News Column

"Multimeric Biotinidase Resistant Multimodality Probes" in Patent Application Approval Process

July 12, 2014



By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- A patent application by the inventors Bhushan, Kumar Ranjan (St Louis, MO); Misra, Preeti (St Louis, MO), filed on December 13, 2012, was made available online on June 26, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Patents).

This patent application has not been assigned to a company or institution.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The high affinity (strept)avidin-biotin binding system is widely utilized in pre-clinical diagnostic applications and is under evaluation as a molecular component for imaging and tumor-targeted cancer therapeutic {Rusckowski, 1996; van Gog, 1998}. It is believed that the biotinidase enzyme, which is present in serum and tissue of both animals and humans in nanomolar concentrations, cleave the biotinamide bond linking biotin (vitamin H) and lysine in biocytin, such that this essential vitamin can be recycled {Foulton, 1998; Hymes, 1996}. For in vivo studies, though, targeted biotin derivatives must have serum stability and aqueous solubility {Wilbur, 1997}. The nonspecific nature of the cleavage of biotinamide bonds in biotin conjugates has made it imperative that biotin derivatives employed in vivo be designed in a manner that blocks the enzyme activity.

"The multimodality imaging of direct or pre-targeting (strept)avidin-biotin strategy has been elusive. The detection of biotin on molecules is facilitated greatly by the wide variety of (strept)avidin-based technologies exploiting the extremely strong noncovalent interaction of (strept)avidin for biotin, with a K.sub.a of 10.sup.15 M. Usually in a pre-targeting strategy, a monoclonal antibody-(strept)avidin conjugate is injected into a patient, allowed to localize at the tumor over 24-48 h, followed by the clearing of excess reagent from blood and administration of the radiolabeled biotin derivative."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "Multimodality imaging is becoming increasingly common in both the clinic and the laboratory {Park, 2005; Pietrzyk, 1996; Saoudi, 1999}. By combining both anatomical and functional images, one can, for example, localize uptake of a tracer or contrast agent to a particular tissue or organ within a subject. In the clinic, multimodality imaging can be used for the diagnosis of disease, staging to help select between therapies, or to assess the efficacy of a course of treatment. In the laboratory, multimodality imaging is used to correlate genomic and phenomic types, make quantitative data more reliable, quantify the damage due to induced disease states or injuries, and assess the usefulness of treatment options.

"Rational for multimodal probes: (a) Nuclear and Optical Probes: Nuclear imaging is an established clinical molecular imaging modality that offers good sensitivity at deep tissue sites. However, nuclear imaging techniques remain limited by several factors such as time-consuming procedures, expensive equipment, need for highly skilled personnel and relatively poor spatial resolution {Weissleder, 2001}. On the other hand, optical imaging is a relatively new molecular imaging modality that offers real-time and high-resolution imaging of fluorophores embedded in diseased tissues {Sokolov, 2003; Ntziachristos, 2003; Gurfinkel, 2003}. Of the various optical imaging techniques investigated to date, near-infrared (NIR, 700-900 nm wavelength) fluorescence-based imaging is of particular interest for noninvasive in vivo imaging because of the relatively low tissue absorption and minimal autofluorescence of NIR light {Ntziachristos, 2003; Gurfinkel, 2003}. Considering the importance and advantages of both nuclear and fluorescence imaging, a combination of these two techniques provides an attractive approach for enhancing the imaging accuracy and providing complementary information for improving diagnosis and management of diseases. The strategy to achieve this goal is to develop an optical and nuclear dual labeled imaging agent.

"(b) MR and Optical Probes: In general, optical imaging methods have high sensitivity and are cost effective at the cell/tissue level. However, most optical imaging apparatus lacks the capacity of tomographic image reconstruction, and therefore 3-D localization of signals in intact tissues/organs has rarely been achieved noninvasively {Li, 2004}. Magnetic resonance (MR) imaging offers the advantages of being noninvasive, tomographic, and high resolution. However, MR imaging contrast dependent on endogenous differences in water content and on relaxation time in the tissue of interest. The specificity and sensitivity of MR imaging is enhanced by contrast agents based on paramagnetic metals such as gadolinium. Combining the excellent 3D spatial resolution and unlimited depth penetration of MR imaging with very high sensitivity of NIR imaging should serve to traverse shortcomings of each technology {Massoud, 2003}.

