Design and Chemical Synthesis of a Magnetic Resonance Contrast Agent with Enhancedin VitroBinding, High Blood−Brain Barrier Permeability, andin VivoTargeting to Alzheimer's Disease Amyloid Plaques

Abstract

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-β (Aβ) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer's disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Aβ with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Aβ30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of ±4385 Da determined by electrospray ionization mass spectrometry. This diamine-and gadolinium-substituted derivative of Aβ is shown to have enhanced in vitro binding to Alzheimer's disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PS1 transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.Molecular imaging by magnetic resonance requires a molecular probe containing a contrast agent that is capable of selective tissue targeting in labeling a specific molecular entity within the tissue of interest which can be detected by magnetic resonance imaging (MRI). One of the pathological hallmarks of Alzheimer's disease (AD) is the extracellular accumulation of amyloid-β (Aβ) peptide into plaques. These plaques are essential for the definitive diagnosis of AD, which is usually confirmed only post-mortem. At present, there is no method for direct imaging of individual β-amyloid plaques in humans that would provide a definitive pre-mortem † This work was supported in part by the Alzheimer's Association (J.F.P.), NIA (AG22034) (J.F.P.), and the Mayo Foundation.*To whom correspondence should be addressed. Phone: (507) 284-1784. Fax: (507) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diagnosis of this disease or a method of assessing disease progression. MRI has a spatial resolution of 30-50 μm, which at least theoretically, has the capacity to resolve individual plaques (the neuritic plaque size in an AD patient varies from 2 to 200 μm).Radioiodinated human Aβ40 has been used as a molecular probe which binds to β-amyloid plaques both in vitro and in vivo (1). This in vivo binding of plaques was demonstrated with radioiodinated, polyamine-modified, human Aβ40 following intravenous i...

Selective Contrast Enhancement of Individual Alzheimer’s Disease Amyloid Plaques Using a Polyamine and Gd-DOTA Conjugated Antibody Fragment Against Fibrillar Aβ42 for Magnetic Resonance Molecular Imaging

“…125 I-IgG4.1, 125 I-F(ab′) 2 4.1, 125 I-Gd-DOTA-F(ab′) 2 4.1, 125 I-pF (ab′) 2 4.1, 125 I-Gd-DOTApF(ab′) 2 4.1, or buffer were incubated in vitro with sections of unfixed APP/PS1 mouse brain using a modified procedure as described previously (22). Free iodine was removed by dialyzing each antibody overnight against PBS and then rinsed further in a centrifugal filter (Vivaspin 20, 30,00 MWCO, Cat.…”

“…The linear trend of the curve clearly depicts that F(ab′) 2 4.1 is not saturated with DOTA and more sites are available for modification. One to two DOTA attachments per F(ab′) 2 4.1 are desired as it was previously demonstrated that Gd-DTPA modification of another contrast agent with more than one Gd-DTPA moiety per molecule adversely affected its BBB permeability (22).…”

Production and Characterization of Monoclonal Antibodies to Human Sclerostin

“…NM-015464) were synthesized using f-moc chemistry. (26,27) Sequences were as follows: scl mouse peptide 61-78, 61-GRPPHHPYDAK DVSEYSC-78 (human and mouse sequences are not identical but are similar); and scl human peptide 168-183 plus an extra cysteine for KLH conjugation, 168-KRLTRFHNQSELKDFG-183 þ C (human and mouse sequences are identical).…”

Biological activity of FGF-23 fragments

“…Figure 1) was performed in the Mayo Peptide Synthesis Facility by solid phase methods on an ABI 433A peptide synthesizer (Applied Biosystems Inc., Foster City, CA) using protocols previously described for the synthesis of human amyloid-peptides [36]. Briefly, FGF-23 peptides (0.1 mmol scale) were synthesized on NovaSyn TGA resin (Calbiochem-Novabiochem, San Diego, CA) using HBTU activation of N -9-fluoroenylmethoxy-carbonyl (Fmoc) amino acid derivatives and synthesis protocols provided by the instrument's manufacturer.…”