(T1430-09-62) Characterization of Intravenous Iron Supplements: Injectafer^®

Purpose: Intravenous (IV) iron supplements have been used to treat iron deficiency anemia (IDA) since the 1950s. There are several iron supplement drug products on the market including Ferrlecit^®, Venofer^®, Injectafer^®, Monoferric^®, Feraheme^®, etc. The iron supplements are usually composed of iron nanoparticles, which are composed of an iron oxide core that is surrounded by a carbohydrate matrix to promote the extended release of the iron. Although these iron supplements have been in use for several decades, the internal structures of the iron nanoparticles have not been fully understood. For example, Injectafer^® has been proposed to have a ferric hydroxide core with carboxymaltose molecules surrounding the iron core. This research focuses on characterizing the composition of Injectafer^®, including the carbohydrate shell, the iron core and interactions between the two.

Methods: Maltodextrins with dextrose equivalent (DE) of 4-7, 8-15, 13-17, 16.5-19.5 (Sigma-Aldrich, St. Louis, MO) were used to synthesize carboxymaltose. ¹H and ¹³C NMR spectra (Bruker Avance-III-800) have been obtained for both maltodextrins and carboxymaltoses. 2D NMR spectra have been obtained for peak assignments for maltodextrin and carboxymaltose. 2D NMR spectra have also been obtained for maltotriose and carboxymaltotriose to help with peak assignments for maltodextrin and carboxymaltose. Carboxymaltose in Injectafer^® was obtained by treating Injectafer^® with phosphate buffer to remove the ferric hydroxide core. The isolated carboxymaltose has been characterized and compared with the lab-synthesized carboxymaltoses. Different amounts of FeCl₂ and FeCl₃ have been added to maltodextrin and carboxymaltose respectively to understand the influence of iron concentration on the shift of the peaks in the NMR spectra.

Results: Figure 1 shows that the peaks in the ¹³C NMR spectrum are shifting to the right as the Injectafer^® is diluted with water. The shift and broadening of the peaks is likely due to the presence of iron(III) in the sample. In order to confirm the changes in chemical shift are due to the presence of iron, either FeCl₂ or FeCl₃ was added to maltotodextrin and carboxymaltose to study the chemical shift changes as a function of either iron(II) or iron(III) content. Both forms of iron showed a similar change in chemical shift as a function of concentration and were consistent with the changes observed of Injectafer^®. The result is that the changes in the shift as a function of concentration is the same for both carboxymaltose and maltodextrin. Thus the changes are due to the overall amount of iron present in the sample, and not specifically how the iron is interacting with the carbohydrate species. In order to confirm which peaks correspond to the carboxylate end in carboxymaltose, 2D NMR spectra including COSY, TOCSY, HMBC and HMQC were acquired. Since maltodextrin with a DE of 4-7 has a relatively longer chain length that could create difficulties in assigning the NMR spectrum, we used maltotriose to help with peak assignment. Figure 2 shows the ¹H COSY spectra of maltotriose and carboxymaltotriose. The projected position of each proton is also shown in the figure. Figure 3 shows a comparison of carboxymaltotriose and carboxymaltose that was isolated from Injectafer^®. The spectra of the two samples are similar to each other, confirming that the structure of the carboxymaltotriose is comparable to that of carboxymaltose from Injectafer^®. In addition, techniques such as size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, and mass spectrometry were used to study both the carboxymaltose and the intact ferric carboxymaltose nanoparticles.

Conclusion: Maltotriose and carboxymaltotriose have similar ¹H and ¹³C NMR spectra as those of maltodextrin and carboxymaltose, and therefore can be used for assisting in peak assignments for carboxymaltose. Peak shift and broadening were observed with different iron concentrations, suggesting that both free iron and the iron core can impact the NMR spectrum. Other techniques such as inductively coupled plasma mass spectrometry, dynamic light scattering, size exclusion chromatography, and transmission electron microscopy have also been used to facilitate the characterization of Injectafer^®

Figure 1. 13C NMR spectra of Injectafer®, Injectafer® diluted 3 times, Injectafer® diluted 9 times, 20 mg/mL maltodextrin with DE 8-15 (from top to bottom).

Figure 2. 1H COSY spectra of maltotriose (red) vs carboxymaltotriose (blue)

Figure 3. 1H COSY spectrum of carboxymaltotriose (red) vs carboxymaltose (blue)
from Injectafer®