Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research paper

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Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model

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Marco Cespi a, Giulia Bonacucina a, Stefania Pucciarelli b, Paolo Cocci c, Diego Romano Perinelli a, Luca Casettari d, Lisbeth Illum e, Giovanni Filippo Palmieri a, Francesco Alessandro Palermo b,c,⇑, Gilberto Mosconi b a

University of Camerino, School of Pharmacy, Camerino, Italy University of Camerino, School of Biosciences and Biotechnology, Camerino, Italy c University of Camerino, Unità Ricerca e Didattica San Benedetto del Tronto (URDIS), San Benedetto del Tronto, Italy d University of Urbino, Department of Biomolecular Sciences, Urbino, Italy e IDentity, Nottingham, United Kingdom b

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Article history: Received 15 May 2014 Accepted in revised form 17 August 2014 Available online xxxx Chemical compounds studied in this article: 17b-Estradiol (PubChem CID: 5757) Poloxamer 407 (PubChem CID: 24751) Keywords: Thermosensitive gel Poloxamer Sustained release Goldfish 17b-Estradiol Vitellogenin

a b s t r a c t The purpose of this study was to develop and evaluate a delivery system comprising a thermosensitive gel for the sustained release of steroidal hormones in fish, over an extended period of time after a single intramuscular (i.m.) injection and for the improved reproductive performance in fish. Controlled delivery systems based on thermosensitive gels are easy to prepare, low cost and high versatility dosage forms, which have been shown to be effective in several animal species for sustained release of hormones. In this work, a thermosensitive gel system based on poloxamer 407 in water:ethanol medium, able to work as a prolonged release carrier for 17b-estradiol (E2), has been developed. Such a system was able to solubilize the lipophilic E2 and to gel at the required water temperature for fish rearing (20 °C). Moreover, the system exhibited the best injection condition at temperatures below 15 °C when the system behaved as a low viscosity Newtonian liquid. The thermosensitive gel system was tested in vivo in the fish model, Carassius auratus, and the results compared with a single i.m. injection of E2 dissolved in corn-oil and other relevant control systems not containing E2. The results were particularly interesting, since fish injected with the E2 thermosensitive gel formulation, showed significantly higher levels of the circulating hormone than corn oil-E2 treated animals at 72 and 96 h after injection. In addition, the thermogel system was able to sustain the plasma level of E2 for about 11 days. The increased plasma levels of E2 were also accompanied by maintained higher values of plasma vitellogenin (VTG), thus suggesting that the thermosensitive polymer based delivery system could prevent rapid hepatic clearance of E2, resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish. Ó 2014 Elsevier B.V. All rights reserved.

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1. Introduction

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The use of pharmaceutical agents in aquaculture is a wellestablished practice for the treatment of fish diseases and for the

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Abbreviations: EO, ethylene oxide; PO, propylene oxide; E2, 17b-estradiol; VTG, vitellogenin; SDS, sodium dodecyl sulfate; MS 222, 3-aminobenzoic acid ethyl ester methanesulfonate; EE2, 17-a-Ethynylestradiol; TEG, triethylene glycol; EIA, enzyme-immunoassay method; ELISA, enzyme-linked immunosorbent assay; BW, body weight. ⇑ Corresponding author. Lungomare A. Scipioni 6, I-63074 San Benedetto del Tronto (AP), Italy. Tel.: +39 0737 404901; fax: +39 0737 404920. E-mail address: [email protected] (F.A. Palermo).

modulation of fish growth and reproduction [1]. The latter point is particularly relevant for all the species reared in captivity inside commercial aquaculture facilities. In these conditions, many animals show evidence of some degree of reproductive dysfunction such as asynchrony in the time of ovulation, absence of volitional spawning, diminished sperm production or even the absolute failure of maturation and the presence of ovulation phases [2–5]. Although in some cases, these concerns can be overcome or at least mitigated by manipulation of various environmental parameters [4,6], in most cases hormonal treatments can be more effective means of controlling fish reproduction.

http://dx.doi.org/10.1016/j.ejpb.2014.08.008 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: M. Cespi et al., Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.08.008

