In: Serum Albumin: Structure, Functions and Health Impact _ISBN: 978-1-62100-231- Editors: RJ. Alekseev and A. L. Rebane ©2012 Nova Science Publishers, In Chapter 6 ALBUMIN IN DRUG DELIVERY Giuliano Siligardi and Rohanah Hussain® Diamond Light Source, Science Division, Harwell Science and Innovation Campus, Chilton, United Kingdom ABSTRACT Albumin is the most abundant protein in human plasma. being present at a concentration of 30-0 g/l Tt has several important functions in the blood including assistance in maintaining osmotic pressure: transport of hormones (in particular fat soluble ones); transportation of fatty acids to the liver; transportation of drugs: and ‘maintenance of pH. It has a long plasma halflife ~ typically 20 days (Peters 1996). This, combination of low biological activity, long half life and ability to bind fatty (and ‘therefore poorly aqueous soluble) molecules makes albumin an attractive candidate for incorporation into drug delivery/formulation systems and its use in this role has been examined over the last twenty years (Peters 1996). More recently there has also been a growing awareness that albumin may, under certain circumstances, be selectively accummlaied by solid tumours (Cloris et al, 1995; Goldshaid et al: 2010). Clearly this accumulation of albumin offers the potential for improved delivery of drugs to specific sites of action, particularly to tumours. The search for effective dnig delivery systems is one of the major challenges facing pharmaceutical research, especially for biomedically important molecules such as proteins, peptide-based vaccines, tumour therapeutic agents, antimicrobials and lipodrugs. A procedure has been developed whereby human serum albumin (HSA) can be use as a delivery vehicle for a aumber of these biological ‘molecules by exploiting its physiological role as the bodies’ main fatty 2cié cartier. Using esseatially fatty acid free HSA (HSA®) it is possible to form stable complexes with compounds that contain alkyl chains (calleé lipo-compounds). Two lipe-compounds have been used to develop this system. 2 novel antimicrobial peptide and the neurotransmitter -amino-t-butyric acid GABA conjugated with C8 or C14 alkyl chains (Hussain and Siligardi 2006. 2010). Both lipo-compounds were evaluated confirming that the formation of HSA-lipo compomd complexes had 2 mutual stabilizing effect on both HSA and lipo-compounds. The protease enzymes pronase was used fo show that the alkyi chains of the lipo-compounds bound to HSAf conferred a similar if not greater “Emil ohana Inwoin damon a ck134 Ginliano Siligardi and Robanah Hussain resistance to enzymatic degradation than the HSA for clinical use such as Baxter Albumin and Zenalbumin, which contain sodium caprylate and acetyl tryptophanate Here we indicated that the use of HSAff as 2 camier for lipidated drugs might confer higher biostability to the lipidated drugs providing a novel formulation method for therapeutic use. The increased stability of the lipo-compounds when formulated using the HSAf? was not observed when HSA containing fatty acid (HSAfa) was used (Hussain and Siligardi 2000 and 2010). Furthermore in the case of the antimicrobial lipopeptide there was an increased in activity with the HSAff formulation suggesting that besides conferring stability it also enhanced bioavailability of compounds that would otherwise exist as insoluble micelles in aqueous media. These findings have allowed us to develop a simple and effective way of delivering lipo containing compounds using fatry acid free HSA as the drug carrier Figure 1. Cartoon representation of HSA (green) complexed with lipo-drugs. FAs are the fatty acid binding sites. Yellow circle (solid front view, striped back view) represents drug coupled to fatty acid (red),Albuaua in Drug Delivery 139 INTRODUCTION Pharmacokinetics, bicavailability and interactions of pharmaceuticals are important features for the drug formulation and dosage determination. The drug delivery system can crucially influence these pharmaceutical properties which axe ‘tal for the drug therapeutic and/or prophylactic action Numerous drug delivery systems are currently being developed with various degrees of success and applicability (Schermmana, 2002). Biological molecules such as proteins, peptide vaccines, peptide-base tumour therapeutic agents. and peptide-base antimicrobials have particular problems such as susceptibility to endogenous proteases and disqibution t9 target sites. The use of lipidic moieties covalently attached to such molecules has been used to improve absorption and transportation in vivo (Hussain, 1992). However this strategy has met with limited success. Biostability to enzymatic degradation still poses 2 problem with the peptide base drugs containing ipidic moieties. To couateract this problem an increased number of lipidic moieties have to be covalentiy-linked to these drugs which poses another formulation problem ic. solubility for i vivo administration. This will limit the amount that can be administered into the body. hence affecting its efficacy. Tn this chapter we present the use of an exogenous preparation of essentially farty- acid free albumin with lipiadted drags as a novel casrier vehicle (Figure 1). A procedure whereby bioactive compounds tagged with C8 and Cl4 alkly chains but not only can be formulated wath exogenous essentially fatty acid-free human serum albumin (HSA) will be discussed. MATERIAL AND METHODS HSAff and HSAfa were obtained from Sigma, Baxter HSA was obtained from Baxter Healthcare Lid and Pronase® Protease, Nuclease free from Streptomyces griseus was cbained from Calbiochem. Delipidised Baxter was prepared using dialysis. The lipo-Gaba, GabaC8 and GabaC 14 were synthesised using ezowa ether chemistry (Hussain, 1992). The lipopeptide RHOL as syathesised using standard Fmoc/TFA solid phase peptide synthesis. The UV and circular dichrossm (CD) data were collected with a Jasco spectropolar:meter 3720 with time constant 4s, bandwidth lnm. using a 0.05cm pathlength cell (Hellma) and Diamond B23 beamline end-station B with ring current 200mA. 0.28mum slitwidth equivalent to 0 8nm bandwidth, scan speed 38nm/min and using 0.02cm pathlength cell (Hellma). Formulation of HSAff with lipo-compounds. The lipo-Gaba compounds were prepared in ethanol, lipopeptide RHO1 (myristoyl FARKGALRQ) in aqueous solutions and HSA added to give the required molar ratios. The mixtures were incubated with gentle agitation. GABase stulies. The GABase assays for GABA was preformed using the method éescribed by Jakoby (1962) with the modification that esterase (7Ounits) was added for the assay of lipo-Gaba, GabaC8 and GabaCl4. The GABase assay monitors the reduction of NADP to NADPH spectroscopically at 340nm, pH 8.6 at 25°C. as a function of time using GABA asa substrate. Pronase studies. The CD signals were monitored at 220nm as a function of time afer pronase addition (1/100 w/w). The rate of albumin degradation in the presence of lipo-compounds were then monitored. The percentage of pronase stability was calculated