Novel iron composition and method of preparation and use thereof

Abstract

The present invention relates to a novel aqueous iron composition. The aqueous iron composition comprises iron sucrose and bicarbonate. The aqueous iron composition of the present invention exhibits an enhanced renal protective effect compared to conventional iron sucrose compositions.

Description

Background Technology

[0001] This application claims priority to Provisional Application No. 62 / 812,028, filed on February 28, 2019, with the title of the invention “Novel Iron Compositions and Methods of Marking and Using the Same,” the full text of which is incorporated herein by reference.

[0002] Background of the Invention

[0003] The intravenous (IV) iron agent is a colloid composed of spheroidal iron-carbohydrate nanoparticles as shown in Fig. 1. An iron-oxyhydroxide gel is present in the core of each particle, and the core is surrounded by a carbohydrate shell that stabilizes the iron-oxyhydroxide (the main function of the ligand is to stabilize the complex and protect it from further multinucleation).

[0004] Iron carbohydrate complexes act as prodrugs because iron must be released from the iron hydroxide(III) core. According to the proposed mechanism, after administration, stable (type 1) complexes such as ferric carboxymaltose and iron dextran are absorbed by endocytosis by macrophages of the reticuloendothelial system (RES). Refer to the literature (Danielson, J. Structure, chemistry, and pharmacokinetics of intravenous iron agents. Am. Soc. Nephrol. 2004, 15, S93-S98).

[0005] In the case of less stable iron(III)-carbohydrates (type 2), a significant amount of unstable iron can be released from the complex, leading to transferrin saturation and consequently to a significant amount of non-transferrin-bound iron (NTBI), particularly upon high-dose administration. This weakly bound Fe3+ can be readily absorbed by cells in an unregulated manner, potentially inducing oxidative stress (Evans, RW; Rafique, R.; Zarea, A.; Rapisarda, C.; Cammack, R.; Evans, PJ; Porter, JB; Hider, RC Nature of non-transferrin-bound iron: studies on iron citrate complexes and the thalassemic era. J. Biol. Inorg. Chem. 2008, 13, 57-74).

[0006] The only injectable iron-carbohydrate product currently approved by the FDA is (1) INFED ® Dexferrum ® (Iron dextran), Ferahem ® (Ferum oxytol), Injectafer ® (Ferric Carboxymaltose), Venofer ® (Iron Sucrose), Ferrlecit ® It exists in five types of (sodium ferric gluconate complexes). Venoper ® Iron sucrose sold under the name [is] has a molecular weight of approximately 34,000–60,000 daltons (M w It is formulated as a colloidal suspension having the following molecular formula.

[0007] [Na2Fe5O8(OH)ㆍ3(H2O)] n m(C 12 H 22 O 11 )

[0008] Here, n is the degree of iron polymerization and m is a sucrose molecule (C) that forms a complex with the following multinuclear iron core. 12 H 22 O 11 It is the number of ):

[0009] [Na2Fe5O8(OH)ㆍ3(H2O)] n

[0010] Each 1 mL contains 20 mg of elemental iron as iron sucrose in water for injection. Venoper ® It is available in 5 mL single-dose vials (100 mg of iron per 5 mL) and 10 mL single-dose vials (200 mg of iron per 10 mL). The drug contains about 30% sucrose w / v (300 mg / mL) and has a pH of 10.5-11.1. The product does not contain preservatives. The osmolarity of the injectable is 1,250 mOsmol / L.

[0011] A method for synthesizing iron carbohydrates is described in WO 97 / 11711 (1997) by Lawrence et al., which discloses a ferric oxyhydroxyoxide-dextran composition for treating iron deficiency having elliptical particles having a preferred molecular weight range of about 250,000 to 300,000 daltons.

[0012] Recently, iron sucrose has been used in combination with tin protoporphyrin (SnPP) to induce acquired cellular tolerance without causing organ damage. Refer to US Patent No. 9,844,563 by Zager et al. The inventors have recognized the need for an iron sucrose formulation that can be readily combined with tin protoporphyrin (SnPP), is stable, and can be injected into a patient alone or in combination with other agents such as SnPP to treat iron deficiency or for renal protective effects.

[0013] Summary of the Invention

[0014] The present invention relates to an aqueous iron sucrose composition having desirable characteristics. In one aspect, the aqueous iron sucrose composition comprises iron sucrose and bicarbonate. In one aspect, the present invention relates to an aqueous iron pharmaceutical composition comprising iron sucrose; bicarbonate; and a pharmaceutically acceptable aqueous carrier. In another aspect, the present invention relates to a method for the prevention or treatment of kidney disease or disorder comprising administering an aqueous iron composition comprising iron sucrose and bicarbonate in a therapeutically effective amount intravenously. Brief explanation of the drawing

