. The proportion of LTPT over the total TPT amount was close to 100% in the exposed areas, whereas the LTPT proportion in the contralateral eye was 50% or lower. 
Figure 7.
 
TPT accumulation (nanogram of TPT per gram of tissue) in the right eye half exposed to LLv, HL, and HLv implants for 24 or 48 hours compared with the right nonexposed eye half and the left eye. RE, right-exposed half; RNE, right-nonexposed half; L, left eye. Total TPT (ac) and proportion of LTPT over total TPT (df) were assessed in retina (a, d), choroid (b, e), and sclera (c, f). Mean ± SD of n = 3 values are represented. #, LTPT under limit of detection.
Figure 7.
 
TPT accumulation (nanogram of TPT per gram of tissue) in the right eye half exposed to LLv, HL, and HLv implants for 24 or 48 hours compared with the right nonexposed eye half and the left eye. RE, right-exposed half; RNE, right-nonexposed half; L, left eye. Total TPT (ac) and proportion of LTPT over total TPT (df) were assessed in retina (a, d), choroid (b, e), and sclera (c, f). Mean ± SD of n = 3 values are represented. #, LTPT under limit of detection.
Histopathology Findings
Histologic studies in eyes exposed for 48 hours to blank or HLv implants showed nonspecific signs of mild tissue inflammation (scleritis, orbital cellulitis, and uveitis) localized in the exposed area in 2 of 3 eyes exposed to blank implants and in 3 of 3 eyes exposed to HLv implants. HLv implants also induced inflammation in nonexposed areas of the implanted eye. Intraocular alterations in the retinal pigment epithelium, retina, vitreous body, anterior segment, and optic nerve were not observed. No signs of eye toxicity were observed in the contralateral eyes. 
Discussion
Topotecan is an appealing drug for the treatment of intraocular retinoblastoma, but it can cause severe systemic toxicity when administered intravenously at the dosages needed for optimal penetration to the eye. 23,24 Additionally, when administered by local periocular injection in animal models, transscleral diffusion of TPT is limited by systemic absorption. 16 Therefore, we sought to find innovative strategies to maximize transscleral delivery while minimizing systemic concentrations of TPT by using episcleral implants. We demonstrated that high drug concentration gradients induced by the localized release and by the inhibition of the local vasculature were able to saturate the local eye barriers and to increase vitreous concentrations of the drug. 
To achieve high local concentration gradients on the eye surface, coated polymer devices, fibrin-based matrices, and surgically attached reservoirs containing high concentrations of drug may be considered. 2528 For this study, we favored polymer implants geometrically designed to fit between the sclera and the orbital bone. Recent studies have shown that polyethylene devices tightly sutured to the sclera enhance drug diffusion through the indented region of the eye by maximizing contact with the ocular surface. 27 The thickness and convex surface design of our DDS allowed the indentation of the sclera and prevented local migration of the implant without the need for sutures (Fig. 4g). PCL polymer was used in the implant matrix given its proven biocompatibility and its versatility to entrap hydrophilic substances to be released with a controlled and predictable pattern. 20,29 In addition, the slow biodegradability of PCL allowed the size, shape, and hardness of the implants to be retained throughout the in vivo studies, facilitating easy surgical removal from the implantation site at the completion of treatment or in the case of acute local toxicity. 
