Direct nose-to-brain transfer of a growth hormone releasing neuropeptide, hexarelin after intranasal administration to rabbits

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Abstract

The purpose of this study was to investigate the olfactory transfer of a growth hormone releasing neuropeptide, hexarelin to the brain tissues by comparing brain uptake levels after intranasal administration with those after intravenous administration. The hexarelin nasal formulation was prepared using an aqueous cosolvent vehicle consisting of ethanol, propylene glycol, and n-tridecyl-β-d-maltoside as a permeation enhancer. Hexarelin was administered intravenously or intranasally to male rabbits at a dose of 1 mg/kg. Drug concentrations in the plasma, cerebrospinal fluid and six different regions of the brain, i.e., olfactory bulb (OB), olfactory tract (OT), anterior (CB1), middle (CB2), posterior (CB3) cerebrum, and cerebellum (CL) were analyzed by LC/MS method after solid phase extraction. The brain and cerebrospinal fluid levels achieved following intranasal administration were approximately 1.6 times greater than those attained after intravenous administration despite the intranasal plasma levels being significantly lower than the intravenous plasma levels. Intranasal administration resulted in significantly different spatial distribution patterns in various regions of brain with the rank order of COB > COT > CCB1, CB2, CB3 > CCL at 10, 20, and 40 min post-dosing, whereas intravenous administration yielded nearly similar distribution patterns in the brain. The intranasal administration into one nostril (left or right) exhibited markedly greater hexarelin concentrations in olfactory bulb and olfactory tract on the treated-side of brain tissues than those on the non-treated-side of the brain hemisphere. It was demonstrated that the hydrophilic neuropeptide hexarelin was transferred via olfactory pathway to the brain hemispheres and the drug transfer via this route significantly contributed to high brain concentrations after nasal administration to rabbits.

Introduction

Growth hormone (GH—22 kDa), which is secreted by the pituitary gland, stimulates the growth and cell reproduction in humans and other animals. In addition, studies have shown that GH possesses the following effects on metabolic processes: (a) increased rate of protein synthesis occurs in most cells; (b) decreased rate of carbohydrate metabolism in cells; and (c) increased metabolism of free fatty acids and use of fatty acids for energy (Russell-Jones et al., 1993, Bercu and Walker, 1994).

A deficiency in growth hormone production and/or secretion can result in various diseases or conditions, such as dwarfism, profound reduction in lean body mass and concomitant increase in total body fat, reduction in bone density, delayed wound healing, congestive heart failure, and insulin resistance. A lack of GH can also lead to a decrease in skeletal muscle, cardiac muscle mass, and strength that can result in significant decreases in exercise capacity and musculoskeletal frailty, which is typically associated with old age (Juul and Jørgensen, 1996).

Even though administration of recombinant GH is a current effective therapy for growth hormone deficiency, it is not ideal mainly due to its invasive parenteral delivery, adverse side effects, and high cost. Consequently, the potential application of small synthetic molecules in growth hormone replacement therapy is currently the subject of extensive investigation as GH secretagogues (GHS) suitable for nonparenteral administration have recently been synthesized. The growth hormone releasing peptides (GHRP) are a family of synthetic, five to seven amino acid peptides that selectively stimulate GH secretion. In clinical studies, the most potent GH secretagogue of the GHRP family to date is a hexapeptide, hexarelin (HEX), which has the following amino acid sequence: His-d-2-methyl-Trp-Ala-Trp-d-Phe-Lys-NH2. HEX has been shown to possess a strong, long-lasting GH releasing activity both in vitro and in vivo in animals after intravenous (IV) and subcutaneous administration (Cella et al., 1995, Deghenghi et al., 1994).

In general, a common obstacle to routine use of peptide hormones is that they cannot be administered by oral route since they have a low permeability through the gastrointestinal mucosa because of their polar nature. They also undergo chemical and enzymatic inactivation in the gastrointestinal tract and have a significant first pass metabolism in the liver, although some small peptides have shown their pharmacological activities after oral administration.

