PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation

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Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 323 – 333 www.nanomedjournal.com

Original Article: Experimental Nanomedicine, Pharmacology

PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation Madaswamy S. Muthu, MPharm a , Manoj K. Rawat, MPharm b , Amit Mishra, MPharm b , Sanjay Singh, PhD b,⁎ a

Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India b Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, India Received 9 May 2008; accepted 24 December 2008

Abstract The aim of this work was to develop extended-release poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles of risperidone and thermal-responsive in situ gel containing risperidone nanoparticles for parenteral (subcutaneous) delivery and to reduce the dosedependent extrapyramidal side effects of risperidone. PLGA nanoparticles of risperidone were designed by nanoprecipitation method using polymeric stabilizer (Poloxamer 407). The prepared nanoparticles were characterized for particle size by photon correlation spectroscopy and atomic force microscopy. Poloxamer 407–based in situ gel containing PLGA nanoparticles of risperidone was prepared by modified cold method to control the initial rapid release from the nanoparticles. The in vivo efficacy (antipsychotic effect) of prepared formulations (nanoparticles and in situ gel containing nanoparticles) was studied by administering them subcutaneously to mice. Extrapyramidal side effects of the formulations were also studied. The particle size of the prepared nanoparticles ranged between 85 and 219 nm. About 89% to 95% drug encapsulation efficiency was achieved when risperidone was loaded at 1.7% to 8.3% by weight of the polymer. During in vivo studies prepared risperidone formulations showed an antipsychotic effect that was significantly prolonged over that of risperidone solution for up to 72 hours with fewer extrapyramidal side effects. The prolonged effect of risperidone was obtained from the risperidone formulations administered subcutaneously, and this may improve the treatment of psychotic disorders by dose reduction. From the Clinical Editor: The development of extended-release poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles of risperidone is reported in this paper, along with the development of thermal-responsive in situ gel containing risperidone nanoparticles for parenteral (subcutaneous) delivery and to reduce the dose-dependent extrapyramidal side effects. In vivo studies showed a significantly prolonged antipsychotic effect with fewer extrapyramidal side effects. © 2009 Elsevier Inc. All rights reserved. Key words: Atomic force microscopy; Psychopharmacology; Photon correlation spectroscopy; Poloxamer 407; Poly(D,L-lactide-co-glycolide) nanoparticles; Risperidone

Biodegradable polymeric nanoparticles are of interest as vehicles for extended drug delivery and drug targeting.1-3 These nanoparticles have been investigated especially in drug delivery systems for drug targeting because of their particle size (ranging from 10 to 1000 nm) and long circulation in blood.4,5 This research was supported by University Grant Commission, New Delhi, India in terms of a Senior Research Fellowship. ⁎Corresponding author. Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – 221005, India. E-mail address: [email protected] (S. Singh).

Poly(D,L-lactide-co-glycolide) (PLGA), a biodegradable and biocompatible polymer, has been extensively used for developing an array of microparticulate and nanoparticulate drug delivery systems and has several advantages, such as good mechanical properties, low immunogenicity, low toxicity, excellent biocompatibility, and predictable biodegradation kinetics.6-9 Antipsychotic drugs can be of great benefit in a range of psychiatric disorders, including schizophrenia and bipolar disorder, but all are associated with a wide range of potential adverse effects. Generally, atypical antipsychotic agents cause fewer extrapyramidal side effects (EPS) than

1549-9634/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2008.12.003 Please cite this article as: M.S. Muthu, M.K. Rawat, A. Mishra, S. Singh, PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation. Nanomedicine: NBM 2009;5:323-333, doi:10.1016/j.nano.2008.12.003

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conventional antipsychotics. However, some atypical antipsychotics also have a relative risk of EPS.10-12 Risperidone, an atypical antipsychotic agent, has been used in the treatment of psychotic disorders. It has been approved by the U.S. Food and Drug administration (FDA) as an atypical antipsychotic agent, because it entails fewer EPS than conventional antipsychotics. However, EPS are dependent on dose of the risperidone.10 A low-dose risperidone therapy is required to control the psychotic symptoms, and long-term treatment is needed to treat schizophrenia. The use of this drug in the lowest possible effective dosage is recommended for minimizing therisk of the major adverse effects. The drug is practically insoluble in water and undergoes significant “first-pass” metabolism; oral bioavailability is 70% (coefficient of variation = 25%). The active metabolite of risperidone is 9-hydroxy risperidone. The half-lives of risperidone and its metabolite 9hydroxy risperidone are 3 and 21 hours, respectively.13-15 There has been a limited attempt to develop long-acting extended-release preparations of risperidone for the treatment of psychotic disorders. Recently, Rabin et al16 have reported implantable formulations of risperidone prepared using the biodegradable polymer PLGA combined with various drug loads. Implant bioactivity was tested using in vitro release and stability studies, as well as in vivo pharmacokinetic and behavioral studies in mice. This report suggests that implantable formulations are a viable approach to providing long-term delivery of antipsychotic medications based on in vivo animal studies and pharmacokinetics. Risperidone injectable formulation (microparticulate formulation of PLGA) was recently approved by the FDA as the first atypical long-acting antipsychotic medication. Longacting risperidone has been available in the high doses of 25 and 50 mg for intramuscular (IM) injection every 2 weeks. It has the drawback of lack of initial drug release (b1% of dose) for the first 3 weeks after injection, and hence requires oral antipsychotic supplementation for the first 3 weeks. Repeated injections (four injections, one injection every 2 weeks) are required to attain a steady-state level of drug in 6–8 weeks. The repeated injections result in higher incidences of adverse effects (EPS). Moreover, there is no dose reduction with the injection. The risperidone dose of 25–50 mg/14 days IM versus 2–4 mg/day via the oral route suggests that risperidoneloaded microparticulate injection achieves roughly a 1:1 dose relationship for oral to IM. Because this injection will produce no effect during the first 3 weeks of treatment of psychosis, it cannot be used for the short-term management (less than a month's duration) of manifestations of psychotic disorders.17 Presently, the conventional oral formulations of risperidone (2–4 mg/day) are the only available preparation for the shortterm management of manifestations of psychotic disorders. A dramatic dose reduction can be anticipated via parenteral administration of risperidone nanoparticles because of the avoidance of the first-pass effect and extended-release pattern. This has been true for haloperidol, wherein 1.5–3 mg/day IM is equivalent to 6–12 mg/day via the oral route.18 Compared with these conventional oral