Because TPT exerts most of its antiretinoblastoma effect in the first 15 minutes of cellular exposure, 13 we designed implants that initially release high amounts of drug to saturate the local barriers and to achieve high local concentrations of the drug. Long-lasting release at a slower rate achieved with our DDS might have been important in recruiting more retinoblastoma cells at the S-phase of the cycle. In fact, the use of protracted administration schedules of intravenously administered TPT resulted in improved antitumor activity in other pediatric malignancies with a drug sensitivity profile comparable to that of retinoblastoma. 30 Preliminary in vitro experiments showed very slow TPT release rate from implants loaded with pure TPT and no hydrophilic substances (data not shown). We have previously demonstrated that the release of hydrophilic drugs from PCL implants can be correlated with the loaded amount of hydrophilic substance in the PCL matrix. 20 Therefore, we hypothesized that the hydrophilic additive substances (excipients) of the commercial TPT, when homogeneously incorporated in the slow degradation PCL device, would form pores allowing the absorption of fluids and the release of the drug at the desired release rate. This point was confirmed after in vitro incubation by observing the extent of fluid absorption and mass loss of the implants loaded with hydrophilic excipients and the disappearance of the original crystals in the DDS matrix (Fig. 4). To allow comparison with our previous data of TPT vitreous and plasma exposure in rabbits receiving 1 mg/eye (periocular) or 1 mg/rabbit (IV) of TPT solution, 16 the amount of drug loaded in HL and HLv implants was selected to release a total dose of approximately 1 mg in 48 hours, as tested in vitro (Fig. 5). 
The function of the drug released in vitro could not be studied because most of the drug in solution was quickly converted to the inactive carboxylate at pH 7.4 and 37°C. However, we inferred that the stability of the lactone TPT form into the implant might have been favored, both in vitro and in vivo, by the acid excipients (tartaric acid) coloaded in the DDS, which could have provided an acid microenvironment in the polymer matrix. This rationale was supported by the observation that only LTPT was detected in the implants recovered from the rabbits included in the in vivo release study (Fig. 5a). Biodegradable polymers based on poly-D,L-lactide-co-glycolide acid have been reported to stabilize camptothecin lactones because of a hydrolytic process that produces carboxylic acid functional groups, generating a low pH inside the delivery systems. 31 These FDA-approved materials could be an alternative to the also approved PCL polymer we test here; however, the low cost of PCL could be important for the eventual application of similar devices in developing countries, where advanced retinoblastoma is more prevalent. 32  
Our initial in vivo experiments with LL formulations led to selective TPT delivery to the exposed eye and very low plasma exposure. However, these experiments showed relatively low TPT levels in the treated vitreous. Levels in the range of 10 ng/mL are needed for antitumor activity in retinoblastoma, and they were not achieved by these implants. 13 In our previous study, we were able to show in postmortem experiments that the sclera is permeable to TPT, but the drug failed to reach a significant transscleral penetration in vivo, probably because a rapid clearance from the orbit by the regional circulation. 16 Therefore, we hypothesized that vasoconstriction could improve the transscleral flow of TPT; hence, we developed our LLv implants, finding a significant increase in TPT vitreous concentrations compared with the LL implants. However, vitreous TPT concentrations at 24 hours were almost undetectable with low-loaded formulations. Therefore, we increased the TPT load in the implants and studied high loaded formulations (HL and HLv). Surprisingly, even though the TPT load was higher, HL implants did not reach vitreous TPT levels achieved at 5 hours with the LLv approach, highlighting the critical effect of local vasoconstriction. A remarkable increase was observed in local drug penetration with HLv implants compared with HL formulations, further supporting the relevance of a strategy to inhibit local vessels to reduce local clearance of the drug, as previously reported with α-adrenergic selective inhibitors such as oxymetazoline. 33  
