Development of an Intravitreal Peptide (BQ123) Sustained Release System Based on Poly(2-Hydroxyoctanoic Acid) Aiming at a Retinal Vasodilator Response

Abstract

Purpose:

Development of a novel formulation for intravitreal administration, in which the endothelin_A receptor antagonist BQ123 is incorporated in a biodegradable and injectable polymer drug delivery system, poly(2-hydroxyoctanoic acid), aiming at a prolonged retinal vasodilator response.

Methods:

BQ123 was incorporated in poly(2-hydroxyoctanoic acid), leading to an easily injectable, homogenous mixture. In vitro release profiles were obtained in porcine vitreous humor (n=6). The ex vivo biocompatibility was studied by placing the formulation in contact with porcine retinal tissues and performing histology. In a pilot in vivo study, the change in retinal vessel diameter of mini pigs (n=2) was followed over 3 h after an intravitreal injection of the formulation, as well as the release of BQ123 from the polymer system for approximately 7 days (n=6).

Results:

In vitro, a constant release profile was obtained, releasing approximately 91% of BQ123 within 7 days. Histology on the porcine retinal tissues showed good ex vivo biocompatibility. In vivo, a vasodilative response was observed, with a retinal vessel diameter increase from 14% after 15 min, for approximately 39% after 3 h. At t=3 h, the BQ123 concentration in the vitreous humor was 0.7±0.2 μg/mL, followed by 1.5±1.0 and 1.1±0.8 μg/mL after 3 and 7 days, respectively. 39.9%±6.0% of BQ123 was still present in the polymer depot at t=7 days.

Conclusions:

The results show that an intravitreal injection of this drug delivery system leads to a prolonged vasodilative response and a BQ123 release over 7 days, suggesting its therapeutic potential in the management of retinal ischemic conditions.

Introduction

Retinal vein occlusion (RVO) and diabetic retinopathy are sight-threatening conditions, for which no successful treatment exists to date.^1,2 In the United States alone, 4.1 million patients were estimated to have diabetic retinopathy in 2004, of which 0.9 million were vision threatening.³ Due to a predicted increase in prevalence of diabetes mellitus, forecasts suggest that these numbers will triple in the next 40 years.⁴ RVOs are another important cause of visual impairment due to retinal vascular conditions: Worldwide, an estimated 16 million people suffer from vision loss due to a form of RVO.⁵ Current pharmacotherapy is targeting the secondary effects of these disorders, without addressing the cause of the obstruction of the retinal blood vessels, or the prevention of the retinal ischemia.^1,6 Therefore, there is a need to explore alternative strategies that interfere in an earlier stage of the disease and focus on overcoming the vascular occlusion.

A possible approach is the inhibition of the potent vasoconstrictor endothelin-1 (ET-1) that has been associated with both diabetic retinopathy^7,8 and RVO.⁹ ET-1 causes vasoconstriction of the retinal arteries and a reduction in retinal blood flow, predominantly through activation of smooth muscle endothelin_A (ET_A) receptors.¹⁰ The selective ET_A receptor antagonist BQ123 is a small cyclic peptide that inhibits the vasoconstrictor effect of ET-1 on retinal blood flow.¹¹ Moreover, an intravitreal injection of BQ123 caused vasodilation in both healthy mini pig eyes and eyes in which branch RVO was evoked.¹² Thus, the peptide offers promising characteristics as a therapeutic agent for the treatment of diseases in which the retinal arterioles are restricted.^12,13 However, the vasodilative effect is short lived and reaches its maximum of 30% dilation after 6 min, followed by a rapid decline in dilation.¹² To prolong the duration of action, the development of a BQ123 sustained release system is envisaged that slowly releases therapeutic doses of the peptide over a prolonged period of time. The clinical interest of such a system would be to maintain the arteriolar perfusion, in order to prevent vision loss due to the retinal ischemia.

