Use of the chaperone receptor-associated protein (RAP) for the delivery of therapeutic compounds to the brain and other tissues

Inactive Publication Date: 2005-02-03
HORIZON ORPHAN LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] In other aspects, the invention provides methods of treating tissues or organs having proportionately greater, preferably more than two-fold, amounts of LRP receptors on their cells than other tissues or organs. The selective biodistribution of RAP or RAP-polypeptide conjugated active agents can enhance the selective targeting of such conjugated agents to specific organs.
[0023] In a fourth aspect, the invention provides a method for using the RAP/LRP carrier system in the diagnosis of diseases, disorders, or conditions. The present invention provides screening assays for identifying RAP or RAP polypeptide active agent conjugates that can prevent, amelioriate, or trea

Problems solved by technology

The blood-brain barrier (BBB) also impedes access of beneficial active agents (e.g., therapeutic drugs and diagnostic agents) to central nervous system (CNS) tissues, necessitating the use of carriers for their transit.
For example, management of the neurological manifestations of lysosomal storage diseases (LSDs) is significantly impeded by the inability of therapeutic enzymes to gain access to brain cell lysosomes.
LSDs are characterized by the absence or reduced activity of specific enzymes within cellular lysosomes, resulting in the accumulation of undegraded “storage material” within the intracellular lysosome, swelling and malfunction of the lysosomes, and ultimately cellular and tissue damage.
However, the BBB blocks the free transfer

Method used

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  • Use of the chaperone receptor-associated protein (RAP) for the delivery of therapeutic compounds to the brain and other tissues
  • Use of the chaperone receptor-associated protein (RAP) for the delivery of therapeutic compounds to the brain and other tissues
  • Use of the chaperone receptor-associated protein (RAP) for the delivery of therapeutic compounds to the brain and other tissues

Examples

Experimental program
Comparison scheme
Effect test

example ii

Construction, Expression, Purification and Characterization of RAP Fusions

[0250] Expression constructs encoding fusions between the human receptor-associated protein (RAP) and human alpha-glucosidase (GAA), alpha-L-iduronidase (IDU) or glial cell-derived neurotrophic factor (GDNF) were prepared. For this purpose, a sequence that encodes RAP was fused to the 5′-end of sequences that encode the different fusion partners. All sequences were obtained by high-fidelity PCR amplification of human cDNA with the following primers shown in FIG. 2a. The GDNF fusion was designed for expression in bacteria. To this end, primer RAPBACF was substituted for RAPF in the RAP amplification for this construct (FIG. 2b).

[0251] The 5′-end of RAP was truncated to remove the signal peptide sequence. Instead, an in-frame BamHI site, which encodes the dipeptide GS, was added for the mammalian expression construct. Sequence encoding the tetrapeptide MGGS with an NcoI site at the 5′-end was added for the bac...

example iii

Uptake and Distribution of Unconjugated RAP to the Brain

[0263] The distribution of RAP to brain was measured using a mouse in situ perfusion model. Volumes of distribution (Vd) for RAP, the positive control transferrin and the negative control albumin, were determined over a perfusion interval of 5 minutes. In addition, the relative quantities of the test proteins in the vascular and parenchymal fractions of the perfused brain were determined using the capillary depletion technique (Gutierrez, et al., J. Neuroimmunology 47(2):169-76 (1993)). The results shown in FIG. 11 include an observed, corrected Kinflux of 1 μL / g / min for transferrin. RAP had an observed, corrected Kinflux of 2.2 μL / g / min. RAP is taken up into brain.

[0264] A separate experiment was carried out at a single, 5-minute time-point to determine whether RAP is able to traverse the brain vasculature and enter the parenchyma. Brains were harvested as before, but were subjected to a capillary depletion procedure to dete...

example iv

Measurement of Specific Uptake of RAP-GAA into Enzyme-Deficient Patient Fibroblasts

[0265] The uptake of RAGA into cells deficient in GAA was characterized. The cell line used was GM244 (Coriell Cell Repository), a primary cell line isolated from a patient with glycogen storage disorder type II (Pompe's disease). These fibroblasts take up phosphorylated, recombinant GAA via the mannose-6-phosphate receptor, but also have LRPI receptors, which bind RAP. In order to identify the receptors involved in uptake of different test ligands, samples containing excess free RAP or mannose-6-phosphate were prepared.

[0266] Dilutions of RAP-GAA were made in the uptake medium (Dulbecco's Modified Eagle's Medium supplemented with 25 mM HEPES pH 7.0, 2 mM L-glutamine and 250 pg / mL bovine serum albumin) to yield fusion protein concentrations of 33, 11, 3.7, 1.2, 0.4, and 0.1 nM. The effect of 3 mM mannose-6-phosphate, 500 nM RAP and a combination of the two on the uptake of 5 nM RAP-GAA was also assa...

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Abstract

This invention provides compounds of conjugates of therapeutic or active agents with RAP or a RAP polypeptide, their pharmaceutical compositions and methods for using the such compounds and compositions in the diagnosis, prophylaxis, or treatment of diseases and conditions, including particularly diseases of the central nervous system or lysosomal storage diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10 / 206,448, filed on Jul. 25, 2002, which claims the benefit of U.S. Provisional Patent Application No. 60 / 308,002, filed Jul. 25, 2001. The contents of these and all other U.S. patents cited herein are each hereby incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention is related to compositions comprising Receptor-Associated Protein (RAP) linked to a therapeutic and / or diagnostic / investigational agent, and methods of using such compounds. BACKGROUND OF THE INVENTION [0003] The brain is shielded against potentially harmful substances by the blood-brain barrier (BBB). The microvascular barrier between blood and brain is made up of a capillary endothelial layer surrounded by a basement membrane and tightly associated accessory cells (pericytes, astrocytes). The brain capillary endothelium is much less permeable to low-mo...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K39/395A61K48/00C07K14/705
CPCA61K38/00A61K47/48246C07K2319/23C07K2319/00C07K2319/22C07K14/705A61K47/64A61P9/10A61P25/00A61P25/02A61P25/16A61P25/18A61P25/28A61P35/00
Inventor ZANKEL, TODDSTARR, CHRISTOPHERGABATHULER, REINHARD
Owner HORIZON ORPHAN LLC
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