"Nature often takes advantage of multimerization to decrease ligand off-rate and improve affinity of cell surface binders {Kitov, 2003; Mammen, 1998}. There is a general need to find suitable scaffolds for the assembly of multiple targeting ligands and contrast agents in hope that multimerization would improve the performance of cancer specific ligands.

"Several different multivalent scaffolds have been used successfully in past particularly for applications in carbohydrate/lectin interactions {Lindhorst, 2002; Lundquist, 2002} but also for peptide/protein interactions {Wright, 2001} and in context of tumor targeting {Carlson, 2007; Thumshirn, 2003}. Among these scaffolds are small molecules with few conjugation sites (.about.2-10) and larger systems like dendrimers {Voegtle, 2007} and polymers {Haag, 2006}.

"The high affinity (strept)avidin-biotin binding system is widely utilized in pre-clinical diagnostic applications and is under evaluation as a molecular component for imaging and tumor-targeted cancer therapeutic {Rusckowski, 1996; van Gog, 1998}. It is believed that the DOTA-biotin adducts, formed via an amide bond between the sidearm of biotin and the spacer carrying the chelating moiety, are per se easily hydrolyzable by the serum biotinidase, an enzyme which is also able to break biotinyl peptides {Pispa, 1965; Hymes, 1996}. Different ways have been designed in the past to prevent the recognition at the site of the enzymatic attack (i.e., substitution of the carbon adjacent to the amide NH, alkylation of the amide group, incorporation of d-amino acids at the breaking point of the molecule, etc.) {Wilbur, 1997; Foulton, 1997; Wilbur, 2006; Sabatino, 2003} in order to look for a good compromise between serum stability and high affinity of the compounds toward (strept)avidin. Several synthetic steps were necessary for all these modifications, which, in some cases, resulted in a diminished binding constant with the (strept)avidin pockets. This fact prompted us to design and synthesize in a few steps a conjugatable biotin derivative devoid of the amide target site of the biotinidase.

"The present invention describes a development of multimeric biotinidase resistant multimodality probes. For cancer therapeutics and imaging applications, the in vivo stability of labeled biotin derivatives is of major importance to avoid the release of radioisotopes or labeled fragments of the molecule, which may cause non-specific irradiation of normal tissues. Biotinidase blocking of biotinylated compounds are essential for optimal direct or pre-targeting of tumor-targeted cancer therapeutics and imaging. Present invention explores a system that has potential to provide (strept)avidin-biotin based optimal direct or pre-targeting. In particular, the present invention describes a chemical system for the efficient production of a tri-functional agent comprised of a NIR fluorophore for optical imaging, a metal chelate for simultaneous MR/nuclear imaging, and a biotinidase resistant targeting ligand for high affinity (strept)avidin-biotin binding. Multifunctional probes for concurrent imaging applications could traverse shortcomings of each technology and could provide complementary information.

"In one aspect of present invention, an organic chelating ligand is reacted with a trifunctional linker moiety, having primary amine and carboxylic acid functional groups, followed by deprotection of one or more functional groups to yield one or more free functional groups. Chelation of a metal ion on one or more free functional groups results in a metal chelate. Conjugation of a NIR fluorophore on a metal chelate results in a NIR dye containing metal chelate conjugated carboxylic acid precursor. Reaction of a biotinidase resistant targeting ligand {Wilbur, 2006; Sabatino, 2003; FIG. 5} with a NIR dye containing metal chelate conjugated carboxylic acid precursor results in a multimodalilty biotinidase resistant contrast agent (FIG. 1). In such aspect, trifunctional linker moiety 2 is amino acid, polymer, or dendrimer. Metal ion, M is independently selected from Cu, Fe, In, Tm, Yb, Y, Gd, Eu, and a lanthanide. R is t-butyl ester, ester, or hydrogen. R.sup.1 is Boc, Fmoc, Ac, Cbz, Bz, and Bn. In one embodiment, a biotinidase resistant targeting ligand, R.sup.2H is selected from the group of:

"##STR00001##

"In some embodiments, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and L.sub.5 are independently selected from alkane, polyethylene glycol, and polypropylene glycol. In some embodiments, amino acid is natural amino acid. In some embodiments, amino acid is unnatural amino acid. In some embodiments, alkane is C0-C20 straight chain carbon unit. In some embodiments, polyethylene glycol is 1 to 20 ethylene glycol unit. In some embodiments, polypropylene glycol is 1 to 20 propylene glycol unit. In some embodiments, R.sup.3 is independently selected from alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, hydroxyl, carbonyl, aldehyde, carboxylate, ester, ether, amine, nitro, nitrile, and pyridyl. In some embodiments, IRDye is a NIR fluorophore independently selected from the group of IRDye 78, IRDye 800CW, IRDye 700DX, VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750, DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor 680.