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While in the last decades many efforts have been made to improve the efficacy of hormone drugs, leading to a wide variety of synthetic agonists of natural hormones, a lot of work still remains to be done in terms of development of appropriate and effective sustained delivery systems. In fact, to date most often multiple injections of hormones have to be given over the course of several days, or even over several weeks, in order to maintain elevated hormone plasma levels [3,4]. The multiple treatments require repetitive handling of brood stock and consequently labor, time and monitoring, moreover, this procedure is stressful to the fish and sometimes also results in adverse effects [5]. Despite the fact that sustained administration of hormones has been shown to be effective in aquaculture [7–9], at the present time, only minimal research is devoted to this issue and only a few sustained release systems have been developed and tested on fish breeding, such as cholesterol pellets implants [10,11], biodegradable microspheres [12,13], non-degradable monolithic implants [14,15] and more recently, osmotic pumps [16]. The sustained release cholesterol pellets implants are now actually marketed (Ovaplant™, Western Chemical Inc., USA) [17]. Although these systems have generally demonstrated a good performance when administrated to fish, the production and wider distribution on an industrial scale appears improbable due to the high production cost and to the difficulty of administering the delivery system. The aim of this work was the development of a new sustained release system intended for the administration of hormones in aquaculture and characterized by low production cost, easy administration and high versatility. In particular, it was decided to study the possibility of using a thermogelling system, i.e. a system that is liquid at low temperature and gel at the body temperature of fish (20 °C). These particular characteristics should assure an easy administration of the system, as a liquid, and a prolonged drug release once the system has rapidly gelled inside the body of the fish. The use of a parenteral thermogelling systems has previously been shown to be effective for controlled release of hormones when applied in different animal species [18–20]. These formulations increased the relative bioavailability of steroidal hormones and resulted in the sustained release of testosterone after a single subcutaneous (s.c.) injection in rabbits [18]. Moreover there is evidence that thermogelling systems such as poloxamer can protect against local enzyme-mediated drug metabolism and stabilize peptide hormones [18,20,31]. In the present study, 17b-estradiol (E2) was selected as the model hormone [21,22]. The sustained release system was prepared using poloxamer 407, a block copolymer of ethylene oxide (EO) and propylene oxide (PO) showing a thermogelling behavior which is dependent on its concentration and on the presence of other excipients. Suitable systems were prepared after a preliminary rheological characterization intended to define the correct concentration of the polymer and other excipients (i.e. sodium chloride and ethanol), necessary to obtain a thermosensitive gel with an appropriate gelation temperature for the administration in juvenile goldfish (Carassius auratus). The efficiency of the hormone release system was evaluated by monitoring both plasma E2 and vitellogenin (VTG) concentrations over a period of 18 days.

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2. Materials and methods

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2.1. Materials

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Poloxamer 407 (LUTROL F-127, BASF, Burgbernheim DE), sodium dodecyl sulfate (Bio-Rad laboratories, USA), 17b-estradiol and 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma Aldrich, St. Louis USA) were used as supplied. Ethanol (Sigma Aldrich, St. Louis USA) and sodium chloride (Carlo Erba, Rodano, IT) were stan-

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dard reagents grade, at places in the text ethanol has been described as EtOH. Ultrapure water was produced with a laboratory deionizer (Osmo Lab UPW 2, Gamma 3, IT).

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2.2. Methods

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2.2.1. Formulation of the thermosensitive gelling system The thermosensitive gelling formulations were prepared following the ‘‘cold’’ procedure [23]. Briefly, poloxamer powder was slowly dispersed in the required amount of degassed and deionized water under magnetic stirring while keeping the dispersion on an ice bath. Ethanol and NaCl were added at the end of the preparation procedure after the poloxamer was completely solubilized. E2 was dissolved in the ethanol present in the formulation before it was added to the poloxamer water dispersion. The compositions of the different sample preparations are given in Table 1. As can be seen the samples comprised poloxamer 407 in concentrations ranging from 20% to 22% w/v, NaCl from 0.9% to 3.5% w/v and ethanol from 25% to 50% w/v, while the E2 concentration was kept constant at 1.5 mg/mL. All samples were left at 5 °C for 24 h before being analyzed.

