Reinhard Gabathuler

Transport characteristics of a novel peptide platform for CNS therapeutics

New and effective therapeutics that cross the blood‐brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep‐2 peptide, a sequence derived from ligands that bind to low‐density lipoprotein receptor‐related protein‐1 (LRP‐1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep‐2 conjugate and immunohistochemical studies of injected Angiopep‐2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co‐localization with the neuronal nuclei‐selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [125I]‐Angiopep‐2 into brain endothelial cells occurred by a saturable mechanism involving LRP‐1. The primary sequence and charge of Angiopep‐2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.

Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease.

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimers disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimers disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimers disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G-Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimers disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimers disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimers disease.

Efficient One-Pot Synthesis of Doxorubicin Conjugates Through Its Amino Group to Melanotransferrin P97

Abstract The amino group of doxorubicin 1 is reacted with bis-NHS-ester linkers 6, or anhydrides 13 to offer in high yield modified doxorubicins 7–12 and 14–16, respectively. Compounds 7–12 are mono-NHS-esters, and can be directly coupled with melanotransferrin (p97), a useful vector with the ability to cross the blood-brain barrier, to yield the expected doxorubicin-p97 conjugates. Upon activating the carboxylic group with BTTU, compound 14–16 could be used in the same reaction. Structurally, the amino group of doxorubicin is covalently bonded to the amino groups of p97. The conjugates are potential candidates for treatment of brain tumors.

Synthesis of Doxorubicin Conjugates Through Hydrazone Bonds to Melanotransferrin P97

Abstract Through hydrazone bonds, three doxorubicin-p97 conjugates 5, 9, 12 are synthesized using adipic dihydrazine, 4-(4-N-maleimidophenyl)-butyric acid hydrazine 6 and adipic acid mono hydrazine 10 as linkers. These are potential agents for treating brain tumors.

Efficient Synthesis of Doxorubicin Melanotransferrin p97 Conjugates Through SMCC Linker

Abstract Doxorubicin–succinimidyl 4‐[N‐maleimidomethyl]cyclohexane‐1‐carboxylate (SMCC) 3, prepared by treating doxorubicin (1) with SMCC 2, is treated with 2‐mercaptoacetic acid to give doxorubicin–SMCC–sulfo‐acetic acid 5. Treating with benzotriazol‐1‐yl‐N,N,N′,N‐tetramethyluronium tetrafluoroborate (BTTU), the carboxy group of 5 is activated, and reacts efficiently with the amino group of melanotransferrin p97 to afford the expected doxorubicin‐p97 conjugate 6, which is a potential agent capable to cross the blood–brain barrier (BBB) to treat brain tumors.

Synthesis of Doxorubicin Conjugates Through 14-Hydroxy Group to Melanotransferrin P97

Abstract Using Fmoc to protect the amino group, Fmoc-doxorubicin is conjugated through the 14-hydroxy group to the amino group of melanotransferrin p97 by spacer arms such as succinate and glutarate. The Fmoc group is then removed under basic conditions, which affords doxorubicin-p97 conjugates. The resulting bioconjugates are potential agents for treating brain tumors.

Synthesis of novel heterobifunctional isocyanato cross-linkers and their applications for the preparation of 10-hydroxycamptothecin and SN-38 conjugates with melanotransferrin p97

Abstract Novel heterobifunctional cross‐linkers with an isocyanato group, a protected carboxylic group, and a linear chain spacer are synthesized in high yield by coupling monofunctionalized PEG with 1,6‐diisocyanatohexane. The isocyanato groups of those linkers are highly reactive and are efficient reagents to couple with the hydroxy groups of 10‐hydroxycamptothecin and SN‐38 under mild conditions to give a useful precursor for the synthesis of their bioconjugates with proteins such as melanotransferrin p97.

Synthesis of Camptothecin Melantotransferrin (p97) Conjugate Through a Carbamate Bond

Abstract Through its 20‐hydroxy group and a carbamate bond, 20(S)‐camptothecin (1) is chemically linked to melanotransferrin (p97). The key step to modify the steric hindered hydroxy group of camptothecin is achieved by using triphosgene/DMAP in DMF and immediately reacting with tert‐butyl 6‐aminohexanoate (8). The novel camptothecin conjugate 4 is a potential agent for chemotherapy of brain tumor.

Efficient Synthesis of Polyethylene Glycol Mono‐Carboxylate via Michael Conjugate Addition

Abstract Poly(ethylene glycol) (PEG) with one carboxylate group, the very useful precursors for the synthesis of the PEG derived heterobifunctional linkers, are synthesized in high yield in one‐pot via Michael conjugate addition of acrylate esters with PEG and catalyst amount of sodium in THF.