Pharmacokinetics and tissue distribution of deferoxamine-based nanochelator in rats

Abstract

Aim

To characterize the pharmacokinetics of deferoxamine-conjugated nanoparticles (DFO-NPs), a novel nanochelator for removing excess iron.

Materials & methods

The pharmacokinetics of DFO-NPs were evaluated in Sprague–Dawley rats at three doses (3.3, 10 and 30 μmol/kg) after intravenous and subcutaneous administration.

Results

DFO-NPs exhibited a biphasic concentration-time profile after intravenous administration with a short terminal half-life (2.0–3.2 h), dose-dependent clearance (0.111–0.179 l/h/kg), minimal tissue distribution and exclusive renal excretion with a possible saturable reabsorption mechanism. DFO-NPs after subcutaneous administration exhibited absorption-rate-limited kinetics with a prolonged half-life (5.7–10.1 h) and favorable bioavailability (47–107%).

Conclusion

DFO-NPs exhibit nonlinear pharmacokinetics with increasing dose, and subcutaneous administration substantially improves drug exposure, thereby making it a clinically viable administration route for iron chelation.

Plain language summary

Iron is an essential metal nutrient, but excess iron produces toxic effects that damage multiple organs including the heart, liver and pancreas. Deferoxamine (DFO) is a US FDA-approved drug for treating iron overload, but its use is limited by serious adverse effects and an inconvenient daily dose scheme. The recent development of a DFO-based nanomedicine (DFO-NP) has shown promise in treating iron overload in animals and was safer in animals. Before this new drug can be given to humans, how it is absorbed into the body, processed in the body and removed from the body when given in different amounts and dose routes must be determined. In this study, we tested the absorption, distribution and removal of DFO-NPs after intravenous and subcutaneous injection in rats. This study showed that DFO-NPs behave differently when changing the dose and that subcutaneous injection makes the drug stay in the body longer without ill effect, which means it could be given to patients this way.

Iron is an essential metal that plays a critical role in maintaining proper physiological function [1]. Labile iron, however, can produce reactive oxygen species, which can lead to severe organ damage after sustained exposure [2]. Indeed, a number of iron-associated disorders, including hemochromatosis, iron-loading anemias and transfusional iron overload, lead to pathological accumulation of iron in the liver, heart and pancreas, causing a range of health issues, including diabetes, liver cirrhosis, cardiac arrhythmias and even death from cardiac failure [2,3].

There are two types of treatment for iron-overload disorders: phlebotomy and iron chelation therapy (ICT). Phlebotomy is indicated for nonanemic iron overload and is typically associated with the treatment of hemochromatosis [4,5]. ICT is indicated for anemic iron-overload disorders, such as those associated with blood transfusions [6,7]. There are currently three approved small-molecule iron chelators – deferoxamine (DFO), deferiprone and deferasirox – which have been extensively described and reviewed in the literature [6–9]. Although each approved chelator has been shown to ameliorate iron overload, these existing ICTs cannot meet their full therapeutic potential due to dose-limiting toxicities and poor patient compliance due to potentially severe side effects [7].

Nanochelators are a class of novel iron chelators that have received significant attention in the past decade. This is due to the ability of nanoparticles (NPs) to extend the half-life of small molecule chelators and reduce their off-target distribution, which is often associated with toxicities. Readers are encouraged to consult the works of Hamilton et al. [10] and Jones et al. [11] for recent reviews of advances in nanochelator technologies. In particular, Kang et al. reported on a renal clearable DFO-containing nanoparticle (DFO-NP) prepared by conjugating DFO to ε-poly-lysine (EPL) [12]. This promising nanochelator has demonstrated robust iron chelation in vivo and an improved safety profile compared with native DFO [12,13].

To facilitate clinical translation of this novel therapeutic, the pharmacokinetic (PK) profile of the DFO-NP should be fully characterized. Previous PK studies in mice, which evaluated multiple DFO-NP stoichiometries at a subtherapeutic, intravenous (iv.) dose, indicated that DFO-NPs are rapidly eliminated from circulation with half-lives on the order of 30 to 60 min. Therefore, additional studies are required for thorough characterization of the PK of subcutaneous (sc.) administration – the proposed clinical administration route – and to understand the linearity of PK across doses. It is especially important to characterize dose-dependency of PK, as chelator doses can and should be adjusted based on disease severity and iron burden [6]. This study reports on the complete PK characterization of DFO-NPs in rats at multiple relevant therapeutic doses after iv. and sc. administration.