Poly(2-hydroxyoctanoic acid) was selected for the formulation of such a sustained release system.^14,15 In contrast to solid poly(lactide)-based delivery systems, poly(2-hydroxyoctanoic acid) is a viscous liquid at room temperature¹⁵ and can be administered as a conventional intravitreal injection, thereby avoiding invasive and expensive surgery associated with ocular implants.¹⁶ In addition, this polyester is completely degradable by hydrolysis in an aqueous environment such as the vitreous humor. The drug release is governed by diffusion and degradation of the polymer matrix, respectively. Furthermore, incorporation of the therapeutic agent is achieved by simply mixing drug and polymer, without the need of heat or additional solvents.¹⁵ This is beneficial for a peptide such as BQ123, because it prevents the possible occurrence of stability issues.

The aim of this study was to develop a formulation that forms a drug depot in the vitreous humor on an injection, which continuously releases BQ123 over a period of 7 days. The envisaged formulation requires being injectable by a conventional intravitreal injection and biocompatible with the ocular tissues. Besides, since tissue damage was found to occur within hours after experimentally induced branch RVO,¹⁷ the formulation should cause an immediate vasodilative effect. The loading of BQ123 should be sufficient to reach therapeutic levels over a period of 7 days, taking into consideration that only a small volume of the formulation can be injected to avoid a rise in intraocular pressure.¹⁸ To assess whether these requirements are met, the release of BQ123 from the formulation and the degradation of free BQ123 were studied in vitro in porcine vitreous humor. Furthermore, the biocompatibility of the formulation was tested by placing the polymer in direct contact with ex vivo retinal tissues and performing histology on the tissues. Finally, an in vivo proof of concept was performed in mini pigs to investigate (i) whether the formulation is able to cause a vasodilative effect on the retinal arterioles and (ii) whether it releases therapeutic doses of BQ123 over a period of 7 days. This animal model was chosen, as mini pig arterioles show close similarities with human ones.¹⁹

Methods

Preparation of the formulation

Synthesis of 2-hydroxyoctanoic acid was first described by Trimaille et al.¹⁴ In the present study, poly(2-hydroxyoctanoic acid) was prepared by melt polycondensation at 150°C under vacuum as reported by Asmus et al.¹⁵ The polymer was precipitated over NaHCO₃ to remove low-molecular-weight oligomers. Molecular weight (M_w) and polydispersity index (PDI, M_w/M_n) were determined by gel permeation chromatography. A Waters 515 high-performance liquid chromatography (HPLC) pump, Waters 410 injector, Styragel HR 1–4 columns, and Waters 2414 refractive index detector (Waters Corporation, Milford, CT) were used with tetrahydrofuran as the continuous phase. Calibration was performed using polystyrene standards (PSS, Mainz, Germany). The same batch of poly(2-hydroxyoctanoic acid) with a purity >99%, as measured by NMR, was used for all experiments. Two different percentages of BQ123 (purity of 95%) (Peptide 2.0, Chantilly, VA) were incorporated in the polymer formulation by cryomilling (SPEX 6700 freezer/mill; SPEX SamplePrep, Metuchen, NJ) during 5 min, that is, 0.2% and 1.0% w/w BQ123 in poly(2-hydroxyoctanoic acid). The cryomilling process was described in greater detail by Asmus et al.²⁰ This method may have beneficial effects for future studies, as the milling takes place at −196°C, thus controlling bioburden. No additional excipients, such as preservatives, were added. The final formulations were stored at −20°C.

Ex vivo biocompatibility

Fresh porcine eyes were obtained from a slaughterhouse and the anterior segment and vitreous humor were carefully removed to access the retinal tissues, as described in an earlier work.²¹ The posterior cup was cut into 2 separate parts. On one part, 50 μL of poly(2-hydroxyoctanoic acid) were placed in direct contact with the retinal tissues (n=2) and the effects were visually observed over 1.5 h at 4°C. This temperature was selected, to keep the ocular tissues as much intact as possible during the study time. At 37°C, the tissues dry out completely. A positive control (300 mM 2-hydroxyoctanoic acid monomer in 75% v/v aqueous ethanol²¹) was put on the second part of the same eye. Aqueous ethanol was chosen, as it is not possible to dissolve the monomer in pure water or buffer, due to the hydrophobic nature of 2-hydroxy-octanoic acid. This positive control should, therefore, be considered as the worst case scenario. In parallel, 50 μL of polymer were placed in direct contact with the retinal tissues, after which the posterior segment was fixed in 4% v/v formaldehyde in phosphate buffer for histological examination. As negative control, a part of the posterior segment was fixed without polymer. After embedding the fixed tissues in paraffin and cutting them in serial sections of 7 μm, 5 sections were stained with hematoxilin and eosin following a standard protocol.

