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Organophosphorus Hydrolase Pharmacokinetics and Immunogenicity are Improved by Branched dPEG®

Organophosphorus hydrolase  (OPH,EC 8.1.3.1), also known as Aryldialkylphosphatase, is a remarkably stable homodimeric enzyme that can detoxify organophosphate compounds. Organophosphate compounds are the basis of numerous pesticides (e.g., malathion) and chemical warfare weapons (e.g., sarin, VX). Organophosphates act by blocking the action of the enzyme acetylcholinesterase. Overuse and misuse of organophosphate pesticides are major causes of acute pesticide poisoning and death. See also here.

Boris N. Novikov and colleagues with the Department of Biochemistry and Biophysics at Texas A&M University, reported in the Journal of Controlled Release on the effects of modifying organophosphorus hydrolase (often abbreviated OPH) with linear and branched PEG derivatives. The group chose these PEG reagents because conjugates altered by PEGs see “their immunogenicity is reduced and the renal excretion is slowed considerably, leading to prolonged half-life, reduced side effects and increased treatment efficiency,” C.S. Fishburn, The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2008) 4167–4183.

 

 dPEG® Reagents Modifying Organophosphorus Hydrolase

PEG derived conjugates, specifically discrete linear and branched PEGs, were used to help generate data on the biochemical, biophysical, and pharmacological properties or organophosphorus hydrolase. The discrete PEGs chosen were all terminated with a methoxy group at one end of the PEG linker and functionalized with an NHS ester on the other end of the linker to react with surface-accessible lysine residues, forming an amide bond between the PEG linker and organophosphorus hydrolase. The linear, discrete PEGs had spacing lengths of 16.4Å (PEG4), 30.8Å (PEG8), and 44.9Å (PEG12), while the branched, discrete PEG chosen is shown in Figure 1, below. All of the PEG linkers used were made by Quanta BioDesign Ltd. and purchased through Quanta BioDesign distributor Thermo Scientific (formerly Pierce).

 

PN10401, NHS-dPEG®-(m-dPEG®12)3, used to modify organophosphorus hydrolase

Figure 1: The branched discrete PEG used in this research to modify organophosphorus hydrolase.

 

Attaching the PEG linkers altered the biophysical properties of organophosphorus hydrolase. Specifically, the catalytic functionality (kcat) decreased for all PEGylated species, but the enzyme retained substantial activity of 30% - 60% depending on substrate. Moreover, the substrate affinities of the modified enzymes increased (thus, kM decreased), which meant that the overall catalytic efficiencies of the unmodified and modified enzymes were comparatively close.

 

Organophosphorus Hydrolase PEGylation Altered Pharmacokinetics and Immunogenicity

In animal testing, unmodified organophosphorus hydrolase had a circulated half-life of 0.86 hr and a mean residence time of 1.1 hr. PEGylation of the enzyme even with the smallest PEG (PEG4) increased the circulated half-life and mean residence time of the modified enzyme compared to the unmodified enzyme. PEGylation with the large branched PEG (Figure 1, above) resulted in a dramatically longer circulated half-life (32.5 hr) and mean residence time (46.7 hr). See Figure 2, below.

 

Figure 2: Amount of circulating organophosphorus hydrolase (unmodified vs PEGylated) remaining in bloodstream over time
Figure 2: Amount of circulating enzyme (unmodified vs PEGylated) remaining in bloodstream over time

 

Moreover, injection of unmodified organophosphorus  hydrolase into test animals caused formation of antibodies to the enzyme. Injection of the PEG-modified enzymes into test animals slightly reduced, but did not prevent, formation of antibodies to organophosphorus hydrolase. The differences in antibody formation between the linear PEG4, PEG8, and PEG12 were not statistically significant. By contrast, though, antibodies formed to the enzyme modified with the branched PEG12 (Figure 1) were significantly less compared to the unmodified enzyme. See Figure 3, below.

 

Figure 3: Antibodies formed to organophosphorus hydrolase, comparing unmodified and PEGylated enzyme
Figure 3: Antibodies formed to organophosphorus hydrolase, comparing unmodified and PEGylated enzyme

 

In their conclusions, Novikov, et al., observed,

“The ability of the PEG modifications to both prolong the residence time in the vascular system and to lower immunogenicity of the conjugates was shown to be directly correlated with mass of the attached polymer, with maximal effect achieved with the branched PEG12 ....”

They then suggested that “... it is reasonable to expect an enhanced circulatory residence of the bacterial enzyme (OPH) conjugated with PEG12 in ... humans.”