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2.2.1.1. Rheological analysis. Rheological analysis was performed on the developed systems in order to define the optimal composition of the thermosensitive gel in terms of the concentrations of poloxamer 407, ethanol and NaCl for the preparation of a system characterized by a gelation temperature of 20 °C and a stable appropriate gel structure with controlled release characteristics. Rheological analyses were performed in triplicate using a stress control rheometer (Stress-Tech, Reologica Instruments, Lund, Sweden) equipped with a cone-plate geometry (4/40) operating in the oscillation mode. The gap was 150 lm. The samples were studied by using temperature sweep tests in the temperature range 0°– 50 °C at the rate of 1 °C/min, applying a stress of 10 Pa and a frequency of 1 Hz. The cross-over point between the elastic and viscous modulus was considered as the gel point of the different samples. This parameter, also corresponds to the temperature at which the phase angle is characterized by a value equal to 45° [24]. Formulations with thermogelling characteristics suitable for the aim of the work were further characterized using a viscometry test from 5° to 25 °C (Stress-Tech Reologica Instruments). For each temperature, an appropriate amount of the sample was analyzed, increasing the shear stress (r) from 0.1 to 150 Pa and the corresponding shear rate (D) measured. Since the samples showed non-Newtonian behavior, at least in the gel form, all the flow curves obtained were analyzed using the ‘power law model’:

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n

r ¼ k  D þ r0

ð1Þ

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where k is the consistency index, n the power law index and r0 the yield stress. The consistency index k, also called ‘power law viscosity’, is related to the viscosity of the system, the ‘power law index’, n is related to the flow patterns (Newtonian or non-Newtonian) while the ‘yield stress’ represents the critical stress values necessary for the flow of the sample.

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2.2.2. In vitro release of drug Dissolution studies were performed following the previously described ‘paddle over extraction cell method’ [25–27]. All the experiments were carried out at 20 °C using an USP dissolution apparatus 2 (AT7 smart, Sotax, CH) equipped with Teflon Enhancer Cells (Agilent, USA) having a surface area of 4 cm2 and mounted with an 11 lm pore size filter paper (grade 1, Whatman, UK) as a membrane, since a membrane with large pores allows the sample to remain in the enhancer cell during the experiments without interfering with the drug release.

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Sample

Poloxamer (% w/ v)

NaCl (% w/ v)

Water/ethanol ratio in the medium

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20 20 20 20 20 20 20 20.5 21 21 21 21 21.5 22 22 22 22

0 0 0 0 0.9 1.75 3.5 0.9 0 0.9 1.75 3.5 0.9 0 0.9 1.75 3.5

50/50 65/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25 75/25

1 g of sample was placed in the reservoir of the Enhancer Cell, the membrane was placed on the cell cup and blocked with an oring; finally the reservoir was closed with a screw cap. The Enhancer Cell was left to equilibrate at 20 °C for 10 min and then placed in the dissolution vessels containing 1000 mL of a 0.15% sodium dodecyl sulfate (SDS) aqueous solution. The medium was selected according to data reported on the FDA database of dissolution methods [28] and the solubility of SDS at 20 °C. The paddles were rotated at 50 rpm and their position was adjusted to remain 2 cm above the top of the membrane throughout the study. 1 mL of samples was collected at 0, 15, 30, 60, 120, 180, 240, 300, 360 and 480 min and analyzed with the enzyme-immunoassay method (EIA) as described in the following section for the determination of plasma estradiol concentration during the in vivo studies. All the experiments were run in triplicate. 2.2.3. In vivo study in goldfish The sample preparation containing poloxamer 407 at a concentration of 21% w/w, prepared in an hydroalcoholic solution containing E2 (1.5 mg/mL) and 0.9% sodium chloride, was shown from the rheological analysis to be a superior system, and consequently selected for administration in goldfish (C. auratus). Sexually immature goldfish, ranging in size from 4 to 7 cm in length, were provided by a commercial fish farm (COF, Bologna, Italy). Male and female fish were used indistinctively. Before the start of the experiment, the animals were acclimatized for 2 weeks in glass aquaria with constant aeration and a natural photoperiod. Water quality parameters were monitored every 2 days, showing the following values: pH = 7.7, O2 = 4.5–6 ppm and temperature = 18–20 °C; level of nitrites (NO2) and ammonia (NH3) were shown to be undetectable. Fish were fed once daily in the morning with commercial dry pellets for goldfish. Following the acclimatization, experimental fish were selected and randomly divided into four aquaria (120 L) (n = 55 in each aquarium) each containing constantly aerated water, and the same characteristics as described for the acclimatization period. Before the start of the exposure period, fish were anaesthetized by immersion in 3-aminobenzoic acid ethyl ester methanesulfonate (MS 222; 0.1 g/L tap water), weighed and then given an intramuscular (i.m.) administration of E2 (15 lg E2/g body weight (BW)), either in the poloxamer thermosensitive gel system or dissolved in cornoil. The control fish groups received an i.m. injection of either poloxamer 407 or corn-oil without E2. The fish were returned to

