1.       Introduction

Gamma-Hydroxybutyric Acid (GHB) is a short chain fatty acid that occurs naturally in mammalian CNS [1,2]. GHB is formed in the brain from γ-Aminobutyric Acid (GABA)-derived succinic semialdehyde via succinic semialdehyde reductase [3]. GHB play a role in the brain as neurotransmitter or neuromodulator. GHB is a registered drug for the treatment of cataplexy and narcolepsy, and has also showed therapeutic efficacy for treating drug and alcohol dependence [4,5]. Any perturbation in the GABA pathway result in most common disorders such as epilepsy and other neuronal disorders which are mediated through GHB [6]. Therefore, exogenous administration of GHB was observed to produce more pronounced effects and play a significant role to cover the gaps of endogenously produced GHB [1,3]. Poly(γ-hydroxybutyrate) (P(γ-HB)) is more suitable candidate to be utilized in pharmaceutical industries due to its wide medical applications [7]. P(γ-HB) is a homopolymer of γ-hydroxybutyrate (γ-HB), and belongs to a diverse class of materials called polyhydroxyalkanoates (PHAs) produced by microorganisms inside the cells as energy storage materials [8]. P(γ-HB) has certain unique properties such as biocompatibility and rapid in vivo degradation, which differentiate it from others PHA based polymers [9,10]. The products formed after degradation of P(γ-HB) are monomers and oligomers of GHB in which the monomers act as a natural human metabolite and play an important role in brain function [11]. The metabolite resulted after degradation of P(γ-HB) has short half-life (~35 min), therefore, eliminated easily form the body via Krebs cycle [12]. Several studies described that nanoparticles preparation from PHA-mPEG copolymer has shown no toxicity when evaluated in vitro models of animal’s study [13,14]. Moreover, nanoparticles preparations from such polymer were also shown to be internalized in sufficient amount by human prostate cancer cell line [15].

P(γ-HB) has structural similarity to endogenously produced gamma-hydroxybutyric acid in the brain of animals. The difference is only in the length of monomer units which is repeated in the case of P(γ-HB), while a single monomer unit is present in the natural endogenous produced one. GHB is produced endogenously and also administered exogenously for the supplementation of intracellularly produced GHB by GABA in the brain. Exogenously administered GHB concentration was suggested to produce more pronounced neuronal functions than endogenously synthesized [3]. However, the major problem associated with exogenously administration of GHB is its use as abuse drug; producing mild euphoria, muscle relaxation, sedation, and eventually coma with increasing dosage [6,16]. In our study we suggest that P(γ-HB) a homologue of GHB, may induce similar effect in the induction of GABA mediated proteins as does GHB. However, the general structural similarities do not necessarily associate always with the common reaction mechanism. This study was hypothesized to find out any connection of bacterial produced P(γ-HB) with that of naturally produced GHB in the induction of GABA mediated pathways. P(γ-HB) an analogue of GHB was presumed to produced similar effects as that of exogenously administered GHB. Moreover, exogenously administration of P(γ-HB) is suggested to solve the problem associated with the treatment of narcoleptic patient with GHB such as drug abuse.

2.       Materials and Methods

2.1.  Preparation of P(γ-HB)-mPEG polymer

P(4HB) homopolymer was produced using the method of three-step cultivation of Hydrogenophaga pseudoflavia ATCC 33668 [17]. The nutrient broth-grown cultures were transferred to the Luria-Bertani medium and cultivated for 22 h. The cells isolated by centrifugation were incubated in the carbon-free mineral medium (the same mineral composition as in the PHA synthesis medium) containing 0.3 or 0.6 g /L ammonium sulfate for 10 h [18]. The cells recovered from the second cultivation step were transferred to the nitrogen-free PHA synthesis medium containing 2.24 g/L of γ-butyrolactone and cultivated for 36 h. The polyester was extracted with hot chloroform in a Pyrex Soxhlet apparatus.  The mPEG [number average molecular weight (M_n) = 5000], bis(2-ethylhexanoate) tin catalyst, sodium deoxycholate and Dichloromethane (DCM) were purchased from Sigma-Aldrich Korea Ltd. All other chemicals used were of analytical grades. The synthesis of P(γ-HB)-mPEG diblock copolymer was carried out by transesterification method in the melt as previously described [15]. P(γ-HB)-mPEG blank nanoparticles were prepared from the diblock copolymer by a modified emulsification-solvent evaporation method [15].