[0015] Figure 1 shows the structure of an iron carbohydrate. Figure 2 is a Western blot of the kidney 18 hours after administration of the aqueous iron composition. Figure 3 shows the GPC chromatograms of three S1 formulations. Figure 4 shows an enlarged view of Figure 3. Figure 5 shows the GPC chromatograms of three S1 formulations. Figure 6 shows an enlarged view of Figure 5. Figure 7 shows the GPC chromatograms of three S1 formulations. Figure 8 shows an enlarged view of Figure 7. Figure 9 shows a comparison of GPC for S1, S2, and S3. Figure 10 shows an AFM top view and a side view of S1. Figure 11 shows the particle size analysis at position 1 for S1. Figure 12 shows a passive cross-sectional analysis of three particles for S1. Figure 13 shows the FTIR spectra of S1 and Sucrose, the best library match. Figure 14 shows the FTIR spectra of S2 and Sucrose, the best library match. Figure 15 shows the FTIR spectra of S3 and dextran, the best library match. Figure 16 shows the 1H NMR spectrum of S1-preparation 1. Figure 17 shows the 1H NMR spectrum of S2-preparation 1. Figure 18 shows the 1H NMR spectrum of S3-preparation 1. Figure 19 shows the 13C NMR spectrum of S1-preparation 1. Figure 20 shows the 13C NMR spectrum of S2-preparation 1. Figure 21 shows the 13C NMR spectrum of S3-preparation 1. Figure 22 shows a comparison of raw data for three samples (freeze-dried). Figure 23 shows the offset overlay of the data for all three samples (using two replicate groups for S3). Figure 24 shows the TGA temperature records of S1, S2, and S3 under nitrogen purging conditions. Figure 25 shows the DSC temperature records of S1, S2, and S3. Specific details for implementing the invention

[0016] In one embodiment, the present invention comprises a composition of aqueous iron sucrose (FeS) and bicarbonate (FeS-bicarb). The inventors have found that such a composition has beneficial properties. In one aspect, the FeS-bicarb composition of the present invention can be used as a renal protective agent. The inventors have found that the FeS-bicarb composition according to an embodiment of the present invention is preferentially absorbed in the kidney compared to commercially available forms of FeS. Additionally, the inventors have found that FeS-bicarb results in preferential upregulation of renal protective molecule(s) compared to FeS alone. In another aspect, the FeS-bicarb composition of the present invention can be advantageously combined with other renal protective agents, such as tin protoporphyrin (SnPP), to easily form an injectable renal protective agent.

[0017] One advantage of using FeS-bicarb is that such compositions result in an enhanced renal protective effect. Specifically, the inventors found that FeS-bicarb preferentially upregulates renal protective molecules compared to FeS alone. Without being bound by theory, the inventors claimed that, in addition to FeS, bicarb can alter the relative levels of present Fe(III) and Fe(II). Due to the reddish tint observed in FeS-bicarb products, the inventors claimed that the compositions of the present invention may contain high levels of Fe(II). It can be seen that the renal protective effect is enhanced because Fe(II) has higher reactivity than Fe(III).

[0018] One advantage of using FeS-bicarb is that bicarb has a buffering effect. This can be advantageous when using tin protoporphyrin compositions because SnPP is stored best at low pH, thus preventing unwanted dimerization during storage. According to the present disclosure, SnPP compositions may be combined with FeS-bicarb compositions in a SnPP:FeS ratio of about 1:1 or less, e.g., about 1:2, about 1:4, about 1:8, about 1:10, about 1:20, about 1:50, about 1:100, about 1:1000, about 1:10,000, about 1:100,000, about 1:1,000,000, or any integer or sub-range between these.

[0019] In one aspect, the composition has a molecular weight measured using GPC as described in Example 1. Mp is preferably in the range of 25,000 to 35,000 daltons, more preferably in the range of 28,000 to 32,000 daltons, most preferably about 29,000 daltons. Mw is preferably in the range of 25,000 to 45,000 daltons, more preferably in the range of 30,000 to 40,000 daltons, even more preferably in the range of 33,000 to 38,000 daltons, most preferably about 34,000 daltons. Mn is preferably in the range of 15,000 to 30,000 daltons, more preferably in the range of 20,000 to 25,000 daltons, most preferably about 24,000 daltons. The polydispersity (PDI) is preferably in the range of 1.35 to 1.60, more preferably in the range of 1.38 to 1.5, even more preferably in the range of 1.40 to 1.48, and most preferably about 1.4.

[0020] In one aspect, the composition has a stable zeta potential of -3.0 mV or less, more preferably -7.0 mV or less, most preferably about -10 mV. In one aspect, the composition has a total organic carbon of less than 8.5%, preferably less than 8.0%, most preferably about 7.7%. In one aspect, the osmolarity measured according to Example 1 is in the range of 550 to 1600 mOsm / kg, preferably in the range of 1500 to 1580 mOsm / kg, most preferably about 1540 mOsm / kg.

[0021] Example 1

[0022] The present invention relates to a composition prepared by dissolving an iron sucrose complex in water (about 3.5 L) sufficient to provide a solution of 12 mg / mL (expressed as iron) when diluted to 6.0 L. The required amount of iron sucrose was calculated so that the final concentration is 12 mg / mL for a final liquid volume of 6100 mL (6.1 L). In this case, 73.2 g of iron is required. The potency of iron sucrose used is 0.0550. Therefore, 73.2 g / 0.0550 or 1331 g ± 1 g of iron sucrose is required. 1331 g ± 1 g of iron sucrose was weighed directly into a 6.0 L Erlenmeyer flask. About 3-3.5 L of water was added to the Erlenmeyer flask, and the contents of the flask were stirred.