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the aquaria, and for each time point at 0, 4, 8, 24, 48, 72, 96, 168, 264, 336 and 432 h following the injection, about 150 lL of blood was sampled from each fish in each group of 5 fish. For the blood sampling, animals were euthanized with MS 222 (0.5 g/L tap water) within 5 min after capture, and blood was immediately collected into freshly heparinized tubes. Blood samples were stored on ice bath until processed. After centrifugation, the plasma was frozen on dry ice and stored at 70 °C until assay. Animal manipulation was performed according to the recommendations of the University Ethical Committee, to the European Union directive (2010/63/EU) for animal experiments and under the supervision of the authorized investigators.

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2.2.3.1. Enzyme-immunoassay (EIA) for plasma estradiol. Plasma E2 levels were analyzed by an EIA method (Estradiol EIA kit, Cayman, United States), previously validated in the treated species [29]. The sensitivity of the assay was 8 pg/mL. E2 was extracted from the plasma samples with diethyl ether (Cold Spike Extraction) as described by the supplier.

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2.2.3.2. Pharmacokinetic analyses of plasma estradiol. The pharmacokinetic parameters of E2 were described by a non-compartment pharmacokinetic model [30]. The area under the plasma concentration curve to the last quantifiable time point (AUC0–t) was determined by linear trapezoidal integration with the terminal portion of the curve extrapolated to infinity (AUC0–1). AUC0–1 was calculated as AUC0–t + Ct/Kel; where the overall elimination rate constant (Kel) was calculated from the slope of the linear regression of the log-transformed concentration time curve data in the terminal phase. The endogenous plasma E2 concentration (baseline concentration) was subtracted from all after dosing serum E2 concentration.

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2.2.3.3. Plasma vitellogenin determination by enzyme-linked immunosorbent assay (ELISA). The VTG concentration in the plasma was assayed using a heterologous ELISA method previously validated in C. auratus [31]. VGT plasma levels in teleost fish are specifically associated with estrogen stimulation and can be considered a sensitive indicator of sexual development.

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2.2.3.4. Statistical analysis. Statistical comparisons were made using Student’s t-test and one-way analysis of variance (ANOVA). In all cases, a minimum level of significance of p < 0.05 was used. All statistical analyses were performed using ‘R statistical package’ [32]. All data were expressed as means ± S.D.

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3. Results and discussion

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3.1. Formulation of thermogelling systems

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The aim of this study was the development of a sustained release system for the i.m. administration of E2 for optimal sexual development in fish breading. The system should be easily injectable, i.e. it should be in the state of a sol at temperatures lower than 20 °C, but at the same time form a gel and be able to prolong the release of the drug, at temperatures at and higher than 20 °C. For this reason preparation and characterization of various systems based on poloxamer 407 were performed. E2 is very slightly soluble in water and consequently, in this work, a water/ethanol mixture was chosen as the vehicle. It is known from the literature, that ethanol greatly influences the gelling properties of aqueous systems containing poloxamers [33], so it was initially necessary to perform an evaluation of the effect of this solvent on the thermogelling properties of the polymer dispersion at 20% w/w. From previous studies, a 20% w/w poloxamer