formulations, extended-release PLGA nanoparticles of risperidone via the parenteral route will reduce the frequency of administration, as well as dose and dose-dependent EPS during the short-term management of psychotic disorders. Various investigations have been carried out for extended release of drugs through polymeric nanoparticles, but only limited studies have been focused on utilizing the potential of polymeric nanoparticles for extended drug delivery through the parenteral route.19 Some approaches have been used to improve the existing parenteral dosage forms of polymeric nanoparticles to increase their efficiency. One of these approaches is the utilization of Poloxamer 407 (PL-407)–based thermal-responsive in situ gels in combination with drug-loaded polymeric nanoparticles. PL-407 is a block copolymer comprising poly(oxyethylene) and poly(oxypropylene) segments with a molecular weight of approximately 12,500.20 The property of reversal thermal gelation shown by PL-407 aqueous solutions in the 20% to 35% concentration range has been used as a drug delivery system for parenteral use. Additionally, PL-407 can easily be administered as a solution, which forms a rigid semisolid gel (in situ gel) network upon an increase in temperature.21 This kind of “depot-like” extended-release in situ gel can control the rapid drug release from the polymeric nanoparticles intended for parenteral use. In our previous study we have prepared risperidone-loaded poly(ɛ–caprolactone) (PCL) nanoparticles with 5 mg of drug load and particle size ranging from 99 to 304 nm. Risperidone-loaded PCL nanoparticles were characterized using photon correlation spectroscopy (PCS) and transmission electron microscopy. Differential scanning calorimetry, x-ray diffraction, and electron diffraction ring pattern analysis of these nanoparticles showed amorphous state of risperidone in PCL nanoparticles. In vivo efficacy and EPS after intravenous administration of risperidone-loaded PCL nanoparticles showed prolonged antipsychotic effect with fewer EPS.22 In the present study, PLGA nanoparticles of risperidone were prepared with 10 and 5 mg of drug load and particle size ranging from 84 to 219 nm. Also, we reduced the rapid risperidone release using PL-407–based in situ gel system (20% w/v) to improve the in vivo efficacy. PLGA nanoparticles of risperidone were characterized using PCS and atomic force microscopy (AFM). In addition, in vivo efficacy and EPS after subcutaneous (SC) administration of PLGA nanoparticles of risperidone and in situ gel containing PLGA nanoparticles of risperidone were also studied.

Methods Materials Risperidone was obtained from APL Research Centre (Hyderabad, India) as a gift sample. PLGA (lactate and glycolate in the ratio of 85:15), PL-407, apomorphine hydrochloride, and a dialysis bag with a 12,000 molecular weight cutoff were purchased from Sigma-Aldrich

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Chemicals Private Ltd. (Bangalore, India). All other chemicals were of analytical grade. Preparation of nanoparticles Nanoparticles (nanospheres) were prepared by nanoprecipitation according to the method developed by Fessi et al23 PLGA was dissolved in acetone (25 mL) at 45°C. Volume changes due to evaporation of acetone were adjusted at room temperature (25° to 30°C), and then risperidone was added and dissolved. This organic solution was injected at the rate of 48 mL/min in distilled water (50 mL) containing PL-407 (0.25 g) under magnetic stirring at room temperature. Acetone and some proportion of water were eliminated at 40°C under reduced pressure, and the final volume of the aqueous suspension was adjusted to 10 mL. Final aqueous nanosuspension (containing nanoparticles) was used for further characterizations. A total of 0.25 g of PL-407 was used when preparing PLGA nanoparticles of risperidone, and the same amount was present in the final 10 mL of risperidone nanoparticles suspension. Preparation of PL-407 in situ gel containing risperidone PL-407 in situ gel containing risperidone solution was prepared by the cold method.20 A weighed amount of PL-407 (2.5 g) was slowly added to a cold (5° to 10°C) distilled water (9 mL) with gentle mixing until complete dissolution of the polymer occurred. Accurately weighed risperidone (5 mg in 1 mL of distilled water acidified with lactic acid solution) was added slowly in the previously prepared polymer solution with stirring. The final quantity was made up to 12.5 mL with distilled water. The prepared in situ gel (20% w/v of PL-407 in situ gel) was kept for 24 hours at room temperature for complete polymer desolvation. Final concentration of risperidone in the PL-407 in situ gel was 0.4 mg/mL. Preparation of PL-407 in situ gel containing risperidone nanoparticles PL-407 in situ gel containing risperidone nanoparticles was prepared by the modified cold method.20 A weighed amount of PL-407 (2.25 g) was slowly added to a cold (5° to 10°C) risperidone nanosuspension containing 0.25 g of PL-407 (10 mL of batch P2805) for complete dissolution of the polymer. The final quantity was made up to 12.5 mL with distilled water. The prepared in situ gel was kept for polymer desolvation according to the method described above. The final concentration of risperidone in the PL-407 in situ gel containing PLGA nanoparticles of risperidone was 0.4 mg/mL. Nanoparticles characterizations Particle size analysis and polydispersity Particle size analysis of nanoparticles was performed by PCS. This technique yields the mean particle diameter and particle size distribution. To analyze particle size, nanosuspensions were diluted five times with filtered (0.22 μm) ultrapure water. Samples were analyzed using Mastersizer