2.2.  Physiochemical Properties of P(γ-HB)-mPEG NPs

Morphological observation of nanoparticles was carried out using field emission scanning electron microscopy (FE-SEM Model XL30S, Philips, The Netherlands). For FE-SEM observation the nanoparticles were fixed in a 2.5% glutaraldehyde solution for 2h. The samples were washed twice with PBS buffer and then postfixed overnight in 1% osmium tetraoxide. The samples were again washed twice with PBS and dehydrated in a series of ethanol solutions (10%, 50%, 70%, 90%, 95%, 100%) and dried. For the morphological examination of samples for FE-SEM, the dried nanoparticles were dropped onto a carbon studs with carbon tape. The nanoparticles were coated with gold by a sputter coater (JFC-1100E, ion sputtering device, JEOL, Japan) for 300 s under vacuum at a current intensity of 10 mA and observed under the microscope. Particle sizes and size distributions were determined by the light scattering method (DLS-8000; Otsuka Electronics Co., Osaka, Japan). The mean particle size of the NPs was determined in triplicate, and the average values were calculated. The surface charge of the NPs was characterized in terms of zeta potential, which was determined using electrophoretic light scattering (ELS-Z; Otsuka Electronics Co., Osaka, Japan) at a scattering angle of 20^o.

2.3.  Animal Grouping and Drug Treatment

Adult male Sprague-Dawley rats weighing 250 g (n = 6) were housed individually in 45 x 24 x 20 cm plastic cages containing rodent bedding in a colony room maintained on a 12:12 light/dark cycle. Water was available continuously in the home cage. Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Gyeongsang National University, South Korea.  Two different doses of P(γ-HB)-mPEG nanoparticles (25 and 50 mg/kg) was chosen for chronic administration once daily for 14 days intraperitoneally. The P(γ-HB)-mPEG nanoparticles were suspended in sterile saline and sonicated for 10 min before injection in order to make a homogenous suspension. Control rats were injected with blank saline. After 14 days the rats were anesthetized with diethyl ether and subsequently killed. Brain sections were removed almost 14 h after the last injection. The brain portions were frozen on dry ice and stored at -70^oC until use for Western blot analysis.

2.4.  Western Blot Analysis

Western blot analyses were performed according to the previously determined procedures [19]. The brain tissues (cortex and hippocampus) were gently homogenized in 0.2 M PBS containing protease inhibitor cocktail. The homogenate was centrifuged and the supernatant was used for the measurement of protein concentration. The protein concentration was measured by Bradford assay with the Bio-Rad protein assay solution. Proteins (30 μg) were separated on a 12.5% SDS-PAGE and transferred onto a Polyvinylidene Difluoride (PVDF) membrane (Millipore Corporation). Prestained protein marker, broad range: 7-200 kDa (Color plus pre-stained marker, EBM-1035, Elpis, Korea) was run in parallel for detection of the molecular weights of the proteins. Skimmed milk was used for membrane blocking as to reduce non-specific binding. The immunoblotting was performed using rabbit derived GABA_BR1, GABA_BR2, Dopamine D₁/D₂R, polyclonal antibodies (1:500; Santa Cruz Biotechnology, Cell signaling). Rabbit derived anti-β-actin antibody (1:500; Sigma, St. Louis, MO, USA) was taken as control to confirm uniform loading. The membranes were probed with a goat derived horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with 1 x TBST, the blots were developed with super signal Enhanced Chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ, USA). The X-ray films were scanned and optical densities of the bands were measured using computer based Sigma Gel software (Jandel Scientific, San Rafeal, Chicago, USA).