Plasma total TPT exposure in rabbits receiving HL and HLv implants was approximately three times lower than in rabbits receiving 1 mg drug either periocularly or intravenously. 16 Most interestingly, LTPT levels were low or undetectable in the implanted rabbits, whereas 30% and 40% of total TPT exposure in plasma was lactone in the rabbits that received periocular administration and IV administration, respectively. 16 Therefore, episcleral implants significantly decreased systemic exposure to the potentially toxic lactone form of TPT. The significantly lower systemic TPT levels during the earlier time points using HLv implants (Fig. 5d) likely resulted from the inhibition of systemic absorption of the drug by vasoconstriction. 
The proportion of LTPT over total TPT 5 hours after the insertion of HLv implants in the implanted eye vitreous was 12%. After 24 and 48 hours, the proportion increased to 20% and 40%, respectively, as shown in Figures 6e and 6f. This result highlights the capacity of the local barriers to impede local drug penetration even at high concentration gradients and supports the saturation of the barriers over time with continuously released drug. Our findings should be translated with caution to the clinical situation because barriers might be altered in eyes affected by retinoblastoma, and our non–tumor-bearing animal model was therefore limited. 34 In addition, the rabbit eye, though fairly similar to the human eye in size and volume proportion between the intraocular compartments, may account for additional model limitations because of its lack of internal retinal vessels, high peripheral choroidal flow, 35 or absence of orbital fat, 36 among other factors. Interestingly, the scleral thickness reported for rabbit eyes (0.44 mm), 37 though narrower than in adult humans (0.61 mm), 38 is similar to that reported in a 9-day-old infant (0.40 mm), 38 which would support the use of rabbit eyes for our studies aiming at an early childhood disease. The scleral permeability of several drugs has been studied and correlated with their physicochemical properties, showing that molecular size is the most relevant factor limiting the transscleral transit of drugs in vitro. 39,40 Molecules in the size range of the TPT free base (MWt 421 Da) have been found to permeate human and rabbit scleras. 41,42 However, it is likely that the sclera plays a minor role in limiting local drug delivery compared with more specialized tissues, such as the choroid and the Bruch's membrane. 43  
Finally, we provide evidence that the active form of the drug, LTPT, is accumulated in the sclera and choroid exposed to the implants. These tissues seem to play an active role in transscleral drug delivery, acting as a powerful sink that precludes the penetration of LTPT to the retina and the vitreous, either redirecting the drug to the systemic circulation or accumulating it in their own cells. We hypothesize that the drug is taken up by the transport proteins present in ocular tissues. 44 Experimental data support the differential affinity of drug transporters for lactone and carboxylic forms of drugs, 45 which would explain why carboxylate TPT is predominantly found in the vitreous exposed to the implants, whereas LTPT is entrapped in the ocular barriers. 
To conclude, we have developed a technological strategy to enhance and sustain the penetration of TPT to the posterior segment of treated eyes and to reduce potentially toxic systemic drug exposure. The present research also contributes to the understanding of the active role of the ocular tissues, sclera, and choroid in the clearance of TPT during its transient path toward the intraocular compartments. Our observations may help design pharmacologic and technological strategies to overcome the barrier activity of ocular tissues during transscleral drug penetration, which may ultimately contribute to improving current retinoblastoma treatments. 
Footnotes
 Supported by the Fund for Ophthalmic Knowledge; a Postdoctoral Fellowship from the Ministry of Education and Science, Spain (AMC); University of Buenos Aires Grant UBACYT B025; and the Natalie Dafne Flexer Foundation.
Footnotes
 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2008.
Footnotes
 Disclosure: A.M. Carcaboso, None; D.A. Chiappetta, None; J.A.W. Opezzo, None; C. Höcht, None; A.C. Fandiño, None; J.O. Croxatto, None; M.C. Rubio, None; A. Sosnik, None; D.H. Abramson, None; G.F. Bramuglia, None; G.L. Chantada, None