Materials & methods

Synthesis & characterization of DFO-NP

ZW800-EPL was synthesized according to the previously reported method [12]. Briefly, the NIR fluorophore ZW800-1C was conjugated to EPL using the conventional N-hydroxysuccinimide (NHS) ester chemistry. Free amines were subsequently converted to carboxylic acids via reaction with succinic anhydride. To prepare DFO₅-NP, DFO was conjugated to the free carboxylic acid groups on ZW800-EPL following the method established in [14]. In brief, the terminal carboxylate groups on ZW800-EPL were activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and NHS coupling reagents, through which DFO was covalently attached via an amide bond. The final product was purified using gel filtration chromatography and lyophilized to yield the final powder form.

The DFO₅-NPs used in this experiment were characterized according to the previously reported methods [12]. Hydrodynamic radius was determined using gel filtration chromatography calibrated with a series of protein standards. The DFO stoichiometry was confirmed using ¹H-NMR. The dissociation constant (K_D) of DFO₅-NP was assessed using a ferrozine competition assay and calculated using a multiparametric logistic regression [15].

Animal studies

The animal study was performed in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Massachusetts Lowell Animal Care and Use Committee. Animals were housed under standard conditions of 12-h/12-h light cycle with facility chow and water ad libitum.

Dose administration & sample collection

The pharmacokinetics of DFO-NPs were characterized in male Sprague–Dawley rats (300–350 g; 7–9 weeks; Charles River Labs, MA, USA). For iv. studies, DFO-NPs diluted in normal saline were injected at different doses (3.3, 10 or 30 μmol/kg) into the left tail vein (1 ml/kg) using a syringe equipped with a 25-gauge needle (Becton Dickinson, NJ, USA). Blood was collected from the right tail vein at the following time points: 0.017 (1 min), 0.25, 0.5, 1, 2, 4, 8 and 12 h postadministration. For sc. studies, DFO-NPs (3.3, 10 or 30 μmol/kg) were injected at 1 ml/kg in the dorsal flank. Blood was sampled from the tail vein at the following time points: 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h postadministration. For the high-dose group (30 μmol/kg), blood was sampled at the following time points: 0.25, 0.5, 1, 2, 3, 4, 8, 16 and 24 h postadministration. For all groups, blood was collected using a 23-gauge needle with nonheparinized syringe (Becton Dickinson). During the study, urine was collected using metabolic cages (Lab Products LLC, DE, USA) in 4-h intervals for a total of 12 h of urine collection with the collection tubes protected from light. At the end of each interval, the cage floor was rinsed with distilled water to collect residue and the collected rinse volume was recorded. After the last blood sampling, rats were euthanized using isoflurane overdose, followed by exsanguination and thoracotomy to collect tissues of interest. Tissues were frozen at -80°C until analysis.

Sample processing

Collected blood samples were processed into serum by centrifuging at 1500×g for 15 min. Serum and urine were immediately frozen at -20°C and stored until quantified upon completion of the in-life portion of the study. Collected tissue samples were homogenized for the quantitation of DFO-NP concentration. Tissues were dissected and 100–200 mg was transferred to a clean 2-ml tube (VWR; PA, USA), followed by 4× dilution in phosphate-buffered saline. Tissues were homogenized for approximately 60–100 s until visually homogeneous using a handheld homogenizer (FisherBrand Model 150 with 10 mm-diameter flat probe; Thermo Fisher Scientific, MA, USA) set to 25,000–30,000 r.p.m. Solids were separated from the supernatant by centrifuging at 10,000×g for 10 min at 4°C. The supernatant was subsequently analyzed for DFO-NP concentration.

Measurement of DFO-NP concentration

The concentration of DFO-NPs in biological samples was determined using near-infrared (NIR) fluorescence measurements of the ZW800 moiety of the DFO-NPs, as performed previously [12]. To improve measurement throughput, a plate-based method was developed to measure NIR fluorescence (EX/EM 760/788) using a Synergy H1 plate reader (BioTek, VT, USA) with extended range NIR capability. The read height of the laser optics was optimized to 9.0 mm, and the gain settings were optimized for each sample matrix (e.g., serum, urine and tissue homogenates) and concentration range. Standards were prepared in the relevant sample matrix using blank material collected from untreated animals. Standards and samples were analyzed in black 384-well plates (Thermo Fisher Scientific) and the concentration of samples was determined. For all matrices, samples outside the range of standards were diluted into range and the concentration was corrected for dilution.

Microscopic imaging of kidney sections

Resected kidneys were observed under the NIR imaging system (K-FLARE) at 800 nm and embedded in the optimal cutting temperature (OCT) compound and stored at -80°C. The frozen blocks were sectioned into slices (20 μm). Those sections were then observed using the fluorescence microscopic system (Cytation 5, BioTek Instruments).