2.5.  Data Analysis and Statistics

The object bands of Western blot were scanned and analyzed by densitometry using a computer based on program on Sigma Gel System (SPSS Inc., Chicago, IL). Data were presented as mean ± standard error of mean (SEM). Data were analyzed by ANOVA followed by t-test. A level of p < 0.01 and p < 0.05 was considered to be significant. ‘#’ indicates significant difference (p < 0.01), while ‘*’ indicates significant difference (p < 0.05).

3.       Results

3.1.  Structural Resemblance of P(γ-HB) with GHB

P(γ-HB) has structural similarity to endogenously produced gamma-hydroxybutyric acid in the brain of animals. The difference is only in the length of monomer units which is repeated in the case of P(γ-HB), while a single monomer unit is present in the natural endogenous produced GHB (Figure 1).

3.2.  Characterization of P(γ-HB)-mPEG Nanoparticles

P(γ-HB)-mPEG nanoparticles were prepared by emulsification-solvent evaporation technique. Morphological examination under Scanning Electron Microscopy (SEM) showed the formation of smooth spherically shaped nanoparticles as described in (Figure 2).

3.3.  Effect of P(γ-HB)-mPEG Nanoparticles on Expression of Dopamine D₁R and Dopamine D₂R in the hippocampus of Adult Rats

Previous studies have shown that acute treatment of GHB inhibited the dopamine release [20]. We determined the duration of effect of the nanoparticles in the expression of proteins in hippocampus regions of the rat brain. We chose chronic administration of the nanoparticles because the P(γ-HB) polymer is degraded slowly in nature, however a rapid degradation was suggested in vivo producing the monomer units of γ-HB a potential brain metabolite within given time frame [9,10]. As shown in (Figure 3), repeated exposure to P(γ-HB) induced a significant lower expression of dopamine D₁ and D₂ receptors in the hippocampus region of rat brain as compared to vehicle treated rats (control rats).

3.4.  Effect of P(γ-HB)-mPEG Nanoparticles on Expression of GABAB₁R and GABAB₂R in the Hippocampus of Adult Rats

Next, we determine the effect of P(_γ-HB)-mPEG nanoparticles on the protein expression level of GABAB₁R and GABAB₂R in the hippocampus of rat brain. We observed a significant decreased expression of GABAB₂R with either dose i.e. 25 mg/kg or 50 mg/kg in the hippocampus region of rat’s brain. However, GABAB₁R showed significant decrease in the protein expression with chronically treated higher dose (50 mg/kg) of P(γ-HB) mPEG nanoparticles (Figure 4).

No toxicity with mPEG modified P(γ-HB) nanoparticles was observed in all the groups treated nanoparticles in our work (data not shown).

4.       Discussion

GHB is a putative neurotransmitter or neuromodulator in mammalian brain [21, 22]. Exogenously administered it produces a number of physiological, pharmacological and biochemical effects in brain, including anxiolytic effect, sleep, anesthesia and absence seizures [22-24]. Moreover, GHB modifies the levels of different neurotransmitters, most notably dopamine, opioids, glutamate and acetylcholine [21,25,26]. Sufficiently large doses of the GHB metabolite have been approved by FDA for the treatment of cataplexy attacks in patients with narcolepsy [9].  Most of the known physiological effects of GHB have been demonstrated to be a result of either direct or indirect GABA_B receptor activation [27,28]. However, the exact mechanism how the GHB exerts its effect in the CNS is still not clear.