References
Cunha-Vaz JG . The blood-retinal barriers system: basic concepts and clinical evaluation. Exp Eye Res. 2004;78:715–721. [CrossRef] [PubMed]
Wilson TW Chan HS Moselhy GM Heydt DDJr Frey CM Gallie BL . Penetration of chemotherapy into vitreous is increased by cryotherapy and cyclosporine in rabbits. Arch Ophthalmol. 1996;114:1390–1395. [CrossRef] [PubMed]
Shields JA Shields CL Meadows AT . Chemoreduction in the management of retinoblastoma. Am J Ophthalmol. 2005;140:505–506. [CrossRef] [PubMed]
Chintagumpala M Chevez-Barrios P Paysse EA Plon SE Hurwitz R . Retinoblastoma: review of current management. Oncologist. 2007;12:1237–1246. [CrossRef] [PubMed]
Mendelsohn ME Abramson DH Madden T Tong W Tran HT Dunkel IJ . Intraocular concentrations of chemotherapeutic agents after systemic or local administration. Arch Ophthalmol. 1998;116:1209–1212. [CrossRef] [PubMed]
Dunkel IJ Lee TC Shi W . A phase II trial of carboplatin for intraocular retinoblastoma. Pediatr Blood Cancer. 2007;49:643–648. [CrossRef] [PubMed]
Abramson DH Frank CM Dunkel IJ . A phase I/II study of subconjunctival carboplatin for intraocular retinoblastoma. Ophthalmology. 1999;106:1947–1950. [CrossRef] [PubMed]
Chantada GL Fandino AC Carcaboso AM . A phase I study of periocular topotecan in children with intraocular retinoblastoma. Invest Ophthalmol Vis Sci. 2009;50:1492–1496. [CrossRef] [PubMed]
Schmack I Hubbard GB Kang SJ Aaberg TMJr Grossniklaus HE . Ischemic necrosis and atrophy of the optic nerve after periocular carboplatin injection for intraocular retinoblastoma. Am J Ophthalmol. 2006;142:310–315. [CrossRef] [PubMed]
Abramson DH . Periocular chemotherapy for retinoblastoma: success with problems? Arch Ophthalmol. 2005;123:128–129; author reply 129. [CrossRef] [PubMed]
Frangoul H Ames MM Mosher RB . Phase I study of topotecan administered as a 21-day continuous infusion in children with recurrent solid tumors: a report from the Children's Cancer Group. Clin Cancer Res. 1999;5:3956–3962. [PubMed]
Chantada GL Fandino AC Casak SJ Mato G Manzitti J Schvartzman E . Activity of topotecan in retinoblastoma. Ophthalmic Genet. 2004;25:37–43. [CrossRef] [PubMed]
Laurie NA Gray JK Zhang J . Topotecan combination chemotherapy in two new rodent models of retinoblastoma. Clin Cancer Res. 2005;11:7569–7578. [CrossRef] [PubMed]
Athale UH Stewart C Kuttesch JF . Phase I study of combination topotecan and carboplatin in pediatric solid tumors. J Clin Oncol. 2002;20:88–95. [CrossRef] [PubMed]
Hughes PM Olejnik O Chang-Lin JE Wilson CG . Topical and systemic drug delivery to the posterior segments. Adv Drug Deliv Rev. 2005;57:2010–2032. [CrossRef] [PubMed]
Carcaboso AM Bramuglia GF Chantada GL . Topotecan vitreous levels after periocular or intravenous delivery in rabbits: an alternative for retinoblastoma chemotherapy. Invest Ophthalmol Vis Sci. 2007;48:3761–3767. [CrossRef] [PubMed]
Houghton PJ Cheshire PJ Myers L Stewart CF Synold TW Houghton JA . Evaluation of 9-dimethylaminomethyl-10-hydroxycamptothecin against xenografts derived from adult and childhood solid tumors. Cancer Chemother Pharmacol. 1992;31:229–239. [CrossRef] [PubMed]
Gaudana R Jwala J Boddu SH Mitra AK . Recent perspectives in ocular drug delivery. Pharm Res. 2009;26:1197–1216. [CrossRef] [PubMed]
Burke TG Bom D . Camptothecin design and delivery approaches for elevating anti-topoisomerase I activities in vivo. Ann N Y Acad Sci. 2000;922:36–45. [CrossRef] [PubMed]
Carcaboso AM Chiappetta DA Hocht C . In vitro/in vivo characterization of melt-molded gabapentin-loaded poly(epsilon-caprolactone) implants for sustained release in animal studies. Eur J Pharm Biopharm. 2008;70:666–673. [CrossRef] [PubMed]
Rittenhouse KD Pollack GM . Microdialysis and drug delivery to the eye. Adv Drug Deliv Rev. 2000;45:229–241. [CrossRef] [PubMed]
de Lange EC de Boer AG Breimer DD . Methodological issues in microdialysis sampling for pharmacokinetic studies. Adv Drug Deliv Rev. 2000;45:125–148. [CrossRef] [PubMed]