Noncompartmental PK analysis

Noncompartmental analysis (NCA) of serum concentration-time data [16] was performed using the SimBiology application within the MATLAB software (Version#2021a; MathWorks, MA, USA). The maximum concentration (C_max) and time of maximum concentration (T_max) were determined using SimBiology and verified graphically. The area under the concentration time curve (AUC) was determined using the trapezoidal method from time zero to the last measured time (AUC_0-last) and extended to time infinity based on the terminal slope. The area under the first moment curve (AUMC) was calculated using the trapezoidal method from time zero to the last measured time (AUMC_0-last) and extended to time infinity based on the terminal slope. The mean residence time (MRT) was calculated as the ratio of AUMC and AUC (i.e., MRT = AUMC/AUC), and mean absorption time (MAT) after sc. administration was calculated as the difference between MRT after sc. administration (MRT_sc.) and MRT after iv. administration (MRT_iv.). The volume of distribution at steady state (V_ss) was calculated by the product of MRT_iv. and CL. For all data sets, PK parameters were determined for individual animals, and the mean and standard deviation (SD) of parameter estimates subsequently reported. The tissue-to-plasma concentration ratios or partition coefficients (Kp) were calculated for the kidney and liver at terminal timepoints for each individual animal (n = 4). Results were reported as mean ± SD.

Statistical analysis

All experiments were performed with n = 4 replicates and the experimental data are reported as mean ± SD. Statistical testing was performed using GraphPad software version 9.3.1 (CA, USA). Data were first tested for normality before evaluating differences. Significant differences among three doses for a given dose route were assessed using one-way analysis of variance with post hoc analysis using the Tukey's multiple comparison test. Significant differences between two administration routes were assessed for a given dose level using an unpaired, two-tailed t-test with equal variance. Differences were considered significant at p < 0.05.

Results

Preparation & characterization of DFO-NPs

DFO₅-NPs (Figure 1A) were successfully prepared using the optimized synthetic chemistry. Quantitation of the DFO conjugation ratio by ¹H-NMR indicated an average of approximately 5 DFO per NP (Figure 1B), which was an increase from the original formulation [12]. Notably, the hydrodynamic diameter (5.6 nm) and charge were essentially unchanged from the increased DFO load, meaning the optimal size and charge suitable for renal clearance was maintained [17]. The dissociation constant (K_D) of DFO₅-NP to iron was 14-fold lower than that of native DFO, suggesting that the DFO₅-NP has an approximately threefold greater binding affinity than native DFO when normalized to the equimolar concentration of DFO.

Figure 1. . Synthesis and characterization of deferoxamine-conjugated nanoparticles (nanochelator).

(A) Schematic representation of the DFO-NP including chemical structure and expected iron chelation behavior. (Inset) Exact chemical structures of the NIR dye ZW800 and DFO. (B) Physicochemical properties of DFO-NPs including MW, number of DFO/NP as measured by ¹H-NMR, expected charge in physiological fluids, HD as measured by gel filtration chromatography and K_D for iron binding affinity as measured by ferrozine competition assay.

DFO: Deferoxamine; DFO-NP: Deferoxamine-conjugated nanoparticle; HD: Hydrodynamic diameter; K_D: Equilibrium dissociation constant; MW: Molecular weight; NIR: Near-infrared.

Intravenous pharmacokinetics

The pharmacokinetics of DFO-NPs in serum were evaluated at 3.3, 10 and 30 μmol/kg doses. Following iv. injection (Figure 2A), DFO-NPs showed a biphasic concentration-time profile with rapid initial disappearance from serum within 0.5 h (Figure 2B), followed by steady disappearance up to 12 h. NCA indicated that DFO-NPs administered via iv. bolus injection follow nonlinear PK with increasing dose (Table 1 & Supplemental Table 2). Specifically, the elimination half-life (t_½), as determined by the terminal slope (i.e., K_el), ranged from 2.0 to 3.2 h, with a trend of t_½ increasing as dose increased. Statistically significant differences were found when comparing the half-lives of 3.3 versus 30 μmol/kg doses (p = 0.0053) and 10 versus 30 μmol/kg doses (p = 0.0294). The AUC_0-∞ following iv. administration ranged from 30.1 to 168.0 μM·h, with exposure increasing along with dose. A more detailed evaluation of AUC_0-∞ indicates that exposure does not increase proportionally to dose, with a decrease in dose-normalized AUC (i.e., AUC/Dose) observed with increasing dose. Indeed, the differences among three dose groups were statistically significant for all comparisons (p < 0.014). The NCA results show that terminal slope-based extrapolation was a minor contributor to AUC (1–2%; Supplementary Table 2), which indicates the dose-dependency of AUC is not substantially impacted by terminal sampling limitations.

Figure 2. . Dose-dependent pharmacokinetics of deferoxamine-conjugated nanoparticles after intravenous administration in Sprague–Dawley rats.