In this study we demonstrated the modulating effect of P(γ-HB)-mPEG nanoparticles on dopamine and GABAB receptors. Treatment of rats with P(γ-HB) polymer in the form of nanoparticles leads to the suppression of dopamine D₁R and D₂R, and GABA_B1R and GABA_B2R, in the hippocampus as demonstrated by Western blot analysis. The prepared nanoparticles have the ability to remain suspended in aqueous solution without forming aggregate. Such type nanoparticles have already been described to cross the blood brain barrier [29-31]. The mPEG used in the study is nontoxic and approved by FDA for human use [32]. Moreover, surface modification of the polymer with mPEG provide water dispersibility for the P(γ-HB) polymer thus prevent its precipitation [33]. Particulate system is mostly preferred as drug delivery vehicle and most of the hydrophobic drugs are currently tested for their improved therapeutic efficacy [34,35]. In this system the particle itself is supposed to show therapeutic effect without loading of any foreign object that will elicit a response mediated by endogenously produced or exogenously administered GHB. The exact mechanism of P(γ-HB) polymer expression of GHB mediated proteins is not clearly understood. However, most probably the P(γ-HB) polymer is degraded in to its monomer units and then cross the blood brain barrier to reach its target site.

Previously it was also suggested that P(γ-HB) degraded in vivo rapidly to produce the monomer units of γ-HB a potential brain metabolite within given time frame [9,10]. On the other hand, some research studies described that the polymer may enter the brain as whole in the form of particulate system and elicit the response [29-31]. However, in our study no such accumulation of nanoparticles in the brain parts was observed. So, it can be clearly concluded that the nanoparticles enter the brain in the form degraded monomer units and elicit a response.  Our hypothesis demonstrates that the receptors for GHB and P(γ-HB) are the same and they belong to the same family of macromolecular complexes and that they share structural similar ligands for modulation. It has already known that GHB has affinity for two distinct binding sites in the brain, the GHB receptor and, at higher concentrations, the GABA_B receptor sites [36,37].

A recent study by Snead indicates that GHB receptor, like GABA_B receptor, is coupled to a G protein, but it differs from GABA_B receptor, having significantly different anatomical distribution and ontogeny [38,39]. The putative GHB receptor antagonist NCS-382, and different GABA_B agonists and antagonists have been used to discriminate the relative role of GHB and GABA_B receptors, and to understand the functional meaning of endogenous GHB. NCS-382 has been reported to antagonize some effects of GHB such as GHB-induced seizures and GHB self-administration in mice [23,24,40]. Moreover, novel cyclic γ-Hydroxybutyrate (GHB) analogs were synthesized which showed high affinity and stereoselectivity of binding to GHB sites in rat brain [16,41]. Structurally similar molecules to GHB receptor ligand were found to be associated with similar induction of response as that of GHB mediated such as the implication of sleep process was found to be similarly induced by cannabis receptors [42].

As earlier come in our discussion, there are structural analogy between GHB and P(γ-HB) therefore, for both of these brain metabolites it is necessary to decipher the possible role in the activation GH level and their interactions with their receptors. Moreover, it would be easier to develop new compositions and methods to deliver therapeutic amounts of GHB in vivo that may reduce the side effects associated with direct administration of GHB. In order to prove whether it could be used as chemotherapeutic agent for narcolepsy or other alcohol withdrawal, more comprehensive research and clinical trials have to be done.

5.       Conclusion

In this paper, a new insight of research is proposed to correlate any connection of naturally occurring GHB with bacterial produced P(γ-HB). This new approach for targeting GABA receptors using particulate system of P(γ-HB)-mPEG nanoparticles may provide a major breakthrough for the design of drugs for the therapy of narcolepsy, absence seizures, drug addiction and withdrawal etc. Furthermore, the present study strengthens previous reports on the GHB activation of GABA associated proteins and will help focus future studies related to similar polymers. Our findings represent the first report that GHB markedly decrease expression of GABA_B2, dopamine D₁, D₂ in hippocampus via GABA_B receptor-mediated pathway. These findings have revealed totally novel action of P(γ-HB) and thus provide a new insight into its effect on the CNS. The novel technology can be extended to additional agents, so as to target multiple signaling pathways thus providing a major breakthrough as a therapeutic modulator for various diseases.