In vitro BQ123 release and BQ123 stability in the release medium

The BQ123 release from the polymer was measured over time by placing 50 μL of the 0.2% BQ123 in poly(2-hydroxyoctanoic acid) formulation in 2 mL porcine vitreous humor with 0.2% sodium azide to avoid bacterial growth (n=6 separate vials). These volumes were chosen to simulate the in vivo study in mini pigs. Vials were stored under light shaking conditions at 37°C for 7 days, and 50 μL samples of vitreous humor were taken at different time points to measure the amount of BQ123 released from the delivery system. After each sample collection, the sample volume was replaced by fresh vitreous humor.

The results of the BQ123 release study might be influenced by the fact that the released BQ123 degrades with time. To measure the rate at which BQ123 degrades in vitreous humor, 100 μg of free BQ123 was placed in a vial with vitreous humor (n=2 separate vials) and stored under the same conditions as the polymer formulation. This amount of free BQ123 was chosen, as the polymer depot used in the in vitro study contains 100 μg of BQ123 as well. The amount present at t₀ was considered as 100%.

Quantification of BQ123 in the vitreous humor was performed by HPLC (1200 Series; Agilent Technologies, Basel, Switzerland) on a C18 Waters Symmetry column (4.6×30 mm, 3.5 μm) with fluorimetric detection at experimentally determined wavelengths of 278 nm (excitation) and 348 nm (emission). The temperature was maintained at 35°C, and the flow rate was 1.5 mL/min. A mixture of water and acetonitrile was used as mobile phase in gradient mode, with 20% acetonitrile at the start of elution, increasing to 40% at 2 min and back to 20% at 4 min. Each sample was diluted 1:1 with water and injected in triplicate with an injection volume of 5 μL. All values are presented as mean±standard deviation (SD).

The release profile was corrected for degradation in vitreous humor using the following equations: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm BQ } 123_ { { \rm corrected } } = \left( \frac { 100 } { { \rm BQ } 123_ { { \rm non - degraded } } } \right) * { \rm BQ } 123_ { { \rm measured } } \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm BQ}123_{{ \rm non - degraded}} = - 20.051 \ { \rm t} + 100\end{align*} \end{document}

in which

t is the time point expressed in days;

BQ123_measured is the percentage of BQ123 that is released from the polymer depot and measured in the release medium;

BQ123_non-degraded is the percentage of free BQ123 that is still present in the medium;

BQ123_corrected is the released percentage of BQ123 after correction for degradation.

The degradation profile follows a zero-order kinetic during these first 3 days, with a coefficient of determination (r²) of 0.955; after t=3 days, the percentage of BQ123 stays stable. Therefore, the second equation was only applied for the time points between t₀ and t=3 days; for later time points, the BQ123_non-degraded value was fixed at 40%.

