Issue:April 2019

DRUG DEVELOPMENT – Rapid Preparation of Gadolinium & Protamine Complexes With Aurintricarboxylic Polysalicylates: Implications for Drug Development


ABSTRACT

Derivatives of aurintricarboxylic acid (ATA), a commercial reagent for aluminum ion determination, are under consideration for drug development in view of ATA’s diverse pharmacological spectrum. The ability of this agent to form avid complexes with cations is an important consideration when evaluating ATA and derivatives in drug development, particularly with regard to drug disposition and targeting. Protamine sulfate, an FDA-approved polycationic agent with a wide variety of drug targeting and delivery applications, was used in preparation of a protamine aurintricarboxylate (protamine – ATA) complex. The formation of an insoluble protamine – ATA complex was studied by titration curve analysis, providing evidence that complex formation occurs with most of the polyanionic components of ATA. The ability of this agent to form avid complexes with other trivalent metal ions is an important consideration when evaluating ATA and derivatives in drug development, particularly with regard to drug disposition. The ability of gadolinium (III) complexes to facilitate magnetic resonance imaging could be useful in drug disposition studies of ATA and its derivatives. A gadolinium (III) ATA complex was prepared with gadolinium nitrate hexahydrate and aurintricarboxylic acid (sodium salt). The formation of the insoluble Gd (III) ATA complex was studied by titration curve analysis, providing evidence for stoichiometric formation of the complex with one gadolinium ion to one aurintricarboxylate subunit of ATA. Complexation with protamine and gadolinium (III) may provide interesting approaches to study the disposition of polyanionic ATA and its derivatives.


INTRODUCTION

Aurintricarboxylic acid (ATA), as a commercial preparation, is a mixture of polyanionic components, including the triphenylmethane dye (Figure 1).1,2 ATA has an unusually wide spectrum of activity; including potential antiviral and antiplatelet applications.1,2 Although derivatives of ATA may be more suitable candidates for drug development, the polyanionic nature may be retained in many of these derivatives. Counterion complexation of polyanionic ATA may result in a formulation with altered pharmacological targeting and disposition. Among the agents potentially useful for complex formation, the polycation protamine sulfate, approved by the FDA, offers an interesting opportunity to study the disposition and targeting of ATA. Because ATA was originally developed as a reagent for the determination of aluminum ion in various media, the trivalent state of other metals are known to form both soluble and insoluble complexes with aurintricarboxyic acid.3 Among these trivalent metals is gadolinium (III) or Gd (III), an ion that is used for magnetic resonance imaging (MRI) applications as a chelate.4 Complexation of ATA with Gd (III) offers an interesting opportunity to study the disposition of ATA and related agents as gadolinium complexes by MRI. This report outlines the preliminary preparation and characterization of the protamine and Gd (III) complexes through rapid titration curve analysis.

COMPLEXATION PROCEDURES

Protamine-ATA
Aurintricarboxylic acid (sodium salt) and protamine sulfate USP were purchased from Sigma-Aldrich (St. Louis, MO). Both agents were prepared in distilled water for complexation. ATA (0.5 mg in 0.25-ml solution) was added to each microcentrifuge tube containing 1 ml of protamine sulfate solution at various concentrations (0.2 mg/ml to 1.2 mg/ml) at 23°C with vortex mixing. To remove the protamine – ATA complex, the microtubes were centrifuged at 2000 x g for 5 mins. The absorbance of the supernatant was read at 450 nm to evaluate the titration of ATA by protamine sulfate.

Gd (III)-ATA

In addition to ATA, Gd (III) nitrate hexahydrate were purchased from Sigma-Aldrich (St. Louis, MO). Both agents were prepared in distilled water for complexation. ATA (0.5 mg in 0.25-ml solution) was added to each microcentrifuge tube containing 1 ml of gadolinium nitrate solution at various concentrations (0.15 mg/ml to 3.5 mg/ml) at 23°C with vortex mixing. To remove the Gd (III) ATA complex, the microtubes were centrifuged at 2000 x g for 5 min. The absorbance of the supernatant was read at 450 nm to evaluate the titration of ATA by Gd (III) ion.

RESULTS & DISCUSSION

The formation of the protamine – ATA complex was very rapid and resulted in an insoluble complex, with an increase in ATA absorbance at 450 nm in the presence of protamine (typical of other cations).5 The results of the titration are shown in Figure 2. The titration curve reveals that the increased presence of protamine results in a slight increase in absorbance during the formation of an insoluble complex. At approximately 1 mg protamine sulfate per ml, complexation is essentially complete, having removed most of the ATA from solution. While the protamine – ATA is somewhat stable under the conditions of this titration experiment, the behavior of the complex in physiological media, both in vivo and in vitro, remains to be determined. Because protamine complexes are relatively stable in vivo and protamine derivatives have applications for cellular delivery of macromolecules, the protamine – ATA complex offers an attractive option to study the disposition and targeting of ATA and its derivatives in future studies.6

Likewise, the formation of the Gd (III) ATA complex was very rapid and resulted in an insoluble complex, with an increase ATA absorbance at 450 nm in the presence of gadolinium (as noted previously).5 The results of the titration are shown in Figure 3. The titration curve reveals that the increased presence of gadolinium results in an increase in absorbance during the formation of an insoluble complex. At approximately 0.3 mg of gadolinium nitrate hexahydrate per ml, complexation is essentially complete, having removed virtually all of the ATA from solution. This point corresponds to approximately 1 ion of gadolinium to each ATA subunit. While the Gd (III) ATA is somewhat stable under the conditions of this titration experiment, the behavior of the complex in physiological media, both in vivo and in vitro, remains to be determined. Since gadolinium chelates are relatively stable in MRI imagining studies, the Gd (III) ATA complex and related complexes are very attractive options to study the disposition of ATA and its derivatives in future studies.4

REFERENCES

  1. Weinstein M, Vosburgh E, Phillips M, Turner N, Chute-Rose L, Moake J. Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers. Blood 1991;78(9):2291-2298.
  2. Cushman M, Sherman P. Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions. Biochem Biophys Res Commun. 1992 May 29;185(1):85-90.
  3. Yoe, JH. Some observations on reactions between certain metallic ions and the ammonium salt of aurintricarboxylic acid. J Am Chem Soc. 1932;54(3):1022-1023.
  4. Caravan P., Ellison JJ, McMurry, TJ, Lauffer, RB. Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem Rev. 1999;99:2293-2352.
  5. Clark RA, Krueger GL. Aluminon: its limited application as a reagent for the detection of aluminum species. J Histochem Cytochem. 1985;33(7):729-732.
  6. Goldberg M, Langer R, Jia X. Nanostructured materials for applications in drug delivery and tissue engineering. J Biomater Sci Polym Ed. 2007;18(3):241-268.

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Dr. Timothy Smith is currently a Professor of Pharmacology at the Thomas J Long School of Pharmacy and Health Sciences at the University of the Pacific. He earned his BS in Pharmacy from Purdue University and his PhD in Pharmacology from the University of Minnesota. His primary research focus is the study of the pharmacology and disposition of biopolymer complexes.