 

Quanta BioDesign’s Role in dPEG® Research and Manufacturing

Quanta BioDesign, Ltd. is the developer and leading innovator and manufacturer of discrete PEGylation reagents. We look constantly for new collaborations and opportunities for custom synthesis in creating new dPEG® products. Our customers’ ideas drive the creation of many new products and have given us many popular products. We scientists and Quanta BioDesign are committed to developing new dPEG® constructs for all types of applications including diagnostic, therapeutic, theranostic, and nanotechnology uses. All ideas are welcomed, and we look forward to helping your research by providing the PEGylation reagents necessary for your research and manufacturing needs, whether you need milligrams or multiple kilograms.

For information about any of the PEGylation reagents we make, custom synthesis, or to order products visit us at www.QuantaBioDesign.com.

 

References:

B.N. Novikov, et al., Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol, J. Control. Release (2010), doi:10.1016/j.jconrel.2010.06.003

C.S. Fishburn, The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2008) 4167–4183

 

 Additional dPEG® PEGylation Reagents

Carboxyl-dPEG®₄-(m-dPEG®₄)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₁)₃

Carboxyl-dPEG®₄-(m-dPEG®₂₄)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₂)₃

Carboxyl-dPEG®₄ -(m-dPEG®₈)₃

NHS-dPEG®₄-( m-dPEG®₁₂)₃-ester

 

Adam Fulkert, B.S. – Received his B.S. in Chemistry from The Ohio State University in the autumn of 2011. Adam is a Process Development Chemist involved in process development and scale-up activities.

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Synthetic Allergens for Studying Type-1 Hypersensitivity

Incorporating one of Quanta BioDesign's dPEG® linkers, Notre Dame researchers created novel synthetic allergens for investigating mast cell degranulation. Researching type-1 hypersensitivity (allergic reactions) under laboratory conditions in a way that reflects hypersensitivity in the wild is challenging because in natural conditions epitopes (the part of an antigen recognized by an immune system) are heterogeneous, and the IgE antibodies formed to heterogeneous epitopes are themselves variable. To address this issue, Michael W. Handlogten, Tanyel Kiziltepe, and Başar Bilgiçer, all of the University of Notre Dame, designed and synthesized homotetravalent and heterotetravalent synthetic allergens containing Dansyl, DNP, and DNP-Pro haptens (Figure 1) in different arrangements.

 

Haptens used to synthesize heterotetravalent synthetic allergens
Figure 1: Haptens Used to Synthesize Heterotetravalent Synthetic Allergens

Synthetic Allergens Used a dPEG® Linker

Applying standard Fmoc-amino acid chemistry on solid support in the synthesis, the synthetic allergens used lysine to form a branched structure and Fmoc-N-amido-dPEG®8-acid (PN10273, from Quanta BioDesign, Ltd.) to provide spacing between the lysines and the haptens. (See Figure 2, below.)

 

PN10273, Fmoc-N-amido-dPEG®8-acid, was used to synthesize synthetic allergens used in this study.
Figure 2: Fmoc-N-amido-dPEG®8-acid (PN10273) from Quanta BioDesign, Ltd.

 

Quanta BioDesign’s PN10273 was chosen because it “does not form non-specific interactions with proteins, is flexible enough to minimize steric constraints for hapten binding and enhances the solubility of the hydrophobic haptens” (page 93). Moreover, the dPEG®8 linker length when attached to the lysine residues in the branched structure provided what had previously been determined to be the optimal separation distance between haptens (page 93). From each of the haptens in Figure 1, the researchers synthesized three different homotetravalent (HmTA) and two heterotetravalent (HtTA) synthetic allergens, presenting two of the haptens, each with a valence of two. HtTA-1 presented DNP and Dansyl, while HtTA-2 presented DNP-Pro and Dansyl.

 

Homotetravalent vs. Heterotetravalent Synthetic Allergens

Using the monoclonal antibodies IgEDNP and IgEDansyl, the research group confirmed that both antibodies bind simultaneously to the synthetic allergens. They then compared the extent of mast cell degranulation that occurred with the synthetic allergens as compared to known positive controls. (Mast cell degranulation is the first major immune system response in a hypersensitivity reaction.) They found that homotetravalent-DNP-Pro was unable to stimulate any mast cell degranulation, while homotetravalent-DNP and homotetravalent-dansyl both provoked responses that were only marginally better than the positive controls. In contrast, the two heterotetravalent synthetic allergens provoked much stronger responses than the homotetravalent allergens and the positive controls. HtTA-1 (DNP, Dansyl) induced degranulation over a wide concentration range when both antibodies were present, but failed to induce degranulation under conditions where only one antibody was present. HtTA-2 (DNP-Pro, Dansyl) had a similar ability to induce degranulation. The mast cell degranulation response demonstrated a normal (Gaussian) distribution across the concentration range tested for each synthetic allergen. Moreover, for both heterotetravalent allergens, the maximum degranulation response occurred when the total IgE on the mast cell surface was 25% IgEDansyl, 25% IgEDNP, and 50% orthogonal IgE.