[0023] Sodium bicarbonate is added in an amount such that the final sodium bicarbonate concentration becomes 10 mg / mL when diluted to 6.0 L. Weigh 109.8 ± 0.1 g of sodium bicarbonate and add it to a 6.0 L flask.

[0024] Sodium chloride is added in an amount such that the final sodium chloride concentration upon dilution is 9.0 mg / mL. Weigh 54.9 ± 0.1 mg of sodium chloride and add it to a 6.0 L flask. Stir the suspension for 30–120 minutes to provide a black, opaque solution.

[0025] Monitor the pH of the solution with a pH meter while adding 1M sodium hydroxide in small aliquots until the pH reaches 10.30 and remains stable. Add 40.0 ± 0.1 g of sodium hydroxide to a 1.0 L Erlenmeyer flask. Add 1.0 ± 0.1 L of water to the 1.0 L Erlenmeyer flask and stir until all the sodium hydroxide is dissolved. Attach a pH probe to the 6.0 L Erlenmeyer flask to monitor the pH and add sodium hydroxide in aliquots of less than 100 mL until the pH reaches 10.3 ± 0.1. Then, stir the solution for 10 minutes. After 10 minutes, check the pH again and, if necessary, adjust it to within 10.3 ± 0.1.

[0026] Next, transfer the solution to a volumetric precision flask and dilute it to 6.1 L using water. Transfer exactly 4 L of the 10.3 pH solution to a 6 L Erlenmeyer flask using a 2 L volumetric flask twice. Dilute the remaining 10.3 pH solution to 2 L in the volumetric flask and add it to the 6 L Erlenmeyer flask. Using a 100 mL graduated cylinder, add 100 ± 0.1 mL to the 6.0 L Erlenmeyer flask, and stir the resulting solution for 10 minutes.

[0027] The resulting product solution is dark red to brown. Two iron isotopes are present in the sample formulation in a ratio consistent with that of the standard formulation. The resulting substance has a pH of 10.3, which is within the preferred range of 10.1–10.4. The resulting substance contained 11.5 / 11.6 ppt (mg / mL) iron according to SOP 174472 for determining iron via inductively coupled plasma-mass spectrometry.

[0028] Further characteristics of the resulting composition are listed in Table 1 below:

[0029]

[0030]

[0031] The resulting FeS-Bicarb composition has the following stoichiometry, and the physical constants are shown in Table 2 below:

[0032]

[0033] Example 2

[0034] Intravenous administration of the iron sucrose (FeS) bicarb composition of Example 1 was performed for 4 hours, and as a result, Venoper ® Increased renal heme oxygenase 1 (HO-1) was produced compared to a commercially available iron sucrose (FeS) composition sold under the brand name. The results are shown in Table 3 below.

[0035]

[0036] The increase in HO-1 levels observed in the kidneys was not observed in the liver. On the contrary, the HO-1 levels for FeS-bicarb were Venoper ® It was observed that it did not increase compared to what was observed for. The results are shown in Table 4 below.

[0037]

[0038] Plasma BUN and creatinine are FeS, Venoper, as shown in Tables 5 and 6 below. ® It was similar in both FeS-bicarb.

[0039]

[0040]

[0041] Example 3

[0042] The FeS-bicarb composition of Example 1 was filtered and placed in a vial, which had an FeS concentration of 12 mg / mL (CoreRx Lot # 111002-18011). The osmolarity of this 12 mg / mL solution was 831 mOsm. Venoper ® In the case of iron sucrose injection 20 mg / mL, American Regent, Lot # 8243A, the osmolarity was 1742 mOsm. These osmolarity measurements were performed without dilution.

[0043] Example 4

[0044] Western blots of the kidney 18 hours after administration of the aqueous iron composition are shown in Fig. 2 and Table 7:

[0045] Table 7

[0046]

[0047] On the left are heavy chain-specific Western blots of the kidney 18 hours after administration of SnPP, FeS (Venopher), or Fe + SnPP. N = normal control. Glyc is glycerol used as a positive H-chain ferritin control. N = normal sample (control). Clearly, Fe induces an increase in heavy chains in the kidney.

[0048] Example 5

[0049] A patient suffering from chronic kidney disease is treated by intravenously injecting the aqueous iron composition of iron sucrose and bicarbonate of Example 1.

[0050] Example 6

[0051] The patient who has received an organ transplant is treated by intravenously injecting the aqueous iron composition of iron sucrose and bicarbonate of Example 1.

[0052] Example 7

[0053] A patient who has received an organ transplant is treated by intravenously injecting the aqueous iron composition of iron sucrose and bicarbonate of Example 1 in combination with tin protoporphyrin.