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aqueous system was found to possess a gelation temperature of about 20 °C [24,34]. The results from the rheological analyses (Fig. 1) confirmed that the concentration of ethanol is a dominant factor affecting the poloxamer thermogelling properties. In particular, it can be seen that at a poloxamer concentration of 20% w/w, thermogelling was observed only for the lowest concentration of ethanol used, 25% w/w, with a phase-angle of 45° occurring at about 37 °C. For higher concentrations of ethanol (35% and 50% w/w), the system remained liquid-like and therefore unsuitable for the purpose of the study. The 25% w/w ethanol preparation, apart from allowing gelation to occur, also was shown to allow the solubilization of the dosage of E2 used in this study. At a concentration of 20% w/w, poloxamer 407 in combination with 25% w/w ethanol, due to the higher gelation point (37 °C), would not be suitable as thermogelling drug delivery system for species living at 20 °C. For this reason, a range of other concentrations of poloxamer 407 was evaluated maintaining the solvent composition of water/ethanol at 75:25% w/w with the further addition of NaCl at concentrations from 0.9% to 3.5% w/w. It was known that NaCl was able to alter the gelation properties of poloxamer dispersions, in particular the gel point [35]. An example of the temperature sweep traces for all systems prepared with poloxamer 407 at a concentration of 21% w/w and different concentrations of NaCl (0–3.5% w/w) are show in Fig. 2. It is evident, that the concentration of NaCl had a marked effect on the gelling properties of poloxamer 407 in water/ethanol 75:25. At increasing salt concentration, a remarkable reduction of the gel point was found, until the formulation at a NaCl concentration of 3.5% w/w had completely lost the thermogelling ability, behaving at all temperatures as a gel (phase angle always lower than 45°). It should also be noted, that the salt concentration only affected very slightly the complex viscosity of the systems, especially in the sol state. (Fig. 2). Similar results were reported also for poloxamer 407-lactose formulations [23] and could be explained by the interaction between water and poloxamer in the sol state. At temperatures lower than the gel point, molecules of poloxamer 407 exist as unimers or micelles, according to their concentration and temperature of the solution. The viscosity of the dispersion is due to the solvent-micelles or solvent–unimers interactions. It has previously been demonstrated that the presence of salts influences the evolution of the micellization process, while it has negligible effect on the initial and final species involved in the process, i.e. unimers and micelles [36].

Fig. 1. Effect of ethanol concentration on the modification of phase angle and complex viscosity of 20% poloxamer systems during a temperature sweep test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To obtain a better understanding of the combined effects of the different factors on the gelling properties of the formulations, it was decided to construct a contour plot using the data obtained from the temperature sweep traces, particularly the temperature corresponding to a phase angle of 45°. The contour plot (Fig. 3), obtained through the use of the software, OriginPro 8 SR1 (OriginLab Corporation, Northampton, USA), is able to describe in detail the behavior of such systems in the range of the prepared concentrations of poloxamer 407 and NaCl. The contour plot highlighted both the rheological effects and interactions between NaCl and poloxamer 407. In the concentration range used in these experiments the NaCl is the factor that mostly affected the gel point of the formulation, as can be seen by the more rapid change in the different colored bands related to the NaCl concentration as compared to the change in the bands related to poloxamer 407 concentration. It is also evident that there was a strong interaction between the two factors, since the variation of the gel point as a function of NaCl concentration changed much faster for poloxamer 407 concentrations higher than 21% w/w compared to lower concentrations. The most evident effect of this interaction is the presence of formulations showing gelling behavior even at 0 °C (experimental point defined ‘‘always in the gel state’’ in Fig. 3). These results therefore show a synergistic effect, relative to the gel point, between the increase of both poloxamer 407 and NaCl concentrations. The performance of the rheological analysis allowed the selection of the formulation composed of poloxamer 407 at 21% w/w and NaCl at 0.9% w/w as the most promising thermogel system for the delivery of the E2 to goldfish. Furthermore, diffusion of ethanol was never observed during in vitro tests on gelled samples at 20 °C (poloxamer 21% w/w 0.9% w/w NaCl and poloxamer 21% w/w 3.5% w/w NaCl) only in the preparation comprising poloxamer 21% w/w without NaCl (the only preparation that was liquid at the test temperature of 20 °C). Thus, the formulation composed of poloxamer 407 at 21% w/w and NaCl at 0.9% w/w was further characterized in term of rheological behavior through the use of viscometry tests (flow curves), performed in the temperature range from 5 to 25 °C. The results shown in Fig. 4 confirm the general behavior observed in the temperature sweep test in terms of gel point, allowing a deeper understanding of the rheological properties of the formulation. Particularly, the traces presented in Fig. 4 highlighted that the viscosity remained constantly low up to 15 °C (in the Fig. 4 can be observed an increases of viscosity from around 0.3 to 0.45 Pa s, which however has no practical relevance) and slightly increased (up to 2.5 Pa s, 5 times more the viscosity values up to 15 °C) until 20 °C was reached (‘power law viscosity’