(A) Full serum concentration-time course showing biphasic profile (semi-log). (B) Early (<2 h) time-course showing rapid initial distribution phase. Results are representative of n = 4 per group and data were expressed as mean ± standard deviation.

DFO-NP: Deferoxamine-conjugated nanoparticle.

Table 1. . Select noncompartmental pharmacokinetic parameters of deferoxamine-conjugated nanoparticles following intravenous administration in Sprague–Dawley rats.

Parameter	Units	Mean ± SD	Mean ± SD	Mean ± SD	ANOVA p-value
Nominal Dose	μmol/kg	3.3	10	30
Kel	1/h	0.343 ± 0.027	0.313 ± 0.089	0.221 ± 0.022†	0.0301
t½	h	2.03 ± 0.16	2.33 ± 0.54	3.17 ± 0.33†,‡	0.0056
Cmax	μM	74.0 ± 4.5	201.3 ± 16.9	447.4 ± 31.3†,‡	<0.0001
AUC0-∞	μM·h	30.1 ± 2.9	72.3 ± 6.5†	168.0 ± 5.1†,‡	<0.0001
CL	L/h/kg	0.111 ± 0.010	0.139 ± 0.012†	0.179 ± 0.006†,‡	<0.0001
AUMC0-∞	μM·h2	43.9 ± 8.6	110.3 ± 33.5	259.5 ± 41.0	0.0864
MRT	h	1.45 ± 0.15	1.50 ± 0.34	1.54 ± 0.22	0.8707
Vss	L/kg	0.159 ± 0.006	0.206 ± 0.030†	0.275 ± 0.035†,‡	0.0006

Results are representative of n = 4 per group and data were expressed as mean ± standard deviation.

^†Comparison to 3.3 μmol/kg dose: p < 0.05.

^‡Comparison to 10 μmol/kg dose: p < 0.05.

ANOVA: Analysis of variance; AUC_0-∞: Area under the concentration-time curve from time zero to time infinity; AUMC_0-∞: Area under the first moment curve from time zero to time infinity; CL: Clearance; C_max: Maximum serum concentration; K_el: Elimination rate constant; MRT: Mean residence time; SD: Standard deviation; t_½: Half-life; V_ss: Volume of distribution at steady state.

After iv. administration, clearance (0.111–0.179 L/h/kg) increased with increasing dose. Statistically significant differences in clearance were observed for all comparisons (p < 0.007). In addition, the AUMC_0-∞ ranged from 43.9 to 259.5 μM·h², with almost linear increases in dose-normalized AUMC with increasing dose. The MRT ranged from 1.45 to 1.54 h, with no significant differences between groups. The volume of distribution at steady state (V_ss) ranged from 0.159 to 0.275 L/kg. V_ss increased with increasing DFO-NP dose, with significant differences between 3.3 and 30 μmol/kg (p = 0.0005) and between 10 and 30 μmol/kg (p = 0.0137), but not between 3.3 and 10 μmol/kg doses (p = 0.0827).

Subcutaneous pharmacokinetics

After sc. administration (Figure 3), DFO-NPs showed distinct rising and declining phases as expected for extravascular administration. The NCA results indicate that DFO-NPs follow nonlinear PK after sc. administration (Table 2 & Supplementary Table 3). DFO-NP concentrations rose quickly (Figure 3B) and peaked between 2.0 and 3.8 h with no clear trends for different doses. Statistical analysis does indicate a significant difference between 10 and 30 μmol/kg T_max values (p = 0.0101); however, sparse sampling in the relevant 2- to 4-h interval limits further analysis. C_max ranged from 1.2 to 8.7 μM, with a linear increase in C_max with increasing dose as supported by no significant differences in dose-normalized C_max values. The terminal t_½ ranged from 5.7 to 10.1 h, with a trend of increasing t_½ with increasing dose. The increase in t_½ was statistically significant only when comparing 3.3 and 30 μmol/kg doses (p = 0.0306), which reflects on the variability of the terminal slope analysis for the sc. groups (Table 2 & Supplementary Table 3), likely due to prolonged absorption at high dose.

Figure 3. . Dose-dependent pharmacokinetics of deferoxamine-conjugated nanoparticles after subcutaneous administration in Sprague–Dawley rats.

(A) Full pharmacokinetic time course showing sustained exposure of DFO-NPs over sampling interval. High dose sampling interval was modified to capture absorption and elimination phases adequately over 24 h. (B) Early (<2 h) time-course showing rapid absorption of DFO-NPs. Results are representative of n = 4 per group and data were expressed as mean ± standard deviation.

DFO-NP: Deferoxamine-conjugated nanoparticle.

Table 2. . Select noncompartmental pharmacokinetic parameters of deferoxamine-conjugated nanoparticles following subcutaneous administration in Sprague–Dawley rats.