325

2000 (Malvern Instruments, Malvern, UK), which allows sample measurement in the range of 0.020–2000.00 μm. Polydispersity was determined according to the equation below: Polydispersity =

Dð0:9Þ
Dð0:1Þ Dð0:5Þ

where D(0.9) corresponds to particle size immediately above the sizes of 90% of the nanoparticles, D(0.5) corresponds to the particle size immediately above the sizes of 50% of the nanoparticles, and D(0.1) corresponds to the particle size immediately above the sizes of 10% of the nanoparticles.24 Characterization by atomic force microscopy The surface properties of drug loaded nanoparticles were visualized by an atomic force microscope (Solver P-47-PRO, MDT; Moscow, Russia) under normal atmospheric conditions. Explorer atomic force microscope was in tapping mode, using high-resonant-frequency (F0 = 241 kHz) pyramidal cantilevers with silicon probes having force constants of 41 N/m. Scan speeds were set at 2 Hz. The samples were diluted 10 times with distilled water and then dropped onto glass slides, followed by vacuum drying during 24 hours at 25°C. Height measurements were obtained using AFM image analysis software (NT-MDT; Moscow, Russia). Determination of total drug content of nanosuspension The total drug amount in nanosuspension was determined spectrophotometrically (Jasco, Model 7800; Tokyo, Japan). 25 A 0.50-mL aliquot of nanosuspension was evaporated to dryness under reduced pressure at 35°C. The residue was dissolved in dichloromethane and filtered with a 0.45-μm filter to remove any possible insoluble impurities, and risperidone content was assayed spectrophotometrically at 279 nm (λmax). The calculation was performed as follows: Vol: total Vol: aliquot  Drug amount in aliquot

Total drug content ¼

where Vol. total/Vol. aliquot is the ratio of the aliquot to the total nanosuspension volume. Free dissolved drug in the nanosuspensions and encapsulation efficiency of nanoparticles The total drug content in nanosuspension was calculated as described earlier. Free dissolved drug amount present in the nanosuspension was determined by bulk equilibrium reverse dialysis bag technique, as described by Singh and Muthu.26 Briefly, a dialysis bag (cellulose membrane, molecular weight cutoff 12,000) containing 1 mL of distilled water with 25 mg PL-407 was placed directly into 10 mL of nanosuspension. After equilibrium (6 hours), the dialysis bag was withdrawn from the nanosuspension. The sample collected from the dialysis

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bag was assayed spectrophotometrically at 275 nm (λmax) for risperidone content. The calculation was performed as follows: Free dissolved drug ¼ ðTotal volume=Volume of dialysis bagÞ  Drug amount in the dialysis bag where Total volume is the sum of total volume of the nanosuspension and volume of the dialysis bag sample. Encapsulation efficiency was calculated as indicated below:

Table 1 Formulas of nanoparticles used in risperidone studies Batches

Risperidone (mg)

PLGA (mg)

PL-407 (g)

Acetone (mL)

Water (mL)

P12010

10

120

0.25

25

50

P20010

10

200

0.25

25

50

P28010

10

280

0.25

25

50

P1205

5

120

0.25

25

50

P2005

5

200

0.25

25

50

P2805

5

280

0.25

25

50

Encapsulation efficiency ð%Þ =

Total drug content − Free dissolved drug  100 Drug amount used

In vitro drug release studies The dialysis bag diffusion technique was used to study the in vitro drug release of risperidone nanoparticles.27,28 Either 1 mL of risperidone formulations (nanoparticles; in situ gel containing nanoparticles) or risperidone control preparations (solution; in situ gel) were placed in the dialysis bag (cellulose membrane, molecular weight cutoff 12,000), hermetically sealed, and immersed into 50 mL of phosphate-buffered saline (PBS), pH 7.4. The entire system was kept at 37° ± 0.5°C with continuous magnetic stirring at 200 rpm. Samples were withdrawn from the receptor compartment at predetermined time intervals and replaced by fresh medium. The amount of drug dissolved was determined with ultraviolet spectrophotometry at 275 nm. Since risperidone is insoluble in water, risperidone solutions (0.5 mg/mL and 1 mg/mL) in 50% (w/w) mixture of polyethylene glycol 400 and water (acidified with lactic acid) were used as controls for the in vitro drug release studies of PLGA nanoparticles of risperidone. Only 0.4 mg/mL risperidone-containing PL-407–based in situ gel was used as control for the in vitro drug release study of in situ gel containing PLGA nanoparticles of risperidone. Animals and in vivo evaluation of the efficacy of risperidone formulations Animals Swiss albino mice (23 ± 2 g) (Institute of Medical Sciences, Banaras Hindu University, India) were used to study the effect of PLGA nanoparticles of risperidone and in situ gel containing nanoparticles on the controlled inhibition of apomorphine-induced climbing and sniffing. EPS liability of the risperidone solution and its formulations were studied using catalepsy in mice. Before experimentation they were kept in groups of six and fed with standard diet with water ad libitum. All experimental procedures were reviewed and approved by the Animal and Ethics Review Committee of the Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, India.