Nitschke R Parkhurst J Sullivan J Harris MB Bernstein M Pratt C . Topotecan in pediatric patients with recurrent and progressive solid tumors: a Pediatric Oncology Group phase II study. J Pediatr Hematol Oncol. 1998;20:315–318. [CrossRef] [PubMed]
Leger F Loos WJ Bugat R . Mechanism-based models for topotecan-induced neutropenia. Clin Pharmacol Ther. 2004;76:567–578. [CrossRef] [PubMed]
Sasaki H Nagano T Sakanaka K . One-side-coated insert as a unique ophthalmic drug delivery system. J Control Release. 2003;92:241–247. [CrossRef] [PubMed]
Wang G Tucker IG Roberts MS Hirst LW . In vitro and in vivo evaluation in rabbits of a controlled release 5-fluorouracil subconjunctival implant based on poly(D,L-lactide-co-glycolide). Pharm Res. 1996;13:1059–1064. [CrossRef] [PubMed]
Pontes de Carvalho RA Krausse ML Murphree AL Schmitt EE Campochiaro PA Maumenee IH . Delivery from episcleral exoplants. Invest Ophthalmol Vis Sci. 2006;47:4532–4539. [CrossRef] [PubMed]
Tsui JY Dalgard C Van Quill KR . Subconjunctival topotecan in fibrin sealant in the treatment of transgenic murine retinoblastoma. Invest Ophthalmol Vis Sci. 2008;49:490–496. [CrossRef] [PubMed]
Sun H Mei L Song C Cui X Wang P . The in vivo degradation, absorption and excretion of PCL-based implant. Biomaterials. 2006;27:1735–1740. [CrossRef] [PubMed]
Santana VM Furman WL Billups CA . Improved response in high-risk neuroblastoma with protracted topotecan administration using a pharmacokinetically guided dosing approach. J Clin Oncol. 2005;23:4039–4047. [CrossRef] [PubMed]
Shenderova A Burke TG Schwendeman SP . The acidic microclimate in poly(lactide-co-glycolide) microspheres stabilizes camptothecins. Pharm Res. 1999;16:241–248. [CrossRef] [PubMed]
Rodriguez-Galindo C Wilson MW Chantada G . Retinoblastoma: one world, one vision. Pediatrics. 2008;122:e763–770. [CrossRef] [PubMed]
Miller DJ Li SK Tuitupou AL . Passive and oxymetazoline-enhanced delivery with a lens device: pharmacokinetics and efficacy studies with rabbits. J Ocul Pharmacol Ther. 2008;24:385–391. [CrossRef] [PubMed]
Abramson DH Frank CM Chantada GL . Intraocular carboplatin concentrations following intravenous administration for human intraocular retinoblastoma. Ophthalmic Genet. 1999;20:31–36. [CrossRef] [PubMed]
Nork TM Kim CB Shanmuganayagam D Van Lysel MS Ver Hoeve JN Folts JD . Measurement of regional choroidal blood flow in rabbits and monkeys using fluorescent microspheres. Arch Ophthalmol. 2006;124:860–868. [CrossRef] [PubMed]
Davis FA . The anatomy and histology of the eye and orbit of the rabbit. Trans Am Ophthalmol Soc. 1929;27:400 402–441. [PubMed]
Boubriak OA Urban JP Akhtar S Meek KM Bron AJ . The effect of hydration and matrix composition on solute diffusion in rabbit sclera. Exp Eye Res. 2000;71:503–514. [CrossRef] [PubMed]
Olsen TW Edelhauser HF Lim JI Geroski DH . Human scleral permeability: effects of age, cryotherapy, transscleral diode laser, and surgical thinning. Invest Ophthalmol Vis Sci. 1995;36:1893–1903. [PubMed]
Prausnitz MR Noonan JS . Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci. 1998;87:1479–1488. [CrossRef] [PubMed]
Ahmed I Gokhale RD Shah MV Patton TF . Physicochemical determinants of drug diffusion across the conjunctiva, sclera, and cornea. J Pharm Sci. 1987;76:583–586. [CrossRef] [PubMed]
Cruysberg LP Franklin AJ Sanders J . Effective transscleral delivery of two retinal anti-angiogenic molecules: carboxyamido-triazole (CAI) and 2-methoxyestradiol (2ME2). Retina. 2005;25:1022–1031. [CrossRef] [PubMed]
Okabe K Kimura H Okabe J . Effect of benzalkonium chloride on transscleral drug delivery. Invest Ophthalmol Vis Sci. 2005;46:703–708. [CrossRef] [PubMed]
Cheruvu NP Kompella UB . Bovine and porcine transscleral solute transport: influence of lipophilicity and the choroid-Bruch's layer. Invest Ophthalmol Vis Sci. 2006;47:4513–4522. [CrossRef] [PubMed]
Zhang T Xiang CD Gale D Carreiro S Wu EY Zhang EY . Drug transporter and cytochrome P450 mRNA expression in human ocular barriers: implications for ocular drug disposition. Drug Metab Dispos. 2008;36:1300–1307. [CrossRef] [PubMed]
Chen C Mireles RJ Campbell SD . Differential interaction of 3-hydroxy-3-methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1. Drug Metab Dispos. 2005;33:537–546. [CrossRef] [PubMed]
Figure 1.
 