Parameter	Units	Mean ± SD	Mean ± SD	Mean ± SD	ANOVA p-value
Nominal dose	μmol/kg	3.3	10	30
Tmax	h	2.5 ± 1.0	2.0 ± 0.0	3.75 ± 0.50‡	0.0108
Cmax	μM	1.18 ± 0.17	3.59 ± 0.42	8.69 ± 0.80‡	0.0731
Kel	1/h	0.125 ± 0.026	0.103 ± 0.035	0.070 ± 0.012†	0.0461
t½	h	5.71 ± 0.98	7.47 ± 2.83	10.06 ± 1.66†	0.0366
AUC0-∞	μM·h	14.2 ± 1.4	45.2 ± 14.0	179.3 ± 7.4	0.0447
F		0.47	0.63	1.07
AUMC0-∞	μM·h2	135 ± 30	547 ± 351	2811 ± 322†	0.0193
MRT	h	9.4 ± 1.2	11.2 ± 4.1	15.7 ± 1.6†	0.0230
MAT	h	7.9	9.7	14.1

Results are representative of n = 4 per group and data were expressed as mean ± SD.

^†Comparison to 3.3 μmol/kg dose: p < 0.05.

^‡Comparison to 10 μmol/kg dose: p < 0.05.

ANOVA: Analysis of variance; AUC_0-∞: Area under the concentration-time curve from time zero to time infinity; AUMC_0-∞: Area under the first moment curve from time zero to time infinity; C_max: Maximum serum concentration; F: Bioavailability; K_el: Elimination rate constant; MAT: Mean absorption time; MRT: Mean residence time; SD: Standard deviation; t_½: Half-life; T_max: Time of maximum serum concentration.

The AUC_0-∞ values following sc. administration ranged from 14.2 to 179.3 μM·h, with increases in exposure with increased dose. Further analysis showed a trend of increasing dose-normalized AUC with increasing dose; however, a statistically significant difference was not identified. A comparison of the AUC_0-∞ values between equivalent doses suggests sc. administration gives an overall favorable bioavailability (F) of 47–107%, with a clear trend of increasing bioavailability with increasing dose (Table 2). The AUMC_0-∞ ranged from 135 to 2811 μM·h² and showed a clear trend of increasing dose-normalized AUMC with increasing dose, including statistically significant differences between 3.3 and 30 μmol/kg (p = 0.0186). The MRT after sc. administration ranged from 9.4–15.7 h, with a trend of increasing MRT with increasing dose. A statistically significant difference was observed when comparing the 3.3 and 30 μmol/kg doses (p = 0.0212), but not for other comparisons. It should be noted that for the sc. data, terminal slope-based extrapolation (Supplementary Table 3) contributed moderately to AUC_0-∞ (19.5–32.7%), and substantially to AUMC_0-∞ (47.8–65.7%), which indicates that variability in the terminal slope analysis had an impact on these calculated parameters and MRT by extension. The MAT (7.9–14.1 h) also increased with increasing dose, suggesting that DFO-NPs are slowly absorbed from the sc. site.

Comparison of iv. & sc. PK parameters

Evaluation of the NCA parameters for iv. and sc. administration of DFO-NPs indicates that the change in administration route significantly alters the pharmacokinetics of the DFO-NPs. As expected, C_max demonstrated a strong dependence on administration route, with an approximately 50–60× decrease comparing iv. and sc. (p < 0.0001 for all comparisons). Interestingly, t_½ showed a significant dependence on administration route, with a statistically significant 2.5 to 3× increase comparing equivalent doses of iv. to sc. (p < 0.005 for all comparisons). This is especially apparent when comparing terminal slopes of equivalent doses between iv. and sc. (Supplementary Figure 1). The MRT also showed a strong dependence on administration route, with a statistically significant 6.5–10× increase in MRT when comparing iv. and sc. (p < 0.004 for all comparisons).

Urinary excretion

Urinary drug excretion (Figure 4) was analyzed for the first 12 h after dose administration. Following iv. administration, 38–63% injected dose (ID) was excreted, with statistically significant increases in urinary drug excretion with increasing dose (p < 0.035 for all comparisons). Following sc. administration, 10–40% ID was excreted in urine with a trend of increasing urinary excretion as dose increased. Statistically significant differences were found when comparing 3.3 to 10 μmol/kg doses (p < 0.0001) and 3.3 to 30 μmol/kg doses (p < 0.0001), but not when comparing 10 to 30 μmol/kg doses (p = 0.0849). This is likely due to a substantial fraction of absorption occurring after the 0- to 12-h window for the 30 μmol/kg dose, as evidenced by substantial serum concentrations measured after 12 h (Figure 3). Total urine excretion was lower for the sc. group compared with equivalent doses in the iv. group, which is likely due to absorption continuing past 12 h for all sc. doses.