[0054] Example 8

[0055] Three samples of iron-sucrose (S1, S2) and iron-dextran (S3) were characterized using various analytical techniques. S1 was prepared according to Example 1 above. S2 was Venoper, a commercially available product. ® It is (iron sucrose injection). S3 is a commercially available product infed ® It is (iron dextran injection). The results are summarized in Table 8 below.

[0056]

[0057]

[0058]

[0059] Finally, the hydroxyl value of the iron-sucrose injection solution was determined by titrating the obtained sample S1 three times repeatedly with dilute HCl. The titration endpoint was pH = 7.0. Assuming that all titrated basic chemical species originated from hydroxylases associated with the ferric oxyhydroxyl core, the H₂ used in the titration + The total moles of OH - It was assumed to be equal to the moles of . Considering TOC and Mw (or Mn) by GPC, the molecular formula of iron sucrose in S1 was calculated as follows:

[0060] Mw-based calculation: [Na6Fe5O8(OH)5 · 3H2O]13 · 73(C12H22O11) Mn-based calculation: [Na6Fe5O8(OH)5 · 3H2O]9 · 51(C12H22O11). Table 9 below shows details regarding sample formulations and identification.

[0061]

[0062] Sample formulation:

[0063] Unless otherwise noted, samples were freeze-dried before analysis to obtain dried residues.

[0064] Gel Permeation Chromatography (GPC):

[0065] GPC is used to determine the molecular weight distribution of polymers. In GPC analysis, a polymer solution is passed through a column filled with a porous gel. The samples are separated according to molecular size, with larger molecules eluting more rapidly than smaller molecules. The retention time of each component is determined and compared with a calibration curve, and the resulting data is then used to calculate the molecular weight distribution of the samples.

[0066] The molecular weight distribution, rather than the intrinsic molecular weight, is a characteristic of all types of synthetic polymers. To characterize this distribution, statistical averages are used. The most common of these averages are "number average molecular weight" (Mn) and "weight average molecular weight" (Mw).

[0067] Number-average molecular weight is similar to the standard arithmetic mean associated with a group of numbers. When applied to polymers, number-average molecular weight represents the average molecular weight of the molecules contained within the polymer. Number-average molecular weight is calculated by assigning equal importance to each molecule, regardless of their individual molecular weights. Number-average molecular weight is calculated by the following formula, where Ni is the number of molecules having the same molar mass as Mi.

[0068]

[0069] The weight-average molecular weight (Mw) differs slightly in calculation and significantly in meaning. Weight-average molecular weight is another statistical term for molecular weight distribution that gives greater importance to larger molecules than to smaller molecules in the molecular weight distribution. The following formula shows the statistical calculation of the weight-average molecular weight.

[0070]

[0071] For GPC, samples were prepared by diluting them in phosphate buffer (according to the USP method) and analyzed to determine the molecular weight distribution of each sample. The results are summarized in Tables 10-12 below. Representative chromatograms obtained by the analysis are presented in Figures 3-9.

[0072] There are two general reasons for weight-average molecular weight. First, for example, when comparing toughness, longer molecules have a greater influence on the toughness of the polymer distribution than shorter molecules. The calculation of weight-average molecular weight focuses on these longer molecules and provides a relative figure that can explain the relative contribution of long molecules to the molecular weight distribution. Weight-average molecular weight is also a figure directly related to the determination of the polymer's molecular weight by light scattering, small-angle neutron scattering (SANS), and sedimentation velocity.

[0073] Secondly, the weight-average molecular weight provides insight into the shape of the molecular weight distribution. This value, in conjunction with the number-average molecular weight, allows for the determination of the ratio of the width of the molecular weight distribution, referred to as the polydispersity index or PI. PI is defined as the ratio of Mw to Mn. The higher the PI, the more dispersed the distribution. The lowest possible value for PI can be 1. This indicates a monodisperse sample, meaning that all molecules in the distributed polymer have the same molecular weight.

[0074] Although not commonly mentioned, the "z-average molecular weight" (Mz) is also provided. This molecular weight average is a value that further explains the molecular weight distribution. This value can be easily determined by sedimentation equilibrium.

[0075] Additionally, the peak molecular weight Mp is sometimes included. The peak molecular weight value is defined as the mode of the molecular weight distribution. It indicates the most abundant molecular weight in the distribution. This value also provides insight into the molecular weight distribution.

[0076] Most GPC measurements are performed based on different polymer standards (typically polystyrene). The accuracy of the results depends on how well the characteristics of the polymer being analyzed match the characteristics of the standards used. The expected error in reproducibility between individually validated, different series of determinations is about 5–10%, which is characteristic of the limiting precision of GPC determinations. Therefore, GPC results are most useful when comparing the molecular weight distributions of different samples during the same series of determinations.