6.       Future perspectives

This new approach for targeting GABA receptors using particulate system may provide a new insight for the design of drugs for the future therapy. Furthermore, the present study may strengthen the previous reports on the GHB activation of GABA associated proteins and will help focus future studies related to similar polymers. The novel technology can be extended to additional agents, so as to target multiple signaling pathways providing a major breakthrough as a therapeutic modulator for various diseases. In order to prove whether it can be used as chemotherapeutic agent for narcolepsy or alcohol withdrawal, more comprehensive research and clinical trials have to be done.

7.       Declaration of Interest

The authors declare no conflict of interest.

8.       Ethical Conduct of Research

The authors state that they have obtained appropriate institutional review board approval. Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Gyeongsang National University, South Korea.

9.       Acknowledgments

This study was supported by grants (grant #: 20090091 489, 2009-0070747, R01-2000-000-00070-0) from the MEST/NRF and in partial grant from directorate of science and technology govt. of KPK, Pakistan. 

Figure 1: Structural resemblance of (A) gamma hydroxybutyrate and (B) poly-gamma hydroxybutyrate.

Figure 2: Morphological characterization of P(γ-HB)-mPEG NPs.

Figure 3: P(γ-HB)-induced decrease expression of dopamine receptors D₁R and D₂R in in hippocampal regions of rat brain. A: Representative Western blot analysis with dopamine D₁R antibody. Mean ± SEM (n=3) of the density of the corresponding proteins of dopamine D1R are presented. B: Representative Western blot analysis with dopamine D₂R antibody. Mean ± SEM (n = 3) of the density of the corresponding proteins of dopamine 2 are presented. Dopamine D1/D2 expression was significantly inhibited in hippocampus following chronic administration of P(γ-HB). *p < 0.05 and #p < 0.01 versus control group.

Figure 4: P(γ-HB)-induced decrease expression of GABA receptors GABA_B1R and GABA_B2R in the hippocampal regions of rat brain. A: Representative Western blot analysis with anti-GABA_B1. Mean ± SEM (n=3) of the density of the corresponding proteins. B: Representative Western blot analysis with anti-GABA_B2. Mean ± SEM (n=3) of the density of the corresponding proteins. GABA_B1R and GABA_B2R expression was significantly inhibited in hippocampus following chronic administration of P(γ-HB). * p < 0.05 and # p < 0.01 versus control group.

1.       Ren X, and Mody I (2003) γ-hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABA_B receptor activation in mouse frontal cortex and hippocampus. Journal of Biological Chemistry 278:  42006-42011.

2.       Volpi R, Chiodera P, Caffarra P, Scaqlioni A, Malvezzi L (2000) Muscarinic cholinergic mediation of the GH response to gamma-hydroxybutyric acid: neuroendocrine evidence in normal and parkinsonian subjects. Psychoneuroendocrinology 25: 179-185.

3.       Wong CGT, Bottiglieri T, Snead OC (2003) GABA, γ-hydroxybutyric acid, and neurological disease. Annals of Neurology 54: S3-S12.

4.       Fuller DE, Hornfeldt CS (2003) From club drug to orphan drug: sodium oxybate (xyrem) for the treatment of cataplexy. Pharmacotherapy 23: 1205-1209.

5.       Gallimberti L, Spella MR, Soncini CA, Gessa GL (2009) γ-hydroxybutyric acid in the treatment of alcohol and heroin dependence. Alcohol 20: 257-262.

6.       Wong CG, Chan KF, Gibson KM, Snead OC (2004) Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug. Toxicological Reviews 23: 3-20.

7.       Pötter M and Steinbüchel A (2005) Poly(3-hydroxybutyrate) Granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6: 552-560.

8.       Anderson AJ, and Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiology Reviews 54: 450-472.

9.       Martin DP, and Williams SF (2003) Medical applications of poly-4-hydroxybutyrate: a strong flexible absorbable biomaterial. Biochemical Engineering Journal 16: 97-105.

10.    Furst UP, Madkour MH, Mayer F,  Steinbüchel A (1994) Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.  Journal of Bacteriology 176: 4328-4337.

