Conjugation of haloperidol to PEG allows peripheral localisation of haloperidol and eliminates CNS extrapyramidal effects

Highlights

• Conjugating haloperidol to PEG prevented haloperidol crossing through the BBB.

• The system retained pharmacological activity towards dopamine D₂ receptors in vitro.

• Conjugated haloperidol did not induce catalepsy in rats after i.v. administration.

• PEGylation would be a new strategy offering compartmental localisation of drugs.

Abstract

We have previously reported the synthesis of a poly(ethylene glycol)-haloperidol (PEG-haloperidol) conjugate that retained affinity for its target D₂ receptor and was stable in simulated physiological conditions. We hypothesised that this polymer-drug conjugate would localise haloperidol's activity either centrally or peripherally, dependent on the location of administration, due to the polymer preventing penetration through the blood-brain barrier (BBB). Herein, we validate this hypothesis using in vitro and in vivo studies. We first demonstrate, via a [³⁵S]GTPγS-binding assay, that drug activity is retained after conjugation to the polymer, supportive of retention of effective therapeutic ability. Specifically, the PEG-haloperidol conjugate (at 10 and 100 nM) was able to significantly inhibit dopamine-induced G-protein activation via D₂ receptors, albeit with a loss of potency compared to the free haloperidol (~18-fold at 10 nM). This loss of potency was further probed and rationalised using molecular docking experiments, which indicated that conjugated haloperidol can still bind to the D₂ receptors, albeit with a flipped orientation in the binding pocket within the receptor, which may explain the reduced activity. Finally, rat catalepsy studies confirmed the restricted permeation of the conjugate through the BBB in vivo. Rats treated intravenously with free haloperidol became cataleptic, whereas normal behaviour was observed in rats that received the PEG-haloperidol conjugate, suggesting that conjugation can effectively prevent unwanted central effects. Taken together these results demonstrate that conjugating small molecules to polymers is effective at prohibiting penetration of the drug through the BBB and is a valid targeting strategy for drugs to facilitate peripheral (or central) effects without inducing side effects in other compartments.

Introduction

Penetration through the blood-brain barrier (BBB) is recognised as a significant challenge when developing therapeutic agents for diseases of the central nervous system, for example, for psychiatric disorders and Alzheimer's disease [1]. What is less well articulated, however, is the requirement to prevent certain peripheral therapeutic drugs from crossing the BBB [2,3]. This need can be demonstrated by considering first-generation antihistamine agents where beneficial peripheral effects can be compromised by unwanted sedative effects when the drug crosses the BBB and acts centrally [4]. Since the size is a key parameter affecting permeability across the BBB, with 500 Da typically being considered as the maximum threshold for BBB permeability, one strategy to reduce penetration through the BBB is to form a polymer-drug conjugate (PDC) [5,6]. A very limited number of studies (three to the best of our knowledge) have previously demonstrated that PDCs can indeed restrict drug activity to peripheral organs and prevent undesired effects in the CNS. A PDC of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and TNP-470, an anti-tumour agent, using a biologically labile tetrapeptide linker, significantly reduced TNP-470-related neurotoxicity in comparison to the free TNP-470 [7]. Movantik®, a PDC in which naloxol (an opioid antagonist) is covalently linked to poly(ethylene glycol) (PEG) via a biologically stable ether linkage, has been used clinically to prevent opioid-induced constipation [8]. Third, and of particular relevance to the work presented here, we have reported the synthesis and characterisation of a non-prodrug PDC in which the D₂ antagonist haloperidol was conjugated to PEG via a biologically stable carbamate linkage (Fig. 1) [9]. We demonstrated stability of the linker in vitro and presented evidence of potential activity by demonstrating that the PEG-haloperidol conjugate retains binding affinity (albeit reduced) to D₂ receptors; we also showed initial evidence that PEG conjugation could prevent haloperidol crossing the BBB through a simple single in silico equation, based on methodology reported in Fu et al. [10].

In the present study, and for the first time, we robustly demonstrate the feasibility of using PEG to prevent penetration of the haloperidol through the BBB using in vitro and in vivo approaches. Specifically, the pharmacological activity of the PEG-haloperidol conjugate was assessed in vitro by measuring the inhibition of dopamine-induced [³⁵S]GTPγS-binding via D₂ receptors. Next, we interpret the retained biological potency of conjugated haloperidol by looking at the effect of PEGylation on haloperidol binding to D₂ receptors using in silico molecular docking studies. Finally, and of particular significance, we extended our study and evaluated the penetration of peripherally administered PEG-haloperidol conjugate through the BBB in vivo. This was carried out by recording catalepsy in rats, as an indication of haloperidol-induced CNS extrapyramidal side effects.

We demonstrate that the PEG conjugation strategy used was capable of preventing the penetration of conjugated haloperidol through the BBB and propose that such strategies can prevent unwanted central side effects of peripherally administered drugs (and/or vice versa), and would form a strong base to re-direct the use of haloperidol, and similar drugs, to treat peripheral non-CNS diseases such as cardiovascular diseases and cancers.