Diagram of the melt-molding compression technique developed to produce one-side coated TPT-loaded PCL implants. Briefly, to produce a PCL coating (drug-free layer of pure polymer), grounded PCL is introduced into the bottom of a stainless steel mold composed of a static platform and a matrix (a cross-section is represented). A homogeneous mixture of PCL and TPT (with hydrophilic excipients) is poured onto the pure PCL layer, and the mold plunger is mounted (left). The filled mold is exposed to a 1.7 kg/m2 pressure and heated (70°C, 1 hour) to melt the polymer and entrap the drug and excipients (middle). The system is cooled (4°C, 0.5 hour) to allow for the solidification of the implants. Implant with dimensions (right). P, pressure.
Figure 1.
 
Diagram of the melt-molding compression technique developed to produce one-side coated TPT-loaded PCL implants. Briefly, to produce a PCL coating (drug-free layer of pure polymer), grounded PCL is introduced into the bottom of a stainless steel mold composed of a static platform and a matrix (a cross-section is represented). A homogeneous mixture of PCL and TPT (with hydrophilic excipients) is poured onto the pure PCL layer, and the mold plunger is mounted (left). The filled mold is exposed to a 1.7 kg/m2 pressure and heated (70°C, 1 hour) to melt the polymer and entrap the drug and excipients (middle). The system is cooled (4°C, 0.5 hour) to allow for the solidification of the implants. Implant with dimensions (right). P, pressure.
Figure 2.
 