11.    Nelson T, Kaufman E, Kline J, Sokoloff L (1981) The extraneural distribution of gamma-hydroxybutyrate.  Journal of Neurochemistry 37: 1345-1348.

12.    Sendelbeck SL, and Girdis CL (1985) Disposition of a 14C-labeled bioerodible polyorthoester and its hydrolysis products, 4-hydroxybutyrate and cis, trans-1,4 bis(hydroxymethyl)cyclohexane, in rats. Drug Metabolism Disposal 13: 291-295.

13.    Shah M, Naseer MI, Choi MH, Kim MO, Yoon SC (2010) Amphiphilic PHA-mPEG copolymeric nanocontainers for drug delivery: Preparation, characterization and in vitro evaluation. International Journal of Pharmaceutics 400: 165-175.

14.    Shah M, Choi MH, Ullah N, Kim MO, Yoon SC (2011) Synthesis and characterization of PHV-block-mPEG diblock copolymer and its formation of amphiphilic nanoparticles for drug delivery. Journal of Nanoscience and Nanotechnology 11: 5702-5710.

15.    Shah M, Choi MH, Ullah N, Kim MO, Yoon SC (2012) Amorphous amphiphilic P(3HV-co-4HB)-b-mPEG block copolymer synthesized from bacterial copolyester via melt transesterification: Nanoparticle preparation, cisplatin-loading for cancer therapy and in vitro evaluation.  European Journal of Pharmaceutics and Biopharmutics 80: 518-527.

16.    Wellendorph P, Høg S, Greenwood JR, Lichtenburg AD, Nielsen B (20050) Novel cyclic γ-Hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain. Journal of Pharmacology Experimental Therapeutics 315: 346-351.

17.    Choi MH, Yoon SC, Lenz RW (1999) Production of poly (3-hydroxybutyric acid-co-4hydroxybutyric acid) without subsequent degradation by Hydrogenphaga pseudoflava. Applied and Environmental Microbiology 65: 1570-1577.

18.    Choi MH, and Yoon SC (1994) Polyester biosynthesis characteristics of Pseudomonas citrollinolis grown on various carbon sources, including 3-methyle branched substrates. Applied and Environmental Microbiology 60: 3245-3254.

19.    Badshah H, Kim TH, Kim MO (2015) Protective effects of anthocyanins against amyloid beta-induced neurotoxicity in vivo and in vitro. Neurochemistry International 80: 51-59.

20.    Howard SG, and Banerjee PK (2002) Regulation of central dopamine by γ-hydroxybutyrate. In: Tunnicliff G, and Cash CD (ed.) Gamma-Hydroxybutyrate, Taylorand Francis, England. Pg No: 111-119.

21.    Maitre M (1997) The γ-hydroxybutyrate signaling system in brain: organization and functional implications. Progress in Neurobiology 51: 337-361.

22.    Bernasconi R, Mathivet P, Bischoff S, Marescaux C (1999) Gamma hydroxybutyric acid: an endogenous neuromodulator with abuse potential?. Trends in Pharmacological Sciences 20: 135-141.

23.    Schmidt C, Gobaille S, Hechler V, Schmitt M, Bourguignon JJ, Maitre M (1991) Anti-sedative and anti-cataleptic properties of NCS-382, a gamma-hydroxybutyrate receptor antagonist. European Journal of Pharmacology 203: 393-397.

24.    Hu RQ, Banerjee PK, Snead III OC (2000) Regulation of Gamma Aminobutyric Acid (GABA) release in cerebral cortex in the Gamma Hydroxybutyric Acid (GHB) model of absence seizures in rat. Neuropharmacology 39: 427-439.

25.    Banerjee PK, Snead III OC (1995) Presynaptic Gamma-Hydroxybutyric Acid (GHB) and gamma-aminobutyric acid (GABAB) receptor-mediated release of GABA and Glutamate (GLU) in rat thalamic ventrobasal nucleus (VB): a possible mechanism for the generation of absence-like seizures induced by GHB. Journal of Pharmacology and Experimental Therapeutics 273: 1534-1543.