"In an another aspect of present invention, a biotinidase resistant targeting ligand {Wilbur, 2006; Sabatino, 2003; FIG. 5} is conjugated with a multivalent scaffold, followed by deprotection of an amino protecting group to generate an amine containing biotinidase resistant targeting ligand conjugated multivalent scaffold. Reaction of an amine containing biotinidase resistant targeting ligand conjugated multivalent scaffold with a NIR dye containing metal chelate conjugated carboxylic acid precursor 5 (FIG. 1) results in a multimeric multimodalilty biotinidase resistant contrast agent (FIG. 2). In such aspect, R.sup.1 is independently selected from Boc, Fmoc, Ac, Cbz, Bz, and Bn. R is t-butyl ester, ester, or hydrogen. M is independently selected from Cu, Fe, In, Tm, Yb, Y, Gd, Eu, and a lanthanide. R.sup.2, R.sup.3 and R.sup.4 are independently selected from:

"##STR00002##

"L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5, L.sub.6, and L.sub.7 are linkers independently selected from alkane, amino acid, --NHCO(CH.sub.2).sub.5--, polyethylene glycol, and polypropylene glycol. In one embodiment, amino acid is natural amino acid. In some embodiments, amino acid is unnatural amino acid. In some embodiments, an alkane is C0-C20 straight chain carbon unit. In some embodiments, polyethylene glycol is 1 to 20 ethylene glycol unit. In some embodiments, polypropylene glycol is 1 to 20 propylene glycol unit. In some embodiments, R.sup.5 is independently selected from alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, hydroxyl, carbonyl, aldehyde, carboxylate, ester, ether, amine, nitro, nitrile, and pyridyl. In some embodiments, IRDye is a NIR fluorophore independently selected from the group of IRDye 78, IRDye 800CW, IRDye 700DX, VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750, DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor 680.

"The present invention describes multimeric multimodality probes. Particularly, the present invention discloses (strept)avidin specific multimeric biotinidase resistant multimodality probes. Mulimodality probes of present invention provide complementary information. The major medical application of present invention is in direct or pre-targeted diagnosis and therapy of tumors by biotinidase blocking agents. Such technology would improve the therapeutic index of tumor by concentrating target molecule at the tumor site, which also has the benefit of producing fewer toxic side-effects in normal organs.

BRIEF DESCRIPTION OF DRAWING

"FIG. 1 represents a multimodalilty biotinidase resistant contrast agent in which a NIR fluorophore is conjugated after a metal ion chelation.

"FIG. 2 represents a multimeric multimodalilty biotinidase resistant contrast agent in which a NIR fluorophore is conjugated after a metal ion chelation.

"FIG. 3 represents a multimodalilty biotinidase resistant contrast agent in which a NIR fluorophore is conjugated before a metal ion chelation.

"FIG. 4 represents a multimeric multimodalilty biotinidase resistant contrast agent in which a NIR fluorophore is conjugated before a metal ion chelation.

"FIG. 5 represents a synthetic scheme for preparation of biotinidase resistant targeting ligand.

"FIG. 6 represents a synthetic scheme for preparation of [Gd-DOTA]-Asp-IRDye-BRBD monomer.

"FIG. 7 represents a synthetic scheme for preparation of [Gd-DOTA]-Asp-IRDye-Ad-BRBD trimer."

URL and more information on this patent application, see: Bhushan, Kumar Ranjan; Misra, Preeti. Multimeric Biotinidase Resistant Multimodality Probes. Filed December 13, 2012 and posted June 26, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2172&p=44&f=G&l=50&d=PG01&S1=20140619.PD.&OS=PD/20140619&RS=PD/20140619

Keywords for this news article include: Tissue Engineering, Biomedical Engineering, Biomedicine, Avidin, Cancer, Alkenes, Patents, Genetics, Oncology, Peptides, Polyenes, Ovalbumin, Amino Acids, Hydrocarbons, Therapeutics, Glycoproteins, Bioengineering, Nanotechnology, Polypropylenes, Carboxylic Acids, Dietary Proteins, Molecular Imaging, Organic Chemicals.

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Source: Obesity, Fitness & Wellness Week


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