The Effect of 2-Hydroxypropyl-ß-Cyclodextrin on the Solubility, Stability and Brain Targeting of Chemical Delivery Systems for Neuropeptides

A general method for brain targeting of neuropeptides such as enkephalins, kyotorphin and various TRH (thyrotropin releasing hormone) analogs, based on their molecular packaging and subsequent, sequential metabolism, was recently described (Bodor et al. 1992, Bodor 1997, Prokai-Tatrai (ntet al., 1996). Some salient aspects of this approach were recently reviewed (Bodor and Buchwald 1998). The main objective of the method is to specifically target neuropeptides to the brain, which otherwise are unaccessible due to their hydrophilic nature, as well as, being excellent substrates of peptidases found in the blood-brain barrier (BBB). Brain delivery, and particularly brain targeting of neuropeptides is very important, as of the some 100 neuropeptides isolated, most, if not all are constituents of the brain and their hyper or hypo production is involved in various CNS diseases. The successful brain targeting of various neuropeptides was achieved by multiple chemical modifications of the target peptides in order to produce lipophilic constructs, which are not recognized by peptidases as peptides or peptide derivatives. But converting a peptide into a lipophilic form would only allow the brain delivery of this disguised peptide construct, which however, would not result in effective brain delivery of the peptide. In order to produce effective targeting, we have introduced a number of other modifications, such as, the use of the earlier described dihydrotrigonellyl “ trigonellyl redox brain targeting system, which is linked to the N-terminal of the peptide through strategically selected spacer functions. In addition, when necessary the C-terminal is further modified in order to allow specific enzymes to produce the active terminal, such as the case of some TRH analogs. The redox targetor system provides a lock-in of the main precursor for a sustained release of the peptide after the strategically planned enzymatic reactions take place. In the same time, the rest of the body is spared from the peptide as the complex CDS is destroyed by various enzymes, primarily in the liver, before the active peptide can be released. The actual brain targeting, the presence of the predicted intermediates and high and sustained pharmacological activity for various “packaged” peptides was demonstrated before. One of the general problems related to these packaged peptide constructs is their intrinsic high lipophilicity. In these constructs in which the peptides represent a perturbation, in order to avoid premature peptidase deactivation, large lipophilic functions on both the C and N-terminals are used. The result is that the original chemical delivery systems (CDS) for these peptides are highly lipophilic, in general their log P is over 6 and consequently they have negligible solubility in water. In addition, due to presence of the 1,4-dihydrotrigonellyl function, these molecules are sensitive to acid catalyzed water addition, as well as, chemical oxidative conversion to the corresponding quaternary pyridinium, trigonellyl salts. We have recently developed a molecular based approach to estimate partition properties for organic solutes (Bodor and Buchwald, 1997), including non-zwitterionic peptides and their derivatives (Buchwald and Bodor, 1998). This method allows us to estimate with excellent accuracy the partition and solubility properties of all potential lipophilic constructs, as well as, their predicted enzymatic intermediates and byproducts. However, the administration of the first, fully packaged form of the peptide is always a problem. Water miscible organic solvents, which would solubilize these molecules, such as DMSO, cannot be used since these highly lipophilic molecules would immediately precipitate upon injection. We have previously found that 2-hydroxypropyl-s-cyclodextrin (HPBCD) is very effective in solubilizing and stabilizing protein drugs (Brewster et al., 1991). Accordingly, we were expecting that the brain targeted peptide derivatives could also benefit from HPBCD. Accordingly, the aim of the present study was to provide a way to convert these highly lipophilic constructs into water soluble forms, and to provide chemical stability for the various sensitive functions. The class of compounds that we are focusing on involves three different types of TRH (Glp-His-Pro-NH2) analogs, in which histidine was replaced by leucine (Leu) or norvaline (Nva), respectively, and the packaging was accomplished by opening the pyroglutamate ring to glutamine to allow the coupling at the N-terminal with Dhtr-Spacer, while the active prolineamide was generated from the additional glycine on the C-terminal, which is oxidatively converted to the prolineamide by peptidyl glycine a-amidating monooxygenase (PAM). In one alternative, the C-terminal proline amide was replaced by pipecolinic acid, which does not require the amide form for activity. All three analogs contained a cholesterol as the ester function, a dihydrotrigonellyl targetor and two L-prolines as the spacer, which provides timely and sustained release of the active peptide.