In vivo BQ123 release and retinal vasodilation

The study was performed on 5 mini pigs (Göttingen breed; Arare Animal Facility, Geneva, Switzerland) with a weight varying from 10 to 12 kg. All experiments were conducted to conform to the ARVO statement for Use of Animals in Ophthalmic and Vision Research and were approved by the local veterinary authorities of Geneva (#1004/3419/2R). The animals were prepared as described in a previous work,²² receiving an intramuscular injection of 3 mL (15 mg) midazolanum maleate (Dormicum^®; Roche Pharma, Reinach, Switzerland), 3 mL (120 mg) azaperone (Stresnil^®; Janssen Pharmaceutica, Beerse, Belgium), and 1 mL (0.5 mg) atropine as premedication. In 2 of the mini pigs, the retinal vasodilative effect was monitored during 3 h after an intravitreal injection of 0.2% BQ123 in poly(2-hydroxyoctanoic acid). The pigs were anesthetized with 2–3 mg ketamine hydrochloride (Ketalar^®; Parke-Davis, Zurich, Switzerland) injected into an ear vein. Thereafter, they were intubated and artificially ventilated as previously described.²² After removal of the eyelids, detachment of the bulbar conjunctiva, and cleaning of the sclera, a metal ring was sutured around the limbus to fix the globe. A sclerotomy was performed 2–3 mm posterior to the limbus, and a contact lens was placed on the cornea. An operating microscope (Carl Zeiss Meditec, GmbH, Oberkuchen, Germany) was used to observe the fundus. Fifty microliters of the 0.2% BQ123 in poly(2-hydroxyoctanoic acid) formulation were injected centrally in the vitreous humor with a Hamilton 250 μL syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) and a 25-gauge needle. The retinal arteriolar diameter was monitored over a period of 3 h with a retinal vessel analyzer (RVA, Imedos GmbH, Jena, Germany).²³ Measurements were performed before the injection and thereafter, every 15 min. Simultaneously, arterial oxygen partial pressure (PaO₂), carbon dioxide pressure (PaCO₂), and pH were measured in blood from the femoral artery with a blood gas analyzer (Labor-systeme Flükiger AG, Menziken, Switzerland) and controlled over the duration of the study. After 3 h, the animal was sacrificed; the eye was enucleated and stored immediately at −20°C.

The remaining 3 pigs received an intravitreal injection of 1% BQ123 in poly(2-hydroxyoctanoic acid) in both eyes, and vitreous humor samples were taken at 3 and 7 days after the injection, to determine the BQ123 concentration in vitreous humor. The 1% formulation was selected to ensure sufficiently high BQ123 concentrations in the vitreous humor for quantification after 7 days. The mini pigs were surveyed on a daily basis over the duration of the study. At t₀, the premedication protocol as described earlier was followed and thereafter, 50 μL of the 1% BQ123 in poly(2-hydroxyoctanoic acid) formulation were injected centrally in the vitreous humor of each eye with a Hamilton 250 μL syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) and a 25-gauge needle. After 3 days, the animals were submitted to the same premedication protocol, before being anesthetized with 2–3 mg ketamine hydrochloride (Ketalar, Parke-Davis, Zurich, Switzerland) injected into a ear vein. A vitrectomy (without activation of the infusion line) was performed to remove approximately 100 μL of vitreous humor from both eyes, in order to quantify the amount of free BQ123 present in the eye. Attention was paid not to touch the polymer depot during this procedure. The vitreous humor samples were stored immediately at −20°C. After 7 days, the animals were sacrificed; the eyes were enucleated and stored at −20°C.

Sample analysis

The BQ123 concentration was determined in the obtained vitreous humor samples, as well as in the enucleated eyes. For the former, the vitreous humor was diluted with water (1:1) before analysis by HPLC with fluorescence detection as described for the in vitro release study. For the latter, the complete vitreous humor with the polymer droplet was separated from the rest of the eye and its total mass was weighed. Two different samples were prepared. For the first sample, the polymer droplet and a small part of the surrounding vitreous humor were removed to quantify the amount of BQ123 that was still present in the polymer bubble. The polymer droplet was completely dissolved in 1 mL isopropanol; thereafter, 9 mL water were added. The sample was centrifuged, and the supernatant was filtered with a 0.22 μm filter. Second, to quantify the amount of BQ123 released from the bubble, the remaining vitreous humor was carefully homogenized and thereafter centrifuged and filtered (0.22 μm). Both samples were diluted 1:1 with water before analysis by HPLC with fluorescence detection. All values are presented as mean±standard deviation (SD).

Results

Characteristics of the formulation

The poly(2-hydroxyoctanoic acid) used in the present study had a molecular weight (M_w) of 3,300 g/mol and a PDI (M_w/M_n) of 1.4. The chemical structure of the polymer is depicted in Supplementary Fig. S1 in Supplementary Data (Supplementary Data are available online at www-liebertpub-com.web.bisu.edu.cn/jop). At 25°C, the viscosity of the injectable was 30 Pa.s as previously described by Asmus.²⁴ The mixture of BQ123 incorporated in the polymer as obtained by cryomilling was observed to be homogenous on visual inspection. An earlier work showed that this single step procedure leads to homogenous mixtures with particle diameters in the micrometer range.²⁰ This resulted in a viscous suspension that was filled into syringes and which remained unchanged in the syringe over time, as well as on injection. The formulations were easily injectable through a 25-gauge needle into the central vitreous humor, as tested and stated by the handling ophthalmologist.