26.    Madden TE and Johnson SW (1998) Gamma-hydroxybutyrate is a GABAB receptor agonist that increases a potassium conductance in rat ventral tegmental dopamine neurons. Journal of Pharmacology and Experimental Therapeutics 287: 261-265.

27.    Lew JY, Espana AG, Lee KY, Carr KD, Goldstein M (1998) Increased site-specific phosphorylation of tyrosine hydroxylase accompanies stimulation of enzymatic activity induced by cessation of dopamine neuronal activity. Molecular Pharmcology 55: 202-209.

28.    Carai MA, Lobina C, Maccioni P, Cabras C, Colombo G, Gessa GL (2008) γ-aminobutyric acid B (GABAB)-receptor mediation of different in vivo effects of γ-butyrolactone. Journal of Pharmacological Sciences 106: 199-207.

29.    Queva C, Danielsen MB, Edlund A (2003) Effects of GABA agonists on body temperature regulation in GABAB₁ -/- mice. British Journal of Pharmacology 140: 315-322.

30.    Patel T, Zhou J, Piepmeier JM, Saltzman WM (2012) Polymeric nanoparticles for drug delivery to the central nervous system. Advanced Drug Delivery Reviews 64: 701-705.

31.    Alam MI, Beg S, Samad A, Baboota S, Kohli K, Ali J (2010) Strategy for effective brain drug delivery. European Journal of Pharmaceutical Sciences 40: 385-403.

32.    Mittal G, Carswell H, Brett R, Currie S, Kumar MNR (2011) Development and evaluation of polymer nanoparticles for oral delivery of estradiol to rat brain in a model of Alzheimer's pathology. Journal of Controlled Release 150: 220-228.

33.    Fishburn CS (2008) The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. Journal Pharmaceutical Sciences 97: 4167-4183.

34.    Knop K, Hoogenboom R, Fischer D, Schubert US (2010) Poly (ethylene glycol) in drug delivery: Pros and cons as well as potential alternatives. Angewadthe Chemie International Edition 49: 6288-6308.

35.    Chellat F, Merhi Y, Moreau A, Yahia LH (2005) Therapeutic potential of nanoparticulate systems for macrophage targeting. Biomaterials 26: 7260-7275.

36.    Vauthier C, Dubernet C, Chauvierre C, Brigger I, Couvreur P (2003) Drug delivery to resistant tumors: the potential of poly (alkyl cyanoacrylate) nanoparticles. Journal of Controlled Release 93: 151-160.

37.    Berton  F, Brancucci A, Beghè F, Cammalleri M, Demuro A, et al. (1999) Gamma-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices. European Journal Pharmacol 380: 109-116.

38.    Snead III OC (1994) The ontogeny of [3H] gamma-hydroxybutyrate and [3H] GABAB binding sites: relation to the development of experimental absence seizures. Brain Research 659: 147-156.

39.    Snead III OC (1996) Relation of the [3H] Gamma-Hydroxybutyric Acid (GHB) binding site to the gamma-aminobutyric acid (GABAB) receptor in rat brain. Biochemical Pharmacology 52: 1235-1243.

40.    Martellotta MC, Balducci C, Fattore L, Cossu G, Gessa GL, et al. (1998) Gamma-hydroxybutyric acid decreases intravenous cocaine self-administration in rats. Pharmacology Biochemistry and Behavior 59: 697-702.

41.    Hog S, Wellendorph P, Nielsen B, Frydenvang K, Dahi IF (2008) Novel high-affinity and selective biaromatic 4-substituted Gamma-Hydroxybutyric Acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies. Journal of Medicinal Chemistry 51: 8088-8095.

42.    Cash CD (1996) What is the role of gamma hydroxybutyrate receptor?. Medical Hypothesis 47: 455-459.