Apomorphine induced climbing and sniffing Animals were divided into four groups of five animals (n = 5). Three groups were administered test compounds (two types of formulations: batch P2805; 20% w/v PL-407– based in situ gel containing batch P2805), one risperidone solution at a dose of 5 mg/kg via SC route, and one group as a saline control. PLGA nanoparticles of risperidone were administered to mice for delivering approximately 5 mg/kg of risperidone during 3 days. This dose was selected based on previous studies using mini-pumps in rodents.29,30 The modified method of Fray et al31 was used for behavioral observation, combined with a time-sampling procedure.32 Each animal was placed into cylindrical wiremesh cages (diameter, 14 cm; height, 13 cm; mesh size, 3 mm) and allowed to adapt for 60 minutes. Thereafter, mice were injected eight times, first with test compound or saline via SC route, followed by SC injection of apomorphine (2.5 mg/kg) at different time intervals (1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours later than the first injection). The first injection consisted of test compounds or saline, and injections 2 to 8 were apomorphine injections. Behavioral observations were made from 11 to 20 minutes after the each apomorphine injection: animals were observed for 10 seconds every minute for the presence or absence of climbing (i.e., all four paws on the cage, above the floor). Sniffing was scored when the animal showed uninterrupted sniffing for at least 3 seconds during this 10-second sampling period. The mice were observed for climbing behavior and scored as follows: 0 = presence of four paws on the floor; 1 = presence of four paws on the cage. The mice were observed for sniffing behavior and scored as follows: 0 = interrupted sniffing; 1 = uninterrupted sniffing for at least 3 seconds. Thus, the score for climbing or sniffing could vary from 0 to 10 for the entire observation period. Catalepsy Catalepsy studies were carried out in Swiss albino mice. They were divided into four groups of five animals (n = 5) and administered two types of formulations (batch P2805; 20% w/v PL-407–based in situ gel containing batch P2805),

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Table 2 Characterization of risperidone nanoparticles Batches P12010

Particle size (nm) (mean ± SD⁎) 84.1 ± 2.2 †

Polydispersity (mean ± SD⁎)

Total drug content (mg) (mean ± SD⁎)

Free dissolved drug (mg) (mean ± S.D ⁎)

Encapsulation efficiency (%) (mean ± SD⁎)

0.7823 ± 0.029

9.90 ± 0.01

0.71 ± 0.01

91.90 ± 0.05

║

P20010

177.3 ± 3.5

0.7419 ± 0.023

9.96 ± 0.03

0.49 ± 0.03

94.70 ± 0.68

P28010

219.1 ± 4.7 †

0.6155 ± 0.056

9.99 ± 0.05 ║

0.48 ± 0.01

95.10 ± 0.76

P1205

85.4 ± 3.3

0.8991 ± 0.029

4.96 ± 0.01

0.63 ± 0.04

86.60 ± 0.72

P2005

173.1 ± 3.1 ‡

0.7417 ± 0.023

4.98 ± 0.02 ¶

0.55 ± 0.02

88.60 ± 1.21

0.7314 ± 0.056

¶

0.52 ± 0.05

89.40 ± 1.24

P2805

216.1 ± 3.6

‡

4.99 ± 0.02

⁎n = 3. † Significant (P b .05) difference in the mean values compared with batch P12010 using Student's t-test. ‡ Significant (P b .05) difference in the mean values compared with batch P1205 using Student's t-test. ║ No significant (P N .05) difference in the mean values compared with batch P12010 using Student's t-test. ¶ No significant (P N .05) difference in the mean values compared with batch P1205 using Student's t-test.

one risperidone solution at a dose of 5 mg/kg via SC route, and one group as a saline control. Catalepsy procedure was measured using the bar test 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after the SC injections (test compound or saline): the forelimbs were placed on a cylindrical bar (diameter, 0.4 cm; 3.5 cm above the table), and the time during which both forelimbs remained on the bar was recorded up to a maximum of 30 seconds. Statistical analysis Results are given as mean ± standard deviation (SD). Mean values of nanoparticles size and total drug content were compared using the Student's t-test. Differences are considered significant at a level of P b .05. In vivo data were analyzed with a one-way analysis of variance (ANOVA) followed by Dunnett's test (control and test compounds) or Tukey's test (between the test compounds). Differences are considered significant at a level of P b .05. Results Preparations, particle size, and polydispersity analysis In the present work PLGA nanoparticles of risperidone were prepared by the nanoprecipitation method. Nanoparticles formulas were established with different risperidone and polymer concentration level so as to obtain higher encapsulation efficiency, desired particle size, and suitable drug release kinetics (Table 1). Concentration of PL-407 was also optimized and selected to obtain stable nanosuspensions. Six replicates were carried out for each formulation batch (n = 6). Three batches were used for particle size analysis, in vitro drug release, and total drug content determination. Three batches were used for free dissolved drug determination. PL-407–based in situ gel containing PLGA nanoparticles of risperidone was prepared by modified cold method to control the initial rapid release from the nanoparticles.