Ex vivo biocompatibility

The biocompatibility of the polymer formulation with the retinal tissues was investigated on ex vivo porcine eyes. On visual inspection, no adverse events were observed after placement of poly(2-hydroxyoctanoic acid) in direct contact with the retinal tissues; whereas the positive control caused white stains on the retinal tissues (Fig. 1A). Histology also showed that the retinal layers remained unaffected on direct contact with the polymer: No differences were observed between the untreated retinal tissues and the ones which were in contact with the formulation (Fig. 1B).

FIG. 1.

Ex vivo poly(2-hydroxyoctanoic acid) biocompatibility. (A) Poly(2-hydroxyoctanoic acid) (left) in direct contact with the retinal tissues does not lead to adverse events. As a positive control, high concentrations of 2-hydroxyoctanoic acid in 75% v/v aqueous ethanol (300 mM) are causing white stains on the retinal tissues due to a damaged ganglion cell layer (right), as was observed by histology in an earlier work.²⁰ (B) Histology of the retinal tissues shows good biocompatibility of poly(2-hydroxyoctanoic acid) (right). No differences were observed in the tissues compared with the nonpolymer-exposed retinal tissues (left).

In vitro BQ123 release and BQ123 stability in the release medium

The in vitro release of BQ123 from the polymer formulation over a period of 10 days showed an initial burst release of 7% during the first 2 h, followed by a constant release of approximately 55%±15% at day 10 (Supplementary Fig. S2A in Supplementary Data). However, these amounts may practically be higher considering that free BQ123 degrades over time in vitreous humor. The degradation profile of free BQ123 in vitreous humor release medium demonstrated that around 60% of the initially present BQ123 degraded within the first 3 days and that a residual 40% of intact peptide was measurable for approximately 7 days (Supplementary Fig. S2B in Supplementary Data). Therefore, the BQ123 release profile was corrected for degradation of free BQ123, revealing an almost complete release after 7 days (Supplementary Fig. S2C in Supplementary Data). The corrected release profile is depicted in Fig. 2, demonstrating an amount between 13 and 17 μg of BQ123 released per day from the polymer depot.

FIG. 2.

In vitro BQ123 release from poly(2-hydroxyoctanoic acid) in porcine vitreous humor, presented in μg/day (mean±SD, n=18: 6 vials, 3 analyses per vial). Data are derived from a correction of the release profile for the degradation of free BQ123 in vitreous humor.

In vivo BQ123 release and retinal vasodilation

The retinal vasodilator response after an intravitreal injection of the BQ123 depot is depicted in Fig. 3 (mean) and Supplementary Fig. S3 (response in both mini pigs separately). A clear vasodilation was observed and remained present over the whole study period (Fig. 4). The onset of the vasodilative effect already occurred in the first 15 min with a vessel diameter increase of 14%. Thereafter, a continuous rise was distinguished, of approximately 39% after 3 h. Figure 5 shows the BQ123 concentration in the vitreous humor at the time points 3 h, 3 days, and 7 days, being 0.7±0.2, 1.5±1.0, and 1.1±0.8 μg/mL, respectively. After 7 days, 40%±6% of the total amount of BQ123 that was injected still remained in the polymer depot, corresponding to 193.3±29.2 μg of BQ123. The drug depot stayed clearly visible over 7 days and was well tolerated during the complete study; no adverse events were observed.

FIG. 3.

Rapid onset of vasodilative effect after intravitreal injection of the polymer drug depot in mini pigs (black curve). The change in vessel diameter (mean, n=2) was measured with a retinal vessel analyzer after an injection of 50 μL 0.2% BQ123 in poly(2-hydroxyoctanoic acid). The vessel diameter that was measured directly before the injection was considered as baseline. In comparison, the change in vessel diameter after a single intravitreal dose of BQ123 (30 μL, 0.61 μg/mL) (grey curve) is short lived, based on Stangos et al.¹²

FIG. 4.