Figure 4. . Dose and administration route-dependent urinary excretion of deferoxamine-conjugated nanoparticles.

Urine was collected over 4-h intervals for 12 consecutive h using metabolic cages, and cumulative amount excreted was determined. Results are representative of n = 4 per group, and data were expressed as mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance and Tukey's post hoc test.

*p < 0.05; **p < 0.01; ***p < 0.001.

DFO-NP: Deferoxamine-conjugated nanoparticle; % ID: % injected dose; iv.: Intravenous; ns: Not significant; sc.: Subcutaneous.

Tissue distribution

To determine the impact of dose and administration route on tissue distribution of DFO-NPs, the concentrations of DFO-NPs were determined in homogenates from tissues of interest (Figure 5), especially highly perfused tissues, including the kidney, liver, spleen, lungs and heart. When evaluated 12 h after iv. administration (Figure 5A), the kidney was the primary site of organ distribution, as expected due to selective renal clearance of DFO-NPs. DFO-NP content in the kidney ranged from 10.2 to 31.7% ID, with a statistically significant decrease in kidney distribution as a function of dose (p < 0.0005 for all comparisons). DFO-NP content in the liver ranged from 3.7 to 6.1% ID, with a statistically significant decrease in liver content as dose increased (p < 0.02 for all comparisons). Negligible amounts of DFO-NPs were found in the spleen (<0.1% ID), lung (<0.02% ID) and heart (<0.02% ID).

Figure 5. . Dose and administration route-dependent tissue distribution of deferoxamine-conjugated nanoparticles.

(A) Tissue distribution evaluated 12 h after intravenous administration. (Inset) Negligible quantities of DFO-NPs accumulate in the spleen, lung and heart after intravenous administration. (B) Tissue distribution evaluated 12 h (3.3, 10 μmol/kg) or 24 h (30 μmol/kg) following subcutaneous administration.

(Inset) Negligible quantities of DFO-NPs accumulate in the spleen, lung and heart after subcutaneous administration. Results are representative of n = 4 per group, and data were expressed as mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance and Tukey's post hoc test.

*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

DFO-NP: Deferoxamine-conjugated nanoparticle; % ID: % injected dose; NQ: Not quantifiable; ns: Not significant.

When evaluated 12 h (3.3, 10 μmol/kg) or 24 h (30 μmol/kg) after sc. administration (Figure 5B), the kidney was the primary site of distribution. DFO-NP content in the kidney ranged from 27.3 to 48.7% ID, with a trend of decreasing DFO-NP content with increasing dose. Statistically significant differences were observed when comparing 3.3 to 30 μmol/kg doses (p = 0.0002) and 10 to 30 μmol/kg doses (p = 0.0067). When compared to the corresponding iv. doses (Supplementary Figure 2A), sc. administration showed a statistically significant increase in DFO-NP kidney content for all doses (p < 0.003 for all comparisons). NIR fluorescence microscopy of sections from sample kidneys demonstrated renal cortex-specific distribution with a clear gradient in signal intensity away from the cortex (Supplementary Figure 4). Minor quantities of DFO-NPs were found in the liver, with no quantifiable concentrations at the 3.3 μmol/kg dose and only 1.3% ID at the 30 μmol/kg dose. Liver drug content was significantly decreased (p < 0.0001 for all comparisons) following sc. administration when compared to corresponding iv. doses (Supplementary Figure 2B). Negligible quantities were found in the spleen (<0.04% ID), lung (0.04%), and heart (<0.01%), with no quantifiable drug in these tissues for the 3.3 μmol/kg dose.

Tissue-to-plasma partition coefficient

The tissue-to-plasma ratio or partition coefficient (Kp) was determined for the final time point in each group (Supplementary Figure 3). Following iv. administration, kidney Kp ranged from 592 to 1797, with a trend of decreasing Kp with increasing dose. However, there were no significant differences among three doses, most likely due to the very high variability in Kp caused by the increased variability in measured terminal serum concentrations. Liver Kp stayed relatively constant in the range of 10.2 to 19.5 with no clear trends and no significant differences (p = 0.5062). Following sc. administration, kidney Kp ranged from 472 to 519, with no trend associated with increasing dose. Liver Kp could not be calculated for the 3.3 μmol/kg dose and increased from 0.45 to 1.00 for the 10 and 30 μmol/kg doses.