The mean particle size and polydispersity of PLGA nanoparticles of risperidone are shown in Table 2. PCS measurements were undertaken in multimodal analysis to obtain a true reflection of particle size distribution. The particle size distribution curves for all the samples were unimodal. Nanoparticles size and population SD were 84.1 ± 2.2, 177.3 ± 3.5 and 219.1 ± 4.7 nm for batches P12010, P20010 and P28010, respectively. The smallest particles of 84.1 ± 2.2 nm were found in batch P12010 (120 mg PLGA content), and the largest particles of 219.1 ± 4.7 nm were seen in batch P28010 (280 mg PLGA content) (Table 2). Differences between particle sizes of these batches were significant (P b .05). A high concentration of PLGA in the organic phase led to an increase in the size of the nanoparticles. A similar trend was found in batches P1205 to P2805 (Table 2). The 10 and 5 milligrams of risperidone-loaded PLGA nanoparticles did not show any difference in particle size when the same quantity of PLGA was used. The polydispersity of the nanoparticles decreased with an increase in PLGA concentration and particle size of the nanoparticles (Table 2). Atomic force microscopy study AFM images of batch P2805 (Figure 1, A and B) show smooth nanoparticle surface without any noticeable pinholes or cracks. AFM also revealed that all nanoparticles were spherical in shape and below 300 nm in size. The nanoparticles size as observed by AFM correlated well with the size measured by PCS. Determination of total drug content, free dissolved drug content of nanosuspension, and encapsulation efficiency of nanoparticles The total drug content in the nanosuspensions varied from 9.90 ± 0.01 to 9.99 ± 0.05 mg (P12010 to P28010) and 4.96 ± 0.01 to 4.99 ± 0.02 mg (P1205 to P2805). The total drug content in the nanosuspension did not show any significant

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Figure 1. AFM image of PLGA nanoparticles of risperidone (A) multiple particles. (B) Three-dimensional image showing size.

difference (P N .05) with increase in the concentration of PLGA (Table 2). The amount of free dissolved drug in the nanosuspensions ranged from 0.48 ± 0.01 mg to 0.71 ± 0.01 mg (P12010 to

P28010) and 0.52 ± 0.05 mg to 0.63 ± 0.04 mg (P1205 to P2805). This was due to the limited solubility of risperidone in the aqueous phase. Free dissolved drug in the nanosuspension decreased with increase in the PLGA concentration

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329

Figure 2. In vitro drug release of (A) risperidone nanoparticles and control risperidone solution (1 mg/mL) in PBS (pH 7.4). (B) Risperidone nanoparticles and control risperidone solution (0.5 mg/mL) in PBS (pH 7.4). Vertical bars represent ± SD (n = 3).

Figure 3. In vitro drug release of (A) risperidone control solution and in situ gel containing risperidone (0.4 mg/mL) in PBS (pH 7.4). (B) Risperidone nanoparticles (batch P2805) and in situ gel containing risperidone nanoparticles (batch P2805) (0.4 mg/mL) in PBS (pH 7.4). Vertical bars represent ± SD (n = 3).

(Table 2). Encapsulation efficiency was also inversely related to free dissolved drug. However, encapsulation efficiency was found to increase with increase in the PLGA amount (Table 2).

(batches from P1205 to P2805) extended the release up to 72 hours (Figure 2, B). The effect of drug loading on risperidone release was also studied. There was a difference in the release pattern of 10 and 5 mg of risperidone-loaded nanoparticles. The t75% values for batches P12010 and P1205 were 4 and 16 hours, respectively in PBS (pH 7.4). Risperidone release was found to extend with decrease in the drug loading (Figure 2, A, B). The PLGA content also affected the drug release kinetics. The increase in PLGA content in the nanosuspensions resulted in slower kinetics. The t75% values in PBS (pH 7.4) were about 4 hours, 8 hours, and 16 hours for batches P12010, P20010, and P28010 containing 120, 200, and 280 mg of PLGA, respectively (Figure 2, A). Similar results were obtained for batches P1205 to P2805 (Figure 2, B). The in vitro release of risperidone from the risperidone control solution and risperidone in the in situ gel formulations

In vitro drug release studies Figure 2, A and B shows the percentage release of risperidone from different batches (P12010 to P28010) and (P1205 to P2805), respectively in PBS (pH 7.4). The release profile of risperidone control solution indicates very rapid diffusion of risperidone with nearly 90% release in 4 hours. The nanosuspensions (batches from P12010 to P28010) initially showed rapid release followed by extended release. After 24 hours of dialysis in PBS (pH 7.4) the percentages of risperidone released were 97%, 91%, and 82% for batches P12010, P20010, and P28010, respectively (Figure 2, A), and for batches P1205, P2005, and P2805 it was 78%, 76%, and 71%, respectively. The nanosuspensions

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Table 3 Ranges of risperidone released during in vitro drug release studies from risperidone solution and risperidone formulations used for in vivo studies

Table 5 Effect of risperidone nanoparticles and in situ gel containing risperidone nanoparticles on inhibition of apomorphine-induced sniffing in mice ⁎

Time (hours)

Up to 1

% Drug release (mean value) Risperidone solution

Batch P2805

Batch P2805 in gel

Time Control and test compounds (via SC route) (hours) Saline Risperidone Batch P2805 control solution (5 mg/kg) (5 mg/kg)