Retinal arteriolar diameter changes in a mini pig. Comparison of the arterioles before injection (left) and 3 h after injection of 50 μL 0.2% BQ123 in poly(2-hydroxyoctanoic acid) (right) demonstrates a visible vasodilative effect. The same magnification was used for both pictures.

FIG. 5.

In vivo BQ123 concentration (μg/mL) in mini pig vitreous humor on an intravitreal injection of BQ123 in poly(2-hydroxyoctanoic acid). Concentrations are presented as mean±SD. Two different formulations were used: t=3 h is the concentration on injection of 0.2% BQ123 w/w in poly(2-hydroxyoctanoic acid) (n=6 samples, 2 separate eyes, 3 analyses per eye), t=3 days and t=7 days on injection of 1% BQ123 w/w in poly(2-hydroxyoctanoic acid) (n=18 samples, 6 separate eyes, 3 analyses per eye).

Discussion

It has been shown earlier that BQ123 elicits a strong, but unfortunately short-lived,¹² vasodilative response on the retinal arterioles. This work aimed at the development of an easily injectable intravitreal sustained release system for BQ123 that is biocompatible with the retinal tissues and which is able to induce an immediate vasodilative effect, in order to overcome the acute obstruction. The therapeutic potential of this formulation is a direct pharmacological effect, in order to improve the oxygen and nutrient supply to the retinal tissues.

The injectable and biodegradable poly(2-hydroxyoctanoic acid) was selected as a potential drug delivery system, as this very hydrophobic polymer forms immediately after injection a single droplet drug depot in the vitreous humor that will be hydrolyzed over time in the aqueous environment, comparable to the injectable polymer poly(ortho ester) described earlier.²⁵ To assess the biocompatibility of poly(2-hydroxyoctanoic acid), a preliminary ex vivo study was conducted, revealing good biocompatibility with the retinal tissues on visual inspection and on histological examinations of the retinal cell layers. During the in vivo study in mini pigs, good biocompatibility of the system with the ocular tissues was observed over a period of 7 days. No adverse events were distinguished. Of course, the present work only provides information on the short-term biocompatibility of the system with the retinal tissues and, therefore, further studies need to investigate the long-term effects. Moreover, it should be noted that this study was intended as an initial “proof of concept,” which is why a limited number of animals were used. The in vivo data should, therefore, be considered preliminary.

The in vitro release results demonstrated the ability of the system to release BQ123 in a controlled manner over a period of 7 days. The initial burst is beneficial, as it leads to an immediate onset of the vasodilative arteriolar response. Despite the observed burst, the overall release for approximately 7 days showed a high correlation for linear regression: A coefficient of determination of 0.98 was found for the noncorrected release profile and of 0.99 when correction for BQ123 degradation was taken into account. Thus, the main release profile follows a zero-order kinetic, leading to a release between 13 and 17 μg of BQ123 per 24 h. The amount of BQ123 released is sufficient to maintain a vasodilator response on the retinal arterioles, based on an extrapolation of an in vivo study by Stangos et al.¹² This study was selected, because the same animal model was used for our novel sustained release formulation. On a single dose of 30 μL BQ123 (0.61 μg/mL), corresponding to an amount of 18.3 ng, a clear vasodilative effect was observed that remained for approximately 15 min, which was the end point of the study.¹² However, extrapolation of the curve leads to the assumption that the effect will have disappeared completely after 30 min. This extremely short vasodilative response is in accordance with another study, in which a single intravitreal injection of different concentrations of BQ123 caused an increase in retinal blood flow in a rat model.⁸ In this study, a maximum was reached after 5 min and return to baseline within 15 min, independent of the concentration of BQ123 injected.⁸ Based on the hypothesis that 18.3 ng of BQ123 triggers an effect for 30 min in mini pigs, it can be calculated that around 0.9 μg would be necessary to maintain a therapeutic effect over 24 h. Our initial in vitro results show that sufficient amounts were released over a period of 7 days. The aims of the following in vivo study were (i) to verify whether the formulation would immediately release sufficient amounts of BQ123 in order to evoke a vasodilative response and (ii) to investigate whether the system releases therapeutic doses of BQ123 over a period of 7 days. On BRVO, a correlation was shown between the poor perfusion of the vascular bed and a constriction of arterioles that lie in the same area.¹² An immediate vasodilative effect on these arterioles is, therefore, of valuable clinical importance, as their constriction leads to a severe drop in oxygen supply to the retinal tissues, or tissue hypoxia in the case of post-BRVO retinal ischemia.^26,27 This hypoxia will induce an up-regulation of vascular endothelial growth factor (VEGF), causing angiogenesis and increased vascular permeability.²⁸ In a rat model, Kaur et al. demonstrated that after 3 h of hypoxia, VEGF concentrations were significantly elevated.²⁹ Moreover, 4 h after experimentally induced branch RVO in a mini pig, the ganglion cell layer showed edema and necrosis.¹⁷ Although vasodilation could exacerbate capillary leakage and edema formation in the area affected by the BRVO, a normal blood supply should potentially help in the formation of venular collateral vessels. It is, therefore, relevant to develop a system that directly interferes with the constriction of the retinal arterioles, in order to maintain arteriolar perfusion post-BRVO, in particular for cases with capillary nonperfusion.