[0077] GPC precision and bias are based on statistical data such as the mean, standard deviation, relative percentage difference, and / or percentage relative standard deviation of the measurements. In the case of quantitative analysis, the quantities listed in the table above are quantitative and refer to known amounts of the standards. A calibration curve was constructed, and information regarding relative standard deviation and relative percentage difference was referenced from the above report. In semi-quantitative analysis, the typical reproducibility determined by the statistical process control of the measurement system is estimated to be approximately 10% (k to 2 at a 95% confidence level). This reproducibility is an estimate of the uncertainty of a single standard measurement over time, and the uncertainty of a specific measurement must be determined on a case-by-case basis. In the case of qualitative analysis, the analytical reference standard was not analyzed to confirm the presence of individual components. In such cases, it is not possible to assign a numerical value to the "uncertainty" of the provided match.

[0078] It should be noted that samples S1 and S2 contain two peaks with distinct molecular weight distributions, whereas sample S3 contains three peaks. Also, it should be noted that Mp could not be calculated for "peak 2" (a small molecule peak, possibly sucrose) because the peak saturated the detector; and that the samples were analyzed at concentrations appropriate for characterizing high molecular weight species at the expense of saturating the detector with low molecular weight species of less interest.

[0079]

[0080]

[0081]

[0082] Dynamic light scattering

[0083] PSD analysis was performed using a laser diffractometer. The volume distribution is calculated from the laser diffraction pattern of the particle cloud by these measurements. This raw dispersion data is then processed using an algorithm and presented based on the equivalent spherical diameter. The results were summarized based on volume (mass) in a histogram providing differential volume percentages smaller or larger than the indicated size.

[0084] Particle size analysis is Malvern ® The analysis was performed on a Zetasizer Nano ZS dynamic light scattering (DLS) instrument. DLS is an ensemble technique that analyzes light scattered by particles moving in Brownian motion and generates a particle size distribution based on the diffusion velocity of the particles. Raw scattering data is processed using complex algorithms and presented based on an intensity-weighted hydrodynamic diameter. The analysis technique is summarized in the literature (ISO 22412:2008 Particle Size Analysis - Dynamic Light Scattering (DLS)) as well as in the literature (ASTM E2490-09(2015) Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy (PCS)).

[0085] The sample obtained as is was water for injection (WFI) and was analyzed by DLS to provide the total physical dimensions of the particles. The intensity- and volume-weighted results obtained from the analysis are presented in Table 13 and Table 14, respectively.

[0086]

[0087] 1 NNLS = negative-exclusive least squares data; 2 Z-mean = average particle size distribution; 3 PDI = Polyvariance Exponential

[0088]

[0089] 1 NNLS = negative-exclusive least squares data; 2 Z-mean = average particle size distribution; 3 PDI = Polyvariance Exponential

[0090] Zeta potential

[0091] Samples were prepared by diluting in buffer for zeta potential (the instrument could not achieve stable readings when diluted in 10 mM NaCl according to the literature (Nanomaterials 2018, 8, 25)). pH and temperature were recorded at the time of zeta potential analysis. The results are summarized in Tables 6 through 8 below. In the case of S2, stable readings could not be obtained. The results for the zeta potential test are reported in Tables 15-17.

[0092]

[0093]

[0094]

[0095] Atomic Force Microscope (AFM)

[0096] The samples obtained as is were diluted 50-fold using MilliQ filtered water (18.2 MΩ / cm, 4 ppb TOC). Approximately 10 μL of this diluted solution was immersed onto freshly cleaved mica fragments and incubated for about 1 minute. The samples were then rinsed 5 times with MilliQ water and dried using nitrogen. For each sample, two 1 μm x 1 μm areas were imaged. Topographical differences in these images are presented by color, where brown indicates low and white indicates high. The z-range is indicated on the vertical scale bar to the right of the image. Perspective views (3-D) of these surfaces are included along with vertical exaggeration indicated in the caption.

[0097] Particle size analysis was performed to characterize the height of particles present within each region. Particles of interest were identified by excluding non-representative features using a height threshold of 0.5 nm. The results for maximum height, minimum height, and average height are summarized in Table 18.

[0098]

[0099] Cross-sectional analysis was performed to manually measure the height of representative particles. The cross-sectional analysis at location 1 for S1 is shown in Figures 10, 11, and 12. The results for S1, S2, and S3, respectively, are summarized in Table 19.

[0100]

[0101] Total Organic Carbon (TOC)

[0102] The total organic carbon (TOC) of the sample was calculated by subtracting inorganic carbon from total carbon (determined using a combustion carbon analyzer). The results are summarized in Table 20 below.

[0103]

[0104] 1 wt% = weight percentage; 2 %RSD = Relative standard deviation

[0105] Osmolarity

[0106] The osmolarity of the sample was measured using the vapor pressure method. The vapor pressure method is a method for determining osmolarity using a sample in a state of natural equilibrium at room temperature. The results of the osmolarity test are summarized in Table 21.

[0107]

[0108] Fe +3 vs Fe +2

[0109] Each sample of a fraction was diluted in concentrated hydrochloric acid according to the reference method provided by Client Gupta 1 et al. The samples were then analyzed according to the method summarized by Stookey 2. The results are shown in Table 22.