Fig. 2. Effect of NaCl concentration on the modification of phase angle and complex viscosity of 21% poloxamer water–ethanol systems (ethanol 25%) during a temperature sweep test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M. Cespi et al., Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.08.008

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Fig. 3. Contour plot showing the effect of poloxamer and NaCl concentration on the gel point of the water–ethanol (75–25) systems. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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curve). From this temperature value a sharp increase of the ‘power law viscosity’ was seen, indicating the beginning of the gelling process. In the gelled sample the viscosity value is around 450 times greater the values measured at temperatures lower than 15 °C and around 75 times higher the values recorded at 20 °C. Furthermore, the results of the flow curve analysis also allowed the monitoring of the evolution of ‘yield stress’ values’ that gives an indication of the critical stress values necessary for the flow of the sample. The trend for this parameter is similar to that observed for the ‘power law viscosity’ although the sharp increase was observed at slightly lower temperature. Moreover, a sudden change of the sample flow behavior from a Newtonian to a nonNewtonian system can be seen in the temperature range 15–20 °C. In fact, the ‘power law index’ moves from values around one, typical of a Newtonian behavior, to values between 0 and 1, characteristic of a non-Newtonian system. Any fluids showing values of ‘‘power law index’’ lower than 1 and values of ‘yield stress’ higher than 0 can be defined as a plastic system, which is a specific case of non-Newtonian fluids. The different parameters recorded during the flow curve analysis suggest that the most suitable temperature range for convenient formulation administration, is from 5 to 10 °C, and that even if up to 15 °C injection of the formulation remains possible.

3.2. In vitro release of estradiol

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The formulation selected from the rheological analysis (poloxamer 21% w/w, NaCl 0.9% w/w in water–ethanol medium) was tested in terms of in vitro estradiol release from the thermogel and the results compared with two other formulations characterized by the same amount of poloxamer and ethanol while varying the percentages of NaCl (0% and 3.5% w/w). These two formulations were selected according to their rheological behavior as reported in Fig. 2. The formulation without NaCl behaved as a liquid solution up to 35 °C, while the other (3.5% w/w NaCl) independent of the temperature always was in the gelled state. The release of estradiol from the three formulations at 20 °C is shown in Fig. 5. The gelled formulations containing 0.9% and 3.5% w/w of NaCl gave comparable release profiles and were able to sustain the drug release up to 6 h. In this case, both the systems were in the gel form when tested, thus it appears that the differences in term of sample viscosity (of around 30%) at 20 °C did not affect the drug release. However, the sample without NaCl, liquid when tested (20 °C) and characterized by a very low viscosity (0.4 Pa s versus values higher than 2000 Pa s for the other systems), showed the slowest estradiol release, with only 70% of the drug being released at 8 h. This result was surprising taking into account the viscosity

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Fig. 4. Effect of temperature on power the law viscosity, power law index and yield stress of the selected system poloxamer 21%, NaCl 0.9% in water–ethanol (75–25) medium. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M. Cespi et al., Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.08.008