This paper provides important insights into hypersensitivity reactions and demonstrates experimentally the importance of allergen valency, affinity, and cooperativity in mast cell degranulation resulting from allergen-IgE binding. Moreover, the authors have provided a valuable, flexible platform on which they and others can build other functionalities (labels, drug conjugates, and so forth) to extend this area of allergy research.

Michael W. Handlogten, Tanyel Kiziltepe, and Başar Bilgiçer. Design of a heterotetravalent synthetic allergen that reflects epitope heterogeneity and IgE antibody variability to study mast cell degranulation. Biochem. J. (2013) 449 (91–99) doi:10.1042/BJ20121088. Also available through PubMed. To see a current publications list from Professor Başar Bilgiçer's lab, click here.

If you need the flexibility of PEG combined with the purity of Quanta BioDesign’s discrete PEG (dPEG®) technology to build your next great, ground-breaking molecule, check out our full range of PEGylation reagents at www.QuantaBioDesign.com today! If you don’t see what you need, contact us for a custom synthesis. We will be very glad to help you find the right product for your needs.

Related Products from Quanta BioDesign, Ltd.

Fmoc-protected amino-dPEG®-acids

Product number 10243,  Fmoc-N-amido-dPEG®2-acid

Product number 10033, Fmoc-N-amido-dPEG®3-acid

Product number 10213, Fmoc-N-amido-dPEG®4-acid

Product number 10053, Fmoc-N-amido-dPEG®5-acid

Product number 10063, Fmoc-N-amido-dPEG®6-acid

Product number 10273, Fmoc-N-amido-dPEG®8-acid

Product number 10283, Fmoc-N-amido-dPEG®12-acid

Product number 10313, Fmoc-N-amido-dPEG®24-acid

Product number 10903, Fmoc-N-amido-dPEG®36-acid

 

Fmoc-protected-amino-dPEG®-NHS esters

Product number 10994, Fmoc-N-amido-dPEG®4-NHS ester

Product number 10995, Fmoc-N-amido-dPEG®8-NHS ester

Product number 10996, Fmoc-N-amido-dPEG®12-NHS ester

 

Fmoc-protected aminooxy-dPEG®-acid

Product number 10849, Fmoc-N-amidooxy-dPEG®12-acid

 

boc-protected amino-dPEG®-acids

Product number 10220,  t-boc-N-amido-dPEG®4-acid

Product number 10760, t-boc-N-amido-dPEG®8-acid

Product number 10761, t-boc-N-amido-dPEG®12-acid

Product number 10763, t-boc-N-amido-dPEG®24-acid

Product number 10902, t-boc-N-amido-dPEG®36-acid

 

CBZ-protected amino-dPEG®-acids

Product number 10268, CBZ-N-amido-dPEG®4-acid

Product number 10066, CBZ-N-amido-dPEG®6-acid

Product number 10276, CBZ-N-amido-dPEG®8-acid

Product number 10286, CBZ-N-amido-dPEG®12-acid

Product number 10316, CBZ-N-amido-dPEG®24-acid

Product number 10906, CBZ-N-amido-dPEG®36-acid

 

Amino-dPEG®-acids

Product number 10244, amino-dPEG®4-acid

Product number 10067, amino-dPEG®6-acid

Product number 10277, amino-dPEG®8-acid

Product number 10287, amino-dPEG®12-acid

Product number 10317, amino-dPEG®24-acid

Product number 10907, amino-dPEG®36-acid

 

If you want to see more peptide modification reagents...

The list above does not cover all of the peptide modification reagents manufactured and sold by Quanta BioDesign, Ltd. If you want to see still more reagents, click here for the complete list, with structures.

 

 Robert H. Woodman, Ph.D.

Robert Woodman earned his B.S. in Microbiology from the University of Southern Mississippi and his Ph.D. in biochemistry from The Ohio State University. He is a Sr. Production Development Scientist and the Quality Control Manager for Quanta BioDesign, Ltd. Robert has used his abilities in organic chemistry to develop new dPEG® products, and is now using his biochemistry training to develop new applications for these products. You can connect with Robert through LinkedIn.

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