An important parameter which distinguishes in vivo- from in vitro- release studies is the fact that clearance of the drug occurs. Although to our knowledge, BQ123 clearance from the vitreous humor has not been investigated, a fast clearance is nonetheless expected based on the low-molecular weight of the peptide (610 g/mol).^30,31 Therefore, it is probable that the amount of BQ123 quantified in the vitreous humor at t=3 h, t=3 days and t=7 days is lower than the amount of BQ123 which was released in vitro. Nevertheless, the BQ123 vitreous concentration was sufficient to evoke an almost immediate vasodilative effect, which was maintained over 3 h. Compared with a single injection of BQ123, this is a 6-fold increase in duration. An earlier work on the same animal model showed that an injection of a physiologic saline solution does not lead to any differences in the retinal vessel diameter, demonstrating that the vasodilative effect is not induced by the injection itself.¹²

At t=3 h, a BQ123 concentration of 0.7±0.2 μg/mL in the vitreous humor led to an increase of 40% in retinal vessel diameter. In order to measure a vasodilative effect on the retinal diameters, the pig should be kept under general anesthesia and controlled conditions in the exact same position. Unfortunately, it was, therefore, not possible to correlate the BQ123 release to a vasodilative effect over a period of several days. However, since the BQ123 concentrations at t=3 days and t=7 days are in the same order of magnitude (1.5±1.0, and 1.1±0.8 μg/mL, respectively), we postulate that these values will be sufficient to maintain the vasodilative effect over a period of 1 week. Since 40% of BQ123 still remained in the formulation droplet after 7 days, a longer duration of the vasodilative effect with this delivery system can be envisaged. Based on these preliminary in vivo results, poly(2-hydroxyoctanoic acid) seems an adequate system for the sustained release of BQ123. Further investigations will be initiated regarding the long-term efficacy of the formulation in future studies.

To summarize, this work was intended as an initial and pivotal investigation of poly(2-hydroxyoctanoic acid) as an intraocular carrier. It demonstrates that the system is a promising drug delivery system for intravitreal application. Good injectability and biocompatibility are observed, as well as an in vitro zero-order release of BQ123 from the polymer system over a period of 7 days. The in vivo data show that the system immediately releases therapeutic doses of BQ123, causing a 6-fold increase in the duration of the vasodilative effect compared with a single intravitreal injection. Moreover, a sustained release of therapeutic doses of BQ123 over a period of 7 days was observed in mini pigs. In conclusion, the data present a proof of concept for the in vivo sustained release of BQ123 from poly(2-hydroxyoctanoic acid), which may eventually result in the development of a therapeutic system for the treatment of retinal ischemic conditions.

Footnotes

Acknowledgments

The authors wish to thank Nicole Gilodi and Manuel Jorge Costa for their help with the in vivo experiments, Marcel Kohler of the “Service de toxicologie de l'environnement bâti” for making available the HPLC with fluorescence detection, and Lena Bagnewski for her help with the histology. Financial support was provided by the Swiss National Science Foundation (#320030-122190).

Author Disclosure Statement

R. Gurny and M. Möller are shareholders of Apidel SA. No competing financial interests exist for the other authors.

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