Surgical insertion of the DDS. (a) The implant is introduced through the conjunctival incision in the superior temporal episcleral zone of the right eye, with the uncoated side facing the sclera. (b) The incision is sutured with 5-0 sutures. (c) Final appearance of the implanted eye.
Figure 2.
 
Surgical insertion of the DDS. (a) The implant is introduced through the conjunctival incision in the superior temporal episcleral zone of the right eye, with the uncoated side facing the sclera. (b) The incision is sutured with 5-0 sutures. (c) Final appearance of the implanted eye.
Figure 3.
 
Diagram of the ocular microdialysis concentric probe designed and manufactured in the laboratory. A 10-mm-long microdialysis membrane (outer diameter [OD] 200 μm, 10,000 molecular weight cutoff; Asahi Medical Co., Japan), an inlet plastic tube (inner diameter, 500 μm), and an outlet silica tube (OD 145 μm) were used to build the probes. Epoxy adhesive (gray dots) was used to glue the components and to seal the end of the dialysis membrane. The probe was mounted in a nitrocellulose semirigid support, which allowed fixing the canula to the sclera with two sutures of 7-0 silk.
Figure 3.
 
Diagram of the ocular microdialysis concentric probe designed and manufactured in the laboratory. A 10-mm-long microdialysis membrane (outer diameter [OD] 200 μm, 10,000 molecular weight cutoff; Asahi Medical Co., Japan), an inlet plastic tube (inner diameter, 500 μm), and an outlet silica tube (OD 145 μm) were used to build the probes. Epoxy adhesive (gray dots) was used to glue the components and to seal the end of the dialysis membrane. The probe was mounted in a nitrocellulose semirigid support, which allowed fixing the canula to the sclera with two sutures of 7-0 silk.
Figure 4.
 
Scanning electron micrographs of LLv implants. (a) Scleral side of an intact LLv implant. (b) Orbital side of the same implant. (c) Image of a fractured implant, showing the porous scleral side (top) and the smooth orbital side (bottom). (d) Detail of the inner implant matrix showing fine needle-like crystals formed by the hydrophilic molecules (TPT + excipients) loaded into the implant. (e) Detail of the matrix after 10 days of incubation in PBS at 37°C; absence of crystals was observed. Original magnification: (a, b) 56×; (c) 24×; (d, e) 2000×. Scale bar: (a, b) 100 μm; (c) 1 mm; (d, e) 10 μm.
Figure 4.
 
Scanning electron micrographs of LLv implants. (a) Scleral side of an intact LLv implant. (b) Orbital side of the same implant. (c) Image of a fractured implant, showing the porous scleral side (top) and the smooth orbital side (bottom). (d) Detail of the inner implant matrix showing fine needle-like crystals formed by the hydrophilic molecules (TPT + excipients) loaded into the implant. (e) Detail of the matrix after 10 days of incubation in PBS at 37°C; absence of crystals was observed. Original magnification: (a, b) 56×; (c) 24×; (d, e) 2000×. Scale bar: (a, b) 100 μm; (c) 1 mm; (d, e) 10 μm.
Figure 5.
 
Drug release profile from TPT-loaded implants. (a) In vitro cumulative release from LLv implants (closed circles; mean ± SD of n = 3 values) and in vivo release values calculated as the difference between the initial TPT load and the unreleased TPT in recovered implants (open circles; individual values). (b) In vitro cumulative release from HLv implants (mean ± SD of n = 3 values). (c) Adrenaline in vitro cumulative release from LLv implants (mean ± SD of n = 3 values). (d) LLv implants scleral side before (left) and after (right) 10 days of in vitro release. (e) Scleral side of LLv implants as preimplanted (left) or recovered from rabbits at 8 hours, 3 days, and 6 days (left to right). (f) Orbital sides of the implants in (d). (g) Enucleated treated (left) and contralateral eyes devoid of conjunctival and muscular tissues of an albino rabbit 8 hours after the insertion of the DDS. (h) Weight loss (percentage of initial weight) of Blank implants and pure PCL implants after in vitro incubation (mean ± SD of three values).
Figure 5.
 
Drug release profile from TPT-loaded implants. (a) In vitro cumulative release from LLv implants (closed circles; mean ± SD of n = 3 values) and in vivo release values calculated as the difference between the initial TPT load and the unreleased TPT in recovered implants (open circles; individual values). (b) In vitro cumulative release from HLv implants (mean ± SD of n = 3 values). (c) Adrenaline in vitro cumulative release from LLv implants (mean ± SD of n = 3 values). (d) LLv implants scleral side before (left) and after (right) 10 days of in vitro release. (e) Scleral side of LLv implants as preimplanted (left) or recovered from rabbits at 8 hours, 3 days, and 6 days (left to right). (f) Orbital sides of the implants in (d). (g) Enucleated treated (left) and contralateral eyes devoid of conjunctival and muscular tissues of an albino rabbit 8 hours after the insertion of the DDS. (h) Weight loss (percentage of initial weight) of Blank implants and pure PCL implants after in vitro incubation (mean ± SD of three values).
Figure 6.
 
