Elsevier

International Congress Series

Volume 1277, April 2005, Pages 171-184
International Congress Series

Development of a potential protein vector (NeuroTrans) to deliver drugs across the blood–brain barrier

https://doi.org/10.1016/j.ics.2005.02.021Get rights and content

Abstract

Purpose

Delivery of chemotherapeutic drugs to the brain is limited by the capillary endothelial cells that form the blood–brain barrier (BBB). In this study, we investigate a novel transport mechanism based on the iron binding protein p97 (or melanotransferrin), which is able to cross the BBB.

Methods

The anticancer drug adriamycin (ADR), which is unable to cross the BBB, was conjugated to p97 to determine if it was possible to deliver a therapeutic dose of ADR to the brains of mice. Conjugates of p97-ADR were prepared and tested for efficacy against subcutaneous rat C6 glioma tumours in athymic mice.

Results

Conjugate and free ADR were shown to be equally effective in inhibiting the growth of these tumours. The ability of p97 to cross into the brain after conjugation to ADR was demonstrated in a mouse model using I125 labeled compounds. Transport of p97 and conjugate were shown to be 6–8 fold higher than BSA or lactoferrin. Conjugates were also tested for efficacy against intracranial rat C6 glioma and human ZR-75-1 mammary tumours in athymic mice.

Conclusion

The conjugate was shown to significantly increase the survival of mice compared to repeated injections of PBS or free ADR. These results demonstrate a marked improvement over existing chemotherapy strategies based on ADR alone. p97 may have significant potential as an effective vehicle for the delivery of therapeutic drugs across the BBB.

Introduction

The effective delivery of chemotherapeutic drugs into the brain is limited by the presence of the blood–brain barrier (BBB). The BBB is made up of specialized capillary endothelial cells that are joined together by tight junctions, which provide a barrier that protects the brain from exposure to unwanted compounds. Unfortunately, the barrier is able to limit the entry and eliminate most chemotherapeutic drugs from the brain [24]. This is achieved by membrane proteins such as P-glycoprotein (Pgp 1 or mdr-1), that are able to eliminate these drugs from the brain, which presents significant problems in treating brain tumours with current chemotherapeutics [33]. Many methods have been developed in an attempt to deliver drugs to the brain to treat brain tumours. However, most have failed to provide significant improvements to long term survival [30]. Radical methods, other than surgery or radiation treatment, have been devised in order to open up the BBB and allow access of the drug to the brain. For example, the permeabilization of the BBB by osmotic shock with mannitol [19] or by specific molecules such as bradykinin analogs has allowed periodic delivery of drugs into the brain [1]. However, this exposes the brain to unwanted and potentially dangerous compounds found in the blood. Small hydrophobic drugs have been designed to allow diffusion through the BBB. However, due to their lipid solubility, charge, low molecular weight, and binding by P-glycoprotein, the transport is extremely inefficient [21], [22]. Another approach is to target the highly specific endogenous transport mechanisms of the BBB [2], [25] where drugs have been conjugated to small hydrophobic peptides [27]. Drugs have also been conjugated to proteins that are able to bind to receptors expressed on the BBB [24], [25]. Unfortunately, in order to overcome the endogenous concentrations of the receptor's ligands the drug must be delivered at very high concentrations. A more promising technology has been developed based on conjugation of drugs to antibodies against these transport mechanisms [4], [18]. For example, antibodies against the transferrin receptor have been used to deliver drugs to tumours in the periphery [17], [32] and across the BBB [9], [18], [23]. Although partially effective, these methods have been limited by saturation of the receptor and low dissociation rate of the antibody. It has also been suggested that the transferrin receptor does not transport transferrin or these antibody conjugates into the brain parenchyma but recycles them back to the blood [20]. In addition severe immune reactions to the antibodies and side effects may be experienced. Overall, these transport mechanisms are expressed throughout the body and render non-specific delivery of the drug.

Novel approaches and improvements to existing therapies are, therefore, required to increase the survival of those suffering from primary and metastasized CNS tumours. As an alternative to using antibodies we have focussed on the iron binding protein p97, or melanotransferrin, a protein closely related to transferrin and lactoferrin [3]. The precise cellular function of p97 and its mechanisms of action have yet to be determined. p97 is found as a soluble form in the blood as well as a membrane bound form [8], which is highly expressed on the surface of melanoma cells, brain capillary endothelial cells [26] and microglial cells associated with Alzheimer's disease amyloid plaques [11], [34]. Since it was noted that the serum levels of Alzheimer patients had elevated p97 [14], [16], it was suggested that p97 may be able to cross the BBB. This was supported by a recent study demonstrating the efficient transcytosis of radiolabelled p97 across the BBB in both in-vitro and in-vivo models [6]. Considering the ability of p97 to cross the BBB, it was proposed that delivery of chemotherapeutic drugs to the brain could by enhanced by conjugation to p97. In this paper we explore the use of p97 to deliver the anthracycline drug adriamycin (ADR), which normally cannot cross the BBB [22], into the brains of mouse tumour models.

Section snippets

Production and purification of p97

The recombinant secreted form of human p97, which was terminated at amino acid position #711, was generated according to Yang, 1999 [35], and transfected in BHK TK-ts13 cells. After 10–12 days of incubation, the supernatant was recovered and centrifuged at 3000 ×g for 20 min at 4 °C.

p97 was affinity purified using a 10 mL column of anti-p97 monoclonal antibody L235 (ATCC HB8446) immobilized on AffiGel-10 (Bio-Rad). p97 concentration was determined using immunofluorescence assay and its purity

Preparation of the p97-ADR conjugates

ADR was conjugated to p97, resulting in relatively pure active conjugates. The purity of the conjugate can be seen in Fig. 1, which shows SYN002 conjugated to [14C] labeled ADR according to Method 1. SYN002 had a molar substitution ratio (MSR) between 4 and 5 with a concentration of 60.62 μg/mL for ADR and 1.8 mg/mL for p97. For SYN018, SYN019 and SYN020 the MSR was between 6 and 7 with an ADR concentration of 17 μg/mL and a p97 concentration of 0.4 mg/mL. The conformation of the p97 after

Discussion

Several studies have shown that p97 may be overexpressed in the brains of those suffering from Alzheimer's Disease [11], [26], [34]. In addition, this overexpression could be detected by increased levels of p97 in the serum of those suffering from the disease [14], [15]. In a more recent study by Demeule et al. 2002 [6], recombinant p97 was shown to be actively transported across an in-vitro BBB model similar to that used by Dehouck et al., 1997 [5]. The transport rate of p97 was 10–15 times

Discussion

Sugiyama

Dr Bill Pardridge in the USA, as you know, published a paper about the OX26 which is the transferrin receptor antibody. In his paper he said the reason why he used the antibody instead of transferrin itself is that the endogenous concentration of transferrin in the circulating blood is high enough to saturate the binding to the transferrin receptor. In your case you used the melanotransferrin. Don't you need to worry about that issue?

Gabathuler

We checked that. Melanotransferrin is found

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