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of the tested samples and could most likely be explained by assuming a separation of the liquid system in a phase rich in poloxamer 407 and a phase rich in ethanol/water had taken place. Consequently, the estradiol (solubilized in the poloxamer micelles) would be released by the poloxamer rich phase which is much more viscous than the other gelled systems. This hypothesis was formulated after the analysis of a dye release from poloxamer systems, as reported in the Supplementary material and should be further evaluated in a more structured study. 3.3. Effect of drug delivery systems on estradiol and vitellogenin plasma levels The mean serum E2 levels after intramuscular (i.m.) administration of both thermogel and corn-oil formulations containing 75 lg of E2 (15 lg E2/g body weight) are shown in Fig. 6. The serum levels of E2 in fish treated with the non-E2 containing control formulations did not show any significant changes during experiments. A significant (p < 0.05) increase in E2 concentration (Cmax 4.20 ± 0.41 ng/mL at 96 h), was found in fish after i.m. injection of the E2 thermogel formulation compared to the oil-E2 formulation at each time point (Fig. 6). Overall, plasma E2 levels found in these experiments were comparable to those of mature female goldfish during vitellogenesis [37]. Furthermore, in the case of the thermogel formulation, a constantly elevated E2 concentration could be observed for up to 11 days, while later the concentration gradually decreased to control levels over the 18-day testing period. In contrast, it can be seen from Fig. 6, that following treatment with the oil-E2 formulation, plasma E2 levels reached the Cmax of 2.03 ± 0.36 ng/mL at 72 h after injection and then decreased rapidly returning to control values at 168 h (7 days). Despite the significant increase in E2 plasma levels observed in both groups of fish after treatments, fish injected with the E2 thermogel formulation showed significantly (p < 0.05) higher levels of the circulating hormone than the oil-E2 treated animals in the time frame of 72–264 h from injection. According to the non-compartmental pharmacokinetic models, we found that the thermogel formulation increased the AUC value compared to the oil-E2 solution group, and hence the relative bioavailability of E2 (Table 2). Previous studies, carried out by Chen et al. [18], using thermosensitive and phase sensitive polymer based delivery systems, showed that these formulations increased the relative bioavailability of steroidal hormones about 38-fold thus resulting in sustained release of testosterone after a single subcutaneous (s.c.) injection in rabbits. However, no information is available in the

Fig. 5. Effect of NaCl concentration on the in vitro release of estradiol from 21% poloxamer water–ethanol (75–25) systems. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Plasma levels of estradiol (E2) in juvenile goldfish injected through intramuscular (i.m.) route with E2 (15 lg E2/g body weight) dissolved either in the polymer solution (gel-E2) or corn-oil (oil-E2). Data are mean ± S.D. (n = 5 at each time point). Asterisks on values at the same time point indicate a significant difference (P < 0.05) between gel-E2 and oil-E2. The control groups (oil-control and gel-control) are shown just for reference and there were no statistically significant differences among the time points in that groups.

literature concerning the effectiveness of such thermosensitive systems as sustained release vehicle for steroidal hormones in fish. Thus, comparison of data with results from other groups is only possible for results derived from different controlled release devices such as implants. Implantation of fish with silastic implants containing E2 at a dose of 20 mg/kg BW for 12 days, resulted in an over twofold elevation of plasma E2 levels, reaching values similar to that of gonadal steroids during final gonadal maturation in sea bass [38]. Yamaguchi et al. [39] have shown that similar results could be obtained in seabream by implantation of silicone capsules containing small amounts of E2 (i.e. 400 lg). The comparison of the poloxamer 407 thermogel system developed here with silastic implants containing E2 shows a similar significant increase in plasma E2 levels around 10 days after implantation. Previous results from other groups have demonstrated that fish administered an intraperitoneal implant of coconut butter containing 10 lg E2/g body weight show a chronic increase in both the circulating levels of E2 and VTG [21,22]. Similarly, the increase in plasma levels of E2 observed in the present study was associated with elevated titers of VTG (Fig. 7) in both gel-E2 and oil-E2 injected fish. In fact, VTG levels increased to 1.2 mg/mL by 16 h and reached a maximum of 1.8 mg/mL within 72 h after gel-E2 injection. This 16-h time span for VTG synthesis was similar to that found in rainbow trout after intravascular injection of 17-aEthynylestradiol (EE2) [40], thus demonstrating that E2 dissolved in the thermogel formulations is rapidly distributed to the liver. Overall, VTG levels remained high over the entire time course suggesting a saturation of VTG formation. In this regard, VTG titers were found to be significantly (p < 0.05) higher after i.m. administration of the thermogel formulation containing E2 in comparison with oil-E2 treated groups at 168 and 336 h following injection. Using male sheepshead minnow, Bowman et al. [41] injected 5 mg/kg BW of E2 in triethylene glycol (TEG) and observed an increase in plasma VTG levels after 24 h, with peak values within 48 h. In the same work the authors also investigated the effect of a double injections of 5 mg/kg BW E2 in TEG showing that a maximum accumulation of plasma VTG was found at 1 day following the 2nd E2 injection. In addition, these data demonstrated a longer half-life of plasma VTG in the double injection experiment compared to the fish injected once. This result is similar to our findings, demonstrating that the thermogel formulation can control the release of E2 in vivo for an extended period of time. Thus, the present study suggests that thermosensitive polymer based deliv-