Discussion

The dose- and administration-route-dependent pharmacokinetics of the nanochelator DFO-NPs were adequately characterized in Sprague–Dawley rats in the present study. In this study, doses of 3.3, 10 and 30 μmol/kg were selected to ensure that serum concentrations could be adequately detected and would be adequately differentiated between doses and to ensure that animals would be exposed to serum concentrations expected to give therapeutic efficacy based on published efficacy data on DFO-NPs [12,18]. As expected, based on published mouse PK data [12,18], DFO-NPs following iv. administration showed a biphasic PK profile. Importantly, we discovered that the DFO-NPs exhibit nonlinear PK, emphasizing the importance of evaluating dose-dependency of PK when developing novel therapeutic modalities. Indeed, as DFO-NP dose increased, there were statistically significant increases in t_½, CL and V_ss, as well as a significant decrease in dose-normalized AUC. Furthermore, there was a trend of nonlinear urinary excretion of the drug, with a significant increase in the fraction of dose excreted in urine found with increasing dose. Similar dose-dependencies were observed for the tissue distribution data, with a decreasing fraction of dose found in the kidney and liver as dose increased. Additionally, the tissue-to-plasma partition coefficient showed a trend of dose-dependent distribution of DFO-NPs only in the kidney, the major site of both DFO-NP distribution and excretion, with decreasing Kp observed as dose increased.

Collectively, the dose-dependent increase in clearance, increase in urinary excretion and decrease in kidney content suggests a potentially capacity-limited reabsorption process occurs during renal elimination. It was previously established [12] that DFO-NPs are cleared selectively through renal filtration – a finding supported by the cumulative DFO-NP recovery of approximately 70% ID in urine and kidney in this study – and thus, the proximal tubule of the kidney is the most likely site of the proposed capacity-limited process. This is feasible given that one of the primary functions of the proximal tubule is the reabsorption of filtered solutes [19,20]. Moreover, the clearance of DFO-NPs determined in this study (0.11–0.18 L/h/kg) is less than the expected glomerular filtration rate of 0.31 L/h/kg in rats [21]. This cannot be accounted for by serum protein binding because it was previously demonstrated that DFO-NPs do not extensively bind to serum proteins (<1%; data not shown). Thus, our findings support the idea that the DFO-NPs are actively reabsorbed in the kidney and that a high dose of DFO-NPs decreases the efficiency of reabsorption due to saturation of this process. Although it is unclear how proximal tubule cells (PTCs) process a biopolymer-based nanochelator, recent studies have shown that PTCs utilize a nonselective fluid-phase endocytosis mechanism in addition to the canonical receptor-mediated uptake pathways [19]. Indeed, PTC-based reuptake of exogenous molecules has been shown for organic molecules including renally clearable dextran [22] and inorganic sodium-borohydride gold nanoclusters [20]. Of particular interest to this work were the findings of Lawrence et al. [20], which showed that PTC-based reuptake could be saturated for renally cleared molecules, including gold nanoclusters that are not expected to interact with receptor specific systems such as FcRn or the megalin–cubulin complex. This proposed mechanism also explains the finding of decreased kidney content with increasing dose and suggests that the DFO-NPs found in the kidney at study termination are in the process of renal recycling. This is supported by the clear localization of DFO-NPs in the renal cortex when viewed using NIR fluorescence microscopy.

The pharmacokinetics of DFO-NPs were also characterized after sc. administration because it is the preferred route of administration for effective iron chelation [12,18] and the proposed route of administration for clinical development. As seen with the iv. group, sc. administration of DFO-NPs resulted in nonlinear PK with increasing dose, as evidenced by increases in apparent t_½, and decreases in dose-normalized AUC and dose-normalized C_max. Importantly for the preclinical evaluation and development of DFO-NPs, NCA quantitatively demonstrated the benefits of sc. administration as evidenced by statistically significant increases in t_½ and MRT when comparing to iv. administration. This outcome suggests that DFO-NPs are subject to absorption rate-limited kinetics with an apparent elimination rate constant during the terminal phase being dictated by a much smaller absorption rate constant [23] (a.k.a., the flip–flop phenomenon). While this is a commonly reported occurrence for biotherapeutics administered via the sc. route [24,25], to the best of our knowledge this phenomenon has not been described for small organic nanoparticles administered sc. This finding suggests that some of the common toxicological concerns raised by nanomaterial accumulation in organs [26,27] could be resolved by engineering nanomaterials that meet the Choi criteria for favorable renal clearance [17] and leveraging the sc. administration route to improve residence time and therapeutic efficacy. This finding is especially important in the context of nanomaterial-based chelation therapies, which need to adequately balance extended circulation time with complete excretion, a challenge that has limited therapeutic efficacy in this field [11].