67.70

38.98

23.10

1

9.4 ± 0.54

Batch P2805 in gel (5 mg/kg)

0 ± 0.00 †

0.8 ± 0.83 †

0.2 ± 0.00 †

†

†

1.2 ± 1.00 †

1–4

21.62

11.76

7.24

4

9.4 ± 0.54 0.8 ± 0.83

4–8

10.51

9.49

11.78

8

9.0 ± 1.41 3.6 ± 2.19 †

1.4 ± 1.14 †

1.6 ± 0.83 †

†

†

1.8 ± 0.83 †

1.0 ± 0.70 †

1.6 ± 1.22 †

8–12

0

4.37

14.74

12

9.0 ± 1.22 5.2 ± 2.38

12–24

0

7.23

5.14

24

9.2 ± 0.83 9.0 ± 0.70

2.0 ± 2.34

1.2 ± 1.64

†, ‡

24–48

0

9.77

6.29

48

9.4 ± 1.34 9.4 ± 0.89

5.0 ± 1.07

48–72

0

10.23

7.43

72

9.2 ± 1.30 9.0 ± 1.22

6.0 ± 0.54 †

Table 4 Effect of risperidone nanoparticles and in situ gel containing risperidone nanoparticles on inhibition of apomorphine-induced climbing in mice ⁎ Time Control and test compounds (via SC route) (hours) Saline Risperidone Batch P2805 control solution (5 mg/kg) (5 mg/kg) 1

10 ± 0.00

Batch P2805 in gel (5 mg/kg)

0 ± 0.0 †

0 ± 0.00 †

0 ± 0.00 †

†

†

†

4

9.4 ± 0.54

8

9.6 ± 0.89 1.6 ± 1.67 †

0 ± 0.0

1.2 ± 1.09 †

0 ± 0.00

1.6 ± 0.54 †

†

†

†

1.4 ± 1.14

0 ± 0.00

12

9.2 ± 0.44 5.6 ± 1.14

24

9.8 ± 0.44 8.8 ± 0.83

2.4 ± 0.54 †

1.2 ± 0.83

0.6 ± 0.54 †

48

9.6 ± 0.44 9.0 ± 0.83

4.8 ± 0.92 †, ‡

2.0 ± 0.41 †, ‡

72

9.6 ± 0.54 9.4 ± 0.54

4.8 ± 0.30 †, ‡

1.6 ± 0.34 †, ‡

⁎Values represent the mean ± SD behavioral score of five animals (n = 5) during observation periods (10 seconds every minute, from 10 to 20 minutes after each apomorphine injection). Apomorphine (2.5 mg/kg SC) was administered 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after different test compounds or saline control. † P b .05 compared with saline control group using Dunnett's test, following significant one-way ANOVA. ‡ P b .05 compared between the batches of P2805 and P2805 in gel using Tukey's test, following significant one-way ANOVA.

are shown in Figure 3, A. The cumulative percentage of risperidone released at 8 hours from risperidone solution and PL-407 in situ gel were 98% and 44%, respectively. The risperidone in situ gel initially showed rapid release (up to 2 hours) followed by controlled release (from 2 to 24 hours) (Figure 3, A). The in vitro release of risperidone from the PLGA nanoparticles of risperidone (batch P2805) and in situ gel containing PLGA nanoparticles of risperidone (batch P2805 in the in situ gel) are shown in Figure 3, B and Table 3. The cumulative percentage of risperidone released at 8 hours from PLGA nanoparticles of risperidone and in situ gel containing PLGA nanoparticles of risperidone were 64% and 42%, respectively. Compared with risperidone nanoparticles (batch P2805), batch P2805 in the in situ gel

0.6 ± 0.39 †, ‡ 0.8 ± 0.70 †, ‡

⁎Values represent the mean ± SD behavioral score of five animals (n = 5) during observation periods (10 seconds every minute, from 10 to 20 minutes after each apomorphine injection). Apomorphine (2.5 mg/kg SC) was administered 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after different test compounds or saline control. † P b .05 compared with saline control group using Dunnett's test, following significant one-way ANOVA. ‡ P b .05 compared between the batches of P2805 and P2805 in gel using Tukey's test, following significant one-way ANOVA.

system controlled the initial rapid release of risperidone from nanoparticles. In vivo evaluation of the efficacy of risperidone formulations Antagonism of apomorphine-induced climbing and sniffing In vivo pharmacodynamic evaluation of PLGA nanoparticles of risperidone and in situ gel containing nanoparticles were carried out with an animal model—Swiss albino mice. The in vivo effects of risperidone administration as nanoparticles, in situ gel containing nanoparticles, and the risperidone solution are presented in Tables 4 and 5. Risperidone solution significantly (P b .05) inhibited apomorphine-induced climbing and sniffing behavior up to 12 hours as compared with saline control (Tables 4 and 5). PLGA nanoparticles of risperidone (batch P2805) significantly (P b .05) inhibited apomorphine-induced climbing and sniffing behavior up to 72 hours as compared with saline control (Tables 4 and 5). PLGA nanoparticles of risperidone in the in situ gel system (batch P2805 in the in situ gel) also significantly (P b .05) inhibited apomorphineinduced climbing and sniffing behavior up to 72 hours as compared with saline control (Tables 4 and 5). Compared with risperidone solution, PLGA nanoparticles of risperidone and PLGA nanoparticles of risperidone in the in situ gel system showed significantly (Pb .05) prolonged inhibition (from 12 hours to 72 hours) of climbing and sniffing (Tables 4 and 5). The control of initial rapid release from the risperidone nanoparticles using in situ gel system (batch P2805 in the in situ gel) changed the therapeutic activity. The significant (P b .05) changes in the climbing and sniffing at 24 hours and 48 hours (using Tukey's test) were observed between the