An alternative administration of peptide hormone is by intranasal (IN) delivery since it has recently been shown to possess several advantages over other methods of administration. A major advantage of IN delivery is that the drug may be administered to achieve a systemic or localized effect, as required, because of the relatively high absorption efficiency of the drugs through the nasal mucosa with a high permeability and relatively lower enzymatic activity. In regard to the absorption efficiency of HEX in human, Ghigo et al. reported that the IN administration provided a significantly greater bioavailability (4.8 ± 0.9%) than that obtained after oral administration (0.3 ± 0.1%) (Ghigo et al., 1994).

Although IN administration produces a relatively high absorption efficiency for HEX as compared to that of the oral route, there is still a formidable problem associated with IN drug administration in that most drug molecules, in particular, hydrophilic peptides diffuse poorly and slowly through the nasal mucosal membrane and thus the desired levels of the therapeutic agent cannot be achieved by means of simple transnasal administration. The low oral and nasal absorption and systemic availability result in high intersubject variability of the GH-releasing effect. Thus, there continues to be a need for development of compositions and methods for convenient, efficient, and effective IN delivery of such substances to animals and humans.

In addition, recently a number of studies have shown the possibility of exploiting the nasal delivery route for direct transport of drugs from nose to brain through the olfactory pathway (Illum, 2000, Vyas et al., 2006, Westin et al., 2006, Nonaka et al., 2008). The extent of direct brain uptake of drugs is highly dependent on the molecular weight, the degree of lipophilicity, the degree of dissociation of the drugs and the type of formulation.

As part of the development studies for the HEX delivery system, the objective of the present study was to investigate simultaneously the plasma pharmacokinetics and brain distribution profiles of the hydrophilic growth hormone releasing peptide, HEX in rabbits after IV and IN administration and to assess whether there is a direct nose-to-brain transport pathway for neuropeptide molecules.

Section snippets

Chemicals

HEX was purchased from GL Biochem Ltd. (Shanghai). Ethanol (EtOH), propylene glycol (PG), glycerol, acetonitrile (HPLC grade), acetic acid, formic acid, sodium ethylenediaminetetraacetate (EDTA-Na), diethylamine, heparin and benzalkonium chloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol (HPLC grade) was purchased from J.B. Baker (Phillipsburg, NJ, USA). n-Tridecyl-β-d-maltoside (TDM) was obtained from Anatrace Inc. Deionized and distilled water was used.

Animals

Male New Zealand

Intranasal absorption of HEX

The time courses of the plasma levels of HEX following IV injection and the IN administration of simple normal saline solution and hydroalcholic cosolvent formulation in rabbits at a dose of 1 mg/kg are presented in Fig. 1. The corresponding non-compartmental PK parameters determined utilizing WinNonlin software are summarized in Table 1. Table 1 also shows the PK parameters determined following IN administration of the cosolvent formation at a dose of 0.5 mg/kg in order to evaluate the

Conclusions

In vivo absorption and brain distribution studies in rabbits revealed that a hydrophilic neuropeptide, HEX could preferentially transfer into the CSF and brain tissues from the nasal cavity; the olfactory bulb and olfactory tract were the essential gateways for this direct pathway. A single administration of HEX solution formulated with a hydroalcholic cosolvent system containing 0.5% n-tridecyl-β-d-maltoside as a permeation enhancer may provide a promising and durable therapeutic option for

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    From these data, two ‘general rules’ emerge: after IN-administration: (1) brain/CSF-levels tend to be higher than the peripheral levels, sometimes higher than the levels obtained after i.v.-administration; (2) peripheral levels rise more slowly and are generally preceded by steeper rising brain/CSF-levels. Such effects have been shown for neuropeptides and hormones like CCK-8 (Pietrowsky et al., 2001, 1996), vascular endothelial growth factor (VEGF) (Yang et al., 2009), MSH/ACTH (Born et al., 2002), insulin-like growth factor-1 (Thorne et al., 2004) and hexarelin (Yu and Kim, 2009), as well as for a variety of other substances like testosterone (Banks et al., 2009), estradiol (Ohman et al., 1980; Wang et al., 2006), ergoloid mesylate (Chen et al., 2008), gastrodin (Wang et al., 2007, 2008), cephalexin (Sakane et al., 1991), tetramethylpyrazine (Feng et al., 2009), and the angiotensin antagonist GR138950 (Charlton et al., 2008). This list can be extended easily but these suffice to illustrate that IN-administration of substances mostly results in faster peaks and/or higher brain concentrations than peripheral administration.

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