[0110]

[0111] %RSD = Relative standard deviation

[0112] Elemental separation by Inductively Coupled Plasma / Mass Spectrometry (ICP / MS) and total iron and sodium content by Inductively Coupled Plasma / Optical Emission Spectrometry (ICP / OES)

[0113] ICP / OES is a spectroscopic technique used to identify and quantify components by element. In ICP, high-frequency energy is transferred via inductive coupling to a fluid of inert gas containing the sample as an aerosol. The energy causes the aerosol to vaporize while simultaneously exciting the resulting free atoms, causing them to emit light. Consequently, the intensity of this light is related to the concentration of the emitted atoms. This technique requires instrument calibration and secondary-source verification before, during, and after completing the analytical run sequence. Additionally, the instrument blanks comply with their respective verification standards. This ensures that no carryover occurs during the analytical sequence. Concentration measurements of major elements performed by ICP typically have an uncertainty in the range of 3 to 5% (95% confidence level). The uncertainty regarding the concentrations of trace elements can be significantly higher.

[0114] Samples S1 to S3 were analyzed by ICP-MS for metals and / or other elements. The samples were also analyzed by ICP-OES to determine the total iron and sodium content. The samples were analyzed three times in their original form. The results are summarized in Tables 23-25.

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] Fourier Transform Infrared Spectroscopy (FT-IR)

[0126] Fourier transform infrared spectroscopy (FT-IR) is a selective tool for identifying substances. In FT-IR, infrared absorption bands are assigned to characteristic functional groups. Based on the presence of multiple such bands, the substance under consideration can be identified. The likelihood of a positive identification is increased if the spectra of known compounds are available. Lyophilized samples were analyzed via horizontal attenuation total reflection (HATR), which is based on the internal reflection of infrared (IR). The FT-IR spectrum of S1 is presented in Figure 13 below, along with a spectrum library match. The data suggest that the substance matches sucrose. The FT-IR spectra of S2 and S3 are presented in Figures 14 and 15. Absorption assignments to functional groups are shown in Tables 26–28.

[0127]

[0128]

[0129]

[0130] 1H Nuclear Magnetic Resonance Spectroscopy (NMR)

[0131] NMR spectroscopy is a very useful method for characterizing materials.

[0132] NMR is a physical phenomenon based on the magnetic properties of atomic nuclei. NMR studies magnetic nuclei (most commonly the nuclei of hydrogen atoms) by aligning them using a very strong external magnetic field and perturbing this alignment using electromagnetic pulses. The response to the perturbation is recorded, and each individual nucleus provides a specific response depending on its chemical, electronic, and spatial environment.

[0133] The freeze-dried sample was reconstituted using deuterium oxide (D2O) and analyzed by 1H NMR spectroscopy.

[0134]

[0135] Chemical formula (I)

[0136] The structure for sucrose is shown above with the hydrogen tin of chemical formula (I). The 1H NMR for S1 is shown in Table 29 below:

[0137]

[0138]

[0139] The structure of dextran is shown above with hydrogen tin of chemical formula (II). Table 30 below shows the 1H NMR for S3.

[0140]

[0141] The NMR spectra of the prepared samples are presented in Figures 16-18. Where possible, the provisional assignments to the major chemical shifts observed in the NMR spectra were based on reference spectra of relevant compounds commonly used in the literature.

[0142] The data suggest that sucrose is present in sample S1 and that the chemical shift matches well with that reported in the literature. However, no peak splitting pattern was observed, which could be due to various reasons, such as the presence of nanoparticles or paramagnetic iron itself.

[0143] The 1H NMR spectrum for sample S2 shows that the peaks are significantly broadened. It is not known whether this is due to particulates creating an increased chemical environment or if the properties of iron in the sample may be the cause of the lack of resolution. Depending on the degree of broadening, peak assignment may not be made. However, the general peak intensity and chemical shift are consistent with those observed for sucrose, as a large and broad response was observed at the chemical shift of 2.5–4.2 ppm and a slight shoulder was observed at the solvent peak near 5.5 ppm.

[0144] 13C Nuclear Magnetic Resonance Spectroscopy (NMR)

[0145] The freeze-dried sample was reconstituted using deuterium oxide (D2O) and analyzed by 13C NMR spectroscopy.

[0146] The results are summarized in Tables 28-30. The NMR spectra of the prepared samples are presented in Figures 19-21. Where possible, the provisional assignments to the major chemical shifts observed in the NMR spectra were based on reference spectra of relevant compounds commonly found in the literature.

[0147] The data suggest that sucrose is present in samples S1 and S2 and that the chemical shift matches well with that reported in the literature. It should be noted that, as with the proton spectrum, sample S2 appears broader than sample S1. Finally, the peak observed in sample S3 matches well with literature values ​​for dextran, which suggests that it is present in the sample.

[0148]

[0149] The structure of sucrose is shown above along with carbon-tin. The 13C NMR results are shown in Table 31 below:

[0150]

[0151]

[0152]

[0153] The structure of dextran is shown above along with the carbon tin of the chemical formula (III). Table 33 below shows the 13C NMR for S3 dextran in D2O.