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Table 2 Pharmacokinetic parameters of estradiol (E2) in juvenile goldfish after a single intramuscular injection of E2 (15 lg E2/g body weight) dissolved either in the polymer solution (gel-E2) or corn-oil. n = 5 fish at each time point. Route of E2 administration

AUC0–t (ng h mL1)

AUC0–1 (ng h mL1)

Cmax (ng mL1)

Tmax (h)

t1/2 (h)

Gel-E2 Oil-E2

620.7 169.5

787.2 203.5

4.20 ± 0.41 2.03 ± 0.36

96 72

166.5 102.9

AUC0–t = area under the concentration–time curve from time 0 to the last measurable concentration; AUC0–1 = area under the concentration–time curve extrapolated to infinity; Cmax = maximum observed concentration; Tmax = observed time to the Cmax; t1/2 = apparent elimination half-time.

Fig. 7. Time course of the effects of intramuscular (i.m.) administration of estradiol (E2; 15 lg E2/g body weight) dissolved either in the polymer solution (gel-E2) or corn-oil (oil-E2) on plasma vitellogenin (VTG) levels of juvenile goldfish. Data are mean ± S.D. (n = 5 at each time point). Asterisks on values at the same time point indicate a significant difference (P < 0.05) between gel-E2 and oil-E2.

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ery systems could prevent rapid hepatic clearance of E2 resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish. Plasma VTG levels in fish are strongly related to the fish’s stage of reproductive development. Because of the specific association between VTG synthesis and estrogen stimulation, VTG is considered a highly sensitive indicator of sexual development in fish. Goldfish undergoes a clear reproductive cycle and spawns once a year in late April or early May. Prior to spawning time gonads are in a growth and maturation phase. In fish, the reproductive cycle is controlled by the reproductive hormones (mainly estrogen) and environmental factors. Endogenous E2, in reproductively active female, stimulates the liver to produce VTG, a yolk precursor protein, which is sequestered by developing oocytes. The liver of both males and females contain the necessary E2 receptors and genetic machinery for the production of VTG that in the plasma of female is present at high levels during egg maturation (early to mid recrudescence). Basically, our results demonstrate that E2 treatment is able to induce VTG synthesis and potential oocytes development in goldfish. The obtained VTG levels remained high for more days (i.e. over the entire time course) compared to the duration observed following both oil-E2 (this study) and double E2 injection experiments [37]. However, induction of VTG is an extremely dynamic process and there is still a lack of knowledge about induction and elimination rates of VTG that makes it difficult to accurately assess dose– response relationships between estrogen/xenoestrogen exposure and VTG formation.

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4. Conclusion

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This work describes the development of a sustained release formulation based on a poloxamer 407 thermogel system intended for E2 administration in fish. The formulation exhibited a gel point at

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20 °C and behaved as a Newtonian low viscosity system at temperature lower than 15 °C. The results demonstrate that a single i.m. injection of the thermogel formulation containing E2 can increase relative bioavailability of the hormone resulting in prolonged presence of high plasma VTG levels. E2 and VTG levels after i.m. injection of gelE2 remained high for more days compared to the duration observed following both oil-E2 (this study) and double E2 injection experiments [37]. Overall, the observed levels are comparable to those of mature vitellogenic female goldfish. Hence, this work demonstrates that the poloxamer 407 thermogelling system show the potential to work as a long-acting sustained release vehicle for steroidal hormones in fish. The fast preparation, the low cost of the main formulation components, the high versatility, together with the much easier administration compared to other prolonged release systems currently studied such as implants, make these systems particularly promising in the field of hormonal treatment in aquaculture.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2014.08.008.

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