TPT vitreous and plasma concentrations in vivo. (a) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous humor aspirated from rabbits implanted in the right eye with LL (n = 4) and LLv implants (n = 5; *P < 0.01 compared with the ipsilateral eye in the LL group; t-test). (b) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous during the 160- to 320-minute period after the insertion of an HL implant in the right eye. Both eyes were sampled by microdialysis (HL group; n = 6). Midpoints of the sample collection time intervals are represented. (c) Total TPT levels (mean ± SD) in right and left vitreous during the 160- to 320-minute period after the insertion of an HLv implant in the right eye. Both eyes were sampled by microdialysis (HLv group; n = 8; *P < 0.05 compared with the left eye; Mann-Whitney rank sum test). (d) Plasma levels of total TPT in the HL and HLv groups. Time is represented in logarithmic scale to discern the early values (mean ± SD of 4–8 values; *P < 0.01 compared with the plasma level value for the HLv group at the same time point; t-test). (e) Total TPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; *P < 0.05 compared with HL and LLv groups; one-way ANOVA with Holm-Sidak multiple comparison; TPT levels in left vitreous were below the limit of detection). (f) LTPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; LTPT levels in left vitreous were below the limit of detection). N.D., not determined.
Figure 6.
 
TPT vitreous and plasma concentrations in vivo. (a) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous humor aspirated from rabbits implanted in the right eye with LL (n = 4) and LLv implants (n = 5; *P < 0.01 compared with the ipsilateral eye in the LL group; t-test). (b) Total TPT levels (mean ± SD at 5 hours) in right and left vitreous during the 160- to 320-minute period after the insertion of an HL implant in the right eye. Both eyes were sampled by microdialysis (HL group; n = 6). Midpoints of the sample collection time intervals are represented. (c) Total TPT levels (mean ± SD) in right and left vitreous during the 160- to 320-minute period after the insertion of an HLv implant in the right eye. Both eyes were sampled by microdialysis (HLv group; n = 8; *P < 0.05 compared with the left eye; Mann-Whitney rank sum test). (d) Plasma levels of total TPT in the HL and HLv groups. Time is represented in logarithmic scale to discern the early values (mean ± SD of 4–8 values; *P < 0.01 compared with the plasma level value for the HLv group at the same time point; t-test). (e) Total TPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; *P < 0.05 compared with HL and LLv groups; one-way ANOVA with Holm-Sidak multiple comparison; TPT levels in left vitreous were below the limit of detection). (f) LTPT in right aspirated vitreous of the LLv, HL, and HLv groups 24 hours after implantation in the right eye (mean ± SD of 3–4 values; LTPT levels in left vitreous were below the limit of detection). N.D., not determined.
Figure 7.
 
TPT accumulation (nanogram of TPT per gram of tissue) in the right eye half exposed to LLv, HL, and HLv implants for 24 or 48 hours compared with the right nonexposed eye half and the left eye. RE, right-exposed half; RNE, right-nonexposed half; L, left eye. Total TPT (ac) and proportion of LTPT over total TPT (df) were assessed in retina (a, d), choroid (b, e), and sclera (c, f). Mean ± SD of n = 3 values are represented. #, LTPT under limit of detection.
Figure 7.
 
TPT accumulation (nanogram of TPT per gram of tissue) in the right eye half exposed to LLv, HL, and HLv implants for 24 or 48 hours compared with the right nonexposed eye half and the left eye. RE, right-exposed half; RNE, right-nonexposed half; L, left eye. Total TPT (ac) and proportion of LTPT over total TPT (df) were assessed in retina (a, d), choroid (b, e), and sclera (c, f). Mean ± SD of n = 3 values are represented. #, LTPT under limit of detection.
Table 1.
 
Characteristics of the TPT-Loaded PCL Implants
Table 1.
 
Characteristics of the TPT-Loaded PCL Implants
Coating Matrix
Formulation (TPT load, mg) PCL (mg) PCL (mg) Hycamtin* (mg) TPT Powder† (mg) Adrenaline (mg) Added Exciplents‡ (mg) Final Weight§ (mg)
LL (0.3) 20 95 5 0 0 0 117 ± 7
LLv (0.3) 20 95 5 0 0.5 0 122 ± 4
HL (2.3) 50 45 5 2 0 0 93 ± 8
HLv (2.3) 50 45 5 2 0.5 0 98 ± 4
Blank (0) 50 45 0 0 0.5 5 94 ± 5
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×