[0154]

[0155] X-ray diffraction (XRD) analysis (freeze-dried material)

[0156] XRD analysis is a method of irradiating crystalline inorganic samples with monochromatic X-rays. The interaction between the sample's lattice structure and these X-rays is recorded, providing information about the crystal structure being irradiated. The characteristic "fingerprint" of the result enables the identification of crystalline compounds present in the sample. Using whole-pattern fitting analysis (Rietveld Refinement), it is possible to perform quantitative analysis on samples containing more than one crystalline compound.

[0157] The freeze-dried samples were analyzed by XRD to characterize the chemical structure and phases present in the samples. The results obtained from the analysis are presented in Table 34. It should be noted that sticky samples were produced by this sample preparation method, particularly in the case of S1 and S2 (S3 was less sticky). In the case of S1 and S2, a drop of methanol was added to the sample, and the material was spread evenly on a sample holder. Sample S3 was ground in a mortar and pestle.

[0158]

[0159] Figure 22 overlays raw XRD data derived from three samples with a small offset for clarity. Sample S2 differs from the other two samples in terms of overall intensity, peak position, as well as peak shape. The broad peak shapes of samples S1 and S3 suggest that these samples consist of a mixture of nanocrystalline and amorphous materials.

[0160] Using the best match obtained by comparing the background-modeled experimental data with the ICDD / ICSD diffraction database for samples S1, S2, and S3, respectively, it was determined that samples S1 and S3 contain a mixture of amorphous and nanocrystalline materials. A sodium iron oxide reference pattern was superimposed on these experimental data. Markers indicate the positions of the expected diffraction peaks for each phase, and marker heights indicate the relative peak intensities for the differentiated and randomly oriented materials. Unlike the other two samples, sample S3 consists mainly of sucrose and amorphous materials.

[0161] Semi-quantitative analysis was performed using WPF (Whole Pattern Fitting), a subset of Rietveld analysis that describes all regions above the background curve. To use this technique, the structure factor and atomic position or reference intensity ratio (a method for comparing the diffraction power of different phases) must be known for all identified phases. In this process, to minimize the R value, which is an estimate of the degree of agreement between the model and experimental data for the entire pattern, the structure factor (related to concentration), lattice parameter (related to peak position), peak width, and peak shape are analyzed for each phase.

[0162] To obtain quantitative results from a sample containing a measurable amount of amorphous material, the density of the amorphous material must be specified to determine how much amorphous material is present. Consequently, the concentration of the amorphous material is uncertain. In such samples, the position of the amorphous peak is assumed to originate from amorphous sucrose with a density of approximately 1.59 g / cm³. Since WPF attempts to account for everything in the sample, any error in the concentration of the amorphous material will result in an error in the crystallinity as well. This means that while the relative concentration of the crystallinity is accurate, the absolute value is erroneous by an amount proportional to the error in the concentration of the amorphous material.

[0163] X-ray diffraction (XRD) analysis (sugar-free material)

[0164] The sample as is was diluted with water, placed in a 10,000 Da molecular weight cutoff (MWCO) filter, and centrifuged to remove small molecules (sugars) from the formulation that caused amorphous material in the previous XRD analysis. Subsequently, the sample was washed five more times with water to remove residual small molecules. The resulting material (passable through the filter) was freeze-dried and analyzed via XRD to characterize the chemical structure and phases present in the sample. Note that sample S3 contains two distinct layers after centrifugation: a thick viscous layer and a thinner upper layer. These layers were separated, freeze-dried individually, and analyzed as two separate samples. The results were averaged to provide the values ​​shown in Table 35, but the individual replicates of each layer are presented in the figure below. The results obtained from the analysis are presented in Table 35.

[0165]

[0166] Weight % = Weight percent, ± 5%; 2 Average of 2 duplicate manufacturings (two layers observed)

[0167] Overlays of XRD patterns from all four samples (two replicate groups in the case of S3) are shown in Fig. 23. The patterns are offset for clarity. Phase identification was performed by comparing the best match between the background-modeled experimental XRD data and the ICDD / ICSD diffraction database for the samples. The reference marker for the phase indicates the location where the expected experimental peak at 2-theta should be situated, and the height of the marker suggests the expected intensity of the experimental peak when the sample is differentiated and randomly oriented. It should be noted that XRD is sensitive to crystal structure but relatively insensitive to elemental or chemical state composition. Phase identification for these samples was difficult due to the nanocrystalline properties of the samples, which significantly broaden the peaks in the XRD patterns.

[0168] The best match for the peaks present in all four samples is the iron oxide phase and the iron oxide hydroxide phase, known as maghemite. The iron oxide hydroxide phase is amorphous because it is formed by heating the beta-phase iron oxide hydroxide to approximately 300°C. Unfortunately, these reference cards do not include the reference intensity ratio (RIR) required for semi-quantitative analysis. However, since the symmetry and composition are similar to those of the iron oxide hydroxide mineral goethite (alpha-FeOOH), the average RIR of goethite was used for the semi-quantitative analysis of the iron oxide hydroxide.