M.S. Muthu et al / Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 323–333 Table 6 Effect of risperidone nanoparticles and in situ gel containing risperidone nanoparticles on reduction in catalepsy in mice

331

Discussion

aqueous medium containing surfactant (PL-407), which resulted in the formation of nanoparticles (polymeric matrix with drug). Polymeric aggregates did not form during the preparation of nanoparticles by nanoprecipitation method. Therefore, the ratios of drug to polymer were the same as the initial ratio. Risperidone was adsorbed on the nanoparticles and/or dispersed into the polymeric matrix of nanoparticles. This may be due to precipitation of drug when put into aqueous phase because of its limited solubility (140 μg/mL at 37°C).22,26 The speed of nanoparticles solidification by nanoprecipitation method allows the drug to be rapidly entrapped, thus preventing its diffusion into the outer phase.33 The presence of some portion of free drug outside the nanoparticles is also indicated by rapid initial in vitro drug release (Figure 2, A, B). Major requirements for nanoparticles formulation intended for parenteral use include biocompatibility of its ingredients and suitable drug carrier size. PL-407 had been used for stabilization of PLGA nanoparticles.34 PL-407 is regarded as nontoxic for parenteral use; therefore, no special purification step is required for its elimination from the final formulation.25 Results of this study show that the nanoparticles size is influenced by formulative variables such as concentration of the polymer. The effect of the polymer concentration on the nanoparticle size may be due to the higher resultant organic phase viscosity, which leads to larger nanodroplet formation.35 In general, the PL-407 in situ gel formation occurs as a result of the progressive dehydration of the polymer micelles as temperature increases, leading to increased chain entanglement. This entanglement is more marked at higher concentration of PL-407, yielding an increase of in situ gel strength and consequently, a decrease of the release rate. The release of risperidone from the in situ gel was observed to follow zero-order release kinetics (between 2 and 24 hours) (Figure 3, A). Zero-order release profiles suggest diffusion as the predominant mechanisms for drug release.21 A total of 2.5 g of PL-407 were used when preparing PL407 in situ gel containing risperidone solution to obtain 20% (w/v) of PL-407 in situ gel. Because the final 10 mL of nanoparticles suspension contain 0.25 g of PL-407, the remaining 2.25 g of PL-407 was used for the preparation of PL-407 in situ gel containing risperidone nanoparticles to make up the final concentration of 20% (w/v).

Preparations, particle size, and polydispersity analysis

In vitro drug release studies

PLGA nanoparticles of risperidone were prepared by the nanoprecipitation method using different formulation variables. The nanoprecipitation protocols utilize an organic phase composed of volatile solvents, whose elimination is readily achieved by evaporation. Risperidone was dissolved in acetone along with the different quantities of PLGA with different drug loading (10 and 5 mg) and introduced into

The release of drug from polymeric nanoparticles was studied by dialysis bag diffusion technique. This is a method commonly used to study the release of drugs from colloidal suspensions.27 Because the aim of the study was to administer the nanoparticles by the SC route, the release studies were carried out with PBS (pH 7.4) to mimic the in vivo conditions.

Time Control and test compounds (via SC route) (hours) Saline Risperidone Batch P2805 control solution (5 mg/kg) (5 mg/kg)

Batch P2805 in gel (5 mg/kg)

1

1.2 ± 0.83

30 ± 0.00

28.4 ± 2.30

27.8 ± 2.16

4

1.0 ± 0.70

30 ± 0.00

19.4 ± 2.30 †, ‡ 12.8 ± 1.32 †, ‡

8

1.4 ± 0.54 17.0 ± 0.70

13.2 ± 1.01 †, ‡

7.2 ± 0.86 †, ‡

†

1.4 ± 1.14 †

12

1.0 ± 0.70 12.8 ± 4.14

0.6 ± 0.54

24

1.0 ± 0.70 10.0 ± 1.58

2.0 ± 1.41 †

1.6 ± 1.14 †

48

1.2 ± 0.44

2.4 ± 1.14

2.6 ± 1.94

2.6 ± 2.07

72

1.4 ± 0.54

2.0 ± 0.70

1.8 ± 1.78

2.0 ± 2.00

⁎Values represent the duration of catalepsy in seconds and are expressed as mean ± SD, (n = 5). Duration of catalepsy was observed 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after administration of different test compounds or saline control. † P b .05 compared with risperidone solution–treated group using Dunnett's test, following significant one-way ANOVA. † P b .05 compared between the batches of P2805 and P2805 in gel using Tukey's test, following significant one-way ANOVA.

formulations of batch P2805 and batch P2805 in the in situ gel system, having a difference in the release rate. Compared with risperidone nanoparticles (P2805), P2805 in the in situ gel system controlled the initial rapid release of risperidone from nanoparticles and showed the maximum inhibition in the apomorphine-induced climbing and sniffing behavior (Tables 4 and 5). Catalepsy The EPS liabilities of risperidone administration as nanoparticles, nanoparticles in the in situ gel system, and solution are presented in Table 6. PLGA nanoparticles of risperidone (batch P2805) and nanoparticles in the in situ gel system (batch P2805 in the in situ gel) significantly (P b .05) reduced catalepsy at 4 hours, 8 hours, 12 hours, and 24 hours as compared with risperidone solution (Table 6). There were significant (P b .05) changes in the catalepsy (using Tukey's test) at 4 hours and 8 hours between the formulations P2805 and P2805 in the in situ gel (Table 6). The possible reason for absence of catalepsy for risperidone solution after 24 hours could be the elimination of active molecules.

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The release rate of the risperidone from the nanoparticles and its appearance in the dissolution medium was governed by the partition coefficient of the drug between the polymeric phase and the aqueous environment in the dialysis bag and by the diffusion of the drug across the membrane as well. The dialysis bag retained the nanoparticles and allowed the diffusion of the drug immediately into the receiver compartment. The nanoparticle size was also associated with changes in drug release kinetics. The smaller sized nanoparticles prepared with lower amounts of PLGA showed higher drug release rates. This release behavior may be explained by a corresponding increase in the total nanoparticles surface, resulting in a larger drug fraction exposed to the leaching medium. Smaller nanoparticles size also leads to a shorter average diffusion path of the matrix-entrapped drug molecules.36 Initial rapid effect on release of risperidone from different nanoparticles batches (P1205 to P28010) (Figure 2, A, B) may be due to the free dissolved drug observed with nanosuspensions and free drug adsorbed on the nanoparticles. The release of drug adsorbed on the nanoparticle surface was considerably faster compared with the encapsulated drug. The increase of PLGA content in the risperidone nanosuspensions results in large size of the polymeric nanoparticles (Table 2), improves the encapsulation of the drug (Table 2), and results in slower drug release (Figure 2, A, B). The batch P2805 containing PLGA nanoparticles of risperidone showed maximum extended drug release. Therefore, batch P2805 was selected for the preparation of in situ gel system, and these preparations (i.e., batch P2805 and batch P2805 in the in situ gel system) were studied for in vivo effects. In vivo evaluation of the efficacy of risperidone formulations The atypical antipsychotic agent risperidone was used because it is among the most appropriate choices for a long-term delivery system based on efficacy, potency, stability, side effect/risk profile, and local subcutaneous in vivo tolerability.16 Pharmacodynamic studies of risperidone and its formulations were carried out to find the antipsychotic effect and any adverse effect after SC adminstration. Atypical antipsychotic drugs are tested for the inhibition of apomorphine-induced climbing and sniffing (for antipsychotic activity) and catalepsy tests (for EPS liability), because these tests have high predictive accuracy. Pharmacodynamic activity by this model is predictive of efficacy against the positive symptoms of psychosis37 and demonstrates prolonged in vivo antagonist activity at dopamine D2 receptors of risperidone-loaded PLGA formulations. Inhibition of apomorphine-induced climbing and sniffing was found to be less for batch P2805 than batch P2805 in the in situ gel system. The initial rapid release of the

risperidone from the batch P2805 leads to changes in the activity of formulation. Antipsychotic effect was observed up to 12, 72, and 72 hours after SC injection of risperidone solution, PLGA nanoparticles of risperidone, and in situ gel containing PLGA nanoparticles of risperidone, respectively. Because the risperidone has a short half-life, the prolonged antipsychotic effect may be due to the role of active metabolite 9-hydroxy risperidone, which has a long half-life. The active metabolite formed may accumulate during the extended release of risperidone from nanoparticles. The batch P2805 in the in situ gel system containing PLGA nanoparticles of risperidone with maximum particle size extended the drug release for 72 hours with minimum rapid initial release (Figure 3, B), showed maximum therapeutic activity (Tables 4 and 5), and resulted in maximum reduction in EPS (Table 6). Tables 3 to 5 show that PLGA nanoparticles of risperidone and in situ gel containing PLGA nanoparticles of risperidone released the drug slowly, and thus the significant antipsychotic effects are extended beyond 12 hours as compared with SC injection of risperidone solution. The cataleptic effect of these two formulations appeared quickly and declined after 8 hours (Table 6). Maximum catalepsy with these two formulations was observed at 1 hour. A possible reason may be the initial burst release of 38.98% and 23.10% at 1 hour from PLGA nanoparticles of risperidone and in situ gel containing PLGA nanoparticles of risperidone, respectively. Also, these formulations showed a beneficial effect beyond 8 hours with minimum EPS due to their slow-release pattern (∼32% of drug released between 8 and 72 hours for both formulations) (Tables 3 to 6). These results confirmed the dose-dependent EPS of risperidone and also showed that a beneficial effect was obtained from the lower therapeutic range.22,38-40 This study confirms that when the nanoprecipitation technique was used to load risperidone into PLGA nanoparticles, high entrapment efficacy was achieved when the polymer precipitated into solid nanoparticles. The nanoparticles size and drug release pattern were shown to be affected by polymer concentrations. When compared with risperidone solution, risperidone formulations showed prolonged inhibition of psychotic behaviors with fewer EPS after SC administration. This may improve the performance of risperidone during treatment of psychotic disorders by dose reduction. In the present study pharmacodynamic studies of risperidone and its formulations were carried out to study the antipsychotic effect and EPS after SC administration. The pharmacokinetics of the parent drug (risperidone) and its active metabolite (9-hydroxy risperidone) will be investigated in our future studies by analyzing blood samples up to 72 hours at different time intervals after administering risperidone formulations into a suitable animal model to support the data obtained by pharmacodynamic studies.

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