[0169] Semi-quantitative analysis was performed using WPF (Whole Pattern Fitting), a subset of Rietveld analysis that describes all intensities on the background curve. To use this technique, the structure factor and atomic position or reference intensity ratio (a method for comparing the diffraction power of different phases) must be known for all identified phases. In this process, to minimize the R value, which is an estimate of the degree of agreement between the model and experimental data for the entire pattern, the structure factor (related to concentration), lattice parameter (related to peak position), peak width, and peak shape are analyzed for each phase.

[0170] Acid decomposition of unstable iron(III) using UV-visible spectroscopy

[0171] UV / Vis spectroscopy is used to determine the concentration of an analyte, either all at once or often over a desired period. This technique measures the absorption of light across ultraviolet and visible wavelengths through a liquid sample. The sample is dispensed into a small vial and placed between the UV / Vis light path and the detector. According to the Beer-Lambert law, using a constant light path length and known absorption coefficients depending on the wavelength, the concentration of the compound in question can be determined from the light absorbed by the sample at that wavelength.

[0172] The samples were analyzed using a method to determine the amount of unstable iron (III) in the samples using UV-visible spectroscopy, adopted from the literature (BS Barot et al. (2014)). The results are summarized in Table 36 below.

[0173]

[0174] Thermogravimetric Analysis (TGA)

[0175] TGA consists of measuring the change in weight of a material as a function of temperature in a controlled atmosphere. This technique requires precise measurements of weight, temperature, and temperature change. Through the temperature chart obtained by analysis, the content of components (e.g., solvents, polymers, inorganic fillers, etc.) and the thermal stability of the polymer can be determined. Typical precision and bias of TGA measurements are discussed under ASTM E2040.

[0176] The freeze-dried samples were analyzed by thermogravimetric analysis (TGA) under nitrogen and air purging. The thermal decomposition of the samples occurs in three separate stages, as shown in Figure 24. The results of these stages are summarized in Table 37.

[0177]

[0178] Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis (DTA)

[0179] The freeze-dried samples were analyzed by differential scanning calorimetry (DSC) under argon purging. Differential scanning calorimetry (DSC) measures the difference in heat flow associated with transitions between a sample and an inert reference as a function of temperature and time. These measurements provide quantitative and qualitative information regarding physical and chemical changes and changes in heat capacity associated with endothermic or exothermic processes. Refer to Figure 25 for the DSC temperature chart. A summary of the DTA is presented in Table 38 below.

[0180]

[0181] n / a = not observed; 2 Possible overlapping transitions

[0182] Determination of hydroxide value and molecular formula by titration

[0183] Sample S1 as obtained was titrated three times with 0.00998N HCl to determine the hydroxyl value of the iron-sucrose injection solution. The titration endpoint was pH = 7.0. Table 39 summarizes these titration results for S1.

[0184] Assuming that all titrated basic species originated from hydroxyls associated with a ferric oxyhydroxyl core, the total moles of H+ used in the titration were assumed to be equal to the moles of OH-. Considering TOC and Mw from GPC, the molecular formula of iron sucrose in S1 was calculated as follows:

[0185] [Na6Fe5O8(OH)5·3H2O]13·73(C12H22O11)

[0186] When Mn is considered in these calculations, the molecular formula is as follows:

[0187] [Na6Fe5O8(OH)5·3H2O]9·51(C12H22O11)

[0188]

[0189] A person skilled in the art will be aware of other embodiments and uses of the invention by considering the specification and practices of the invention disclosed herein. All referenced literature cited herein, including all U.S. and foreign patents and patent applications, is incorporated herein by reference in its entirety. The specification and examples are intended to be regarded merely as illustrative, and the true scope and intent of the invention are implied by the following claims.

Claims

Claim 1 An aqueous iron pharmaceutical composition comprising iron sucrose; bicarbonate; and a pharmaceutically acceptable aqueous carrier, wherein the iron sucrose is present in both iron(II) and iron(III) forms, the pharmaceutical composition has a pH greater than 9, an iron(II) concentration of 0.05% w / v to 0.41% w / v, and the iron sucrose is M according to GPC of 30,000 to 40,000 daltons. w A pharmaceutical composition having an inductive iron. Claim 2 An aqueous iron pharmaceutical composition having a specific gravity of 1.135 to 1.165 at 20℃ in claim 1. Claim 3 An aqueous iron pharmaceutical composition having an iron(II) concentration of 0.10% w / v to 0.20% w / v in claim 1. Claim 4 An aqueous iron pharmaceutical composition according to claim 1, wherein the pH is within the range of 10.1 to 10.

4. Claim 5 An aqueous iron pharmaceutical composition according to claim 1, having an Mw according to GPC of 33,000 to 38,000 daltons. Claim 6 A pharmaceutical composition for treating a disease or disorder comprising the aqueous iron pharmaceutical composition of claim 1 and tin protoporphyrin (SnPP), wherein the aqueous iron pharmaceutical composition and tin protoporphyrin (SnPP) are administered intravenously, and the disease or disorder is organ transplant rejection, chronic kidney disease, or iron deficiency. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete

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