In addition to showing increased half-life, sc. administration gave favorable bioavailability (approximately 50% or higher), with dose-dependent increases in bioavailability. This could be due to a saturable process acting on unabsorbed drug in the sc. space (e.g., nonspecific interactions with matrix proteins or macrophage sequestration), which removes a smaller fraction of the administered dose as the dose amount increases. Processes governing bioavailability of sc. administered therapeutics are poorly understood [24,28] and require more studies in the future. It should also be noted that the calculated bioavailability is strongly influenced by the terminal slope-based extrapolation of AUC, which is impacted by the apparent absorption rate-limited kinetics.

sc. administration also led to similar dose-dependent increase in urinary excretion and decrease in terminal kidney content. Interestingly, overall kidney content was higher for sc. administration compared with iv. administration, which is likely due to a decrease in time spent under saturating conditions brought on by the substantially decreased C_max. Alternatively, relatively lower serum concentrations of DFO-NPs after sc. administration (compared to iv. administration) could promote more efficient tubular reabsorption of DFO-NPs, resulting in increased dose fraction (% ID) of the DFO-NPs remaining in the kidney. Notably, sc. administration substantially decreased liver content of DFO-NPs, which is a promising outcome for translation. Interestingly, opposing trends in dose-dependent liver accumulation were observed for iv. and sc. administration. These results suggest that DFO-NPs found in the liver after high dose iv. administration accumulated due to nonspecific concentration-dependent phenomenon as opposed to total exposure (i.e., related to C_max and not AUC). This finding could be due to residence in the liver sinusoid or nonspecific uptake by liver macrophages that have been shown to take up nonendogenous molecules, especially nanomaterials [29,30], and likely occurring when renal filtration is transiently limited by high concentrations of solutes. Thus, in the case of iv. administration, liver uptake was driven by the high early concentrations, which increased nonlinearly with increasing dose (i.e., C₀ was less than dose-proportional). When presented as dose normalized uptake, there was a trend of decreasing liver uptake with increasing dose. In the case of sc. administration, the maximum concentration in serum was significantly lower than in the iv. group, which accounts for the lower overall liver uptake and more effective renal reabsorption.

Some limitations are noted, given that PK analysis is highly dependent on data quality and assumptions about the behavior of systems. A relatively small sample size (n = 4) was used in this study, which limited the effect size that could be statistically evaluated and contributed to the large error observed for some analyses, especially ratio-based analyses (e.g., determination of Kp). There were also limitations to sample collection for both serum and urine analysis that had outsized impacts on data from the sc. administration groups. In the case of serum analysis, there were practical and ethical limitations on the number and volume of blood samples that could be collected from Sprague–Dawley rats. Since at least 4 samples during the early rising phase were required for adequate characterization of the absorption phase, sampling was sparse during the terminal phase and sampling intervals had to be altered for the 30 μmol/kg dose to capture the longer elimination phase. In the case of urine analysis, animals could not be housed in the metabolic cages for more than 12 h, which limited the ability to interpret urine data for the sc. dose groups given that serum data showed absorption continuing past 12 h. Another limitation related to the serum sampling constraints was the strong influence of sc. absorption on the apparent elimination kinetics in the sc. group, which could not be sampled long enough to find the phase of true steady state. Although the absorption-rate limited kinetics have desirable therapeutic implications, they limit the accuracy of noncompartmental PK parameters that are highly dependent on slopes, potentially causing overestimation when conducting terminal slope-based extrapolation of kinetic parameters including AUC and AUMC. This impacts parameters that rely on AUC or AUMC, such as bioavailability and MRT_sc., and may contribute to the nonlinearity observed for these parameters. Typically, this limitation could be overcome by calculating the elimination rate constant for sc. administration by including the absorption kinetics in the model-independent analysis. However, this approach is limited by the nonlinearity in the PK parameters and the inability to discern whether absorption is first-order or zero-order process from model-independent analysis alone. Model-based analysis, with a focus on PK analysis of sc. absorption using compartment models including potential effect of lymphatic transport, will address this question. An additional limitation is the accuracy of MRT, which assumes that elimination occurs from the central compartment and, by association, influences the accuracy of V_ss (= CL × MRT). Specifically, we note that there were opposing trends of decreasing Kp and increasing V_ss as dose increased. When central elimination occurs, Kp and V_ss should show similar trends. This suggests that elimination of DFO-NPs could take place in the peripheral compartment, which includes the kidney in this scenario, and not in the central compartment [31]. Further comprehensive compartmental modeling in the presence of central or peripheral elimination will help to identify true MRT and V_ss at different doses. Lastly, the analysis of Kp values for the iv. groups was limited by the large variability brought on by an apparent outlier in the 3.3 and 10 μmol/kg dose groups. In both instances, an animal with low plasma concentration and slightly higher tissue concentration substantially increased the Kp values. Additional testing for outliers and normality did not return meaningful results, in large part due to the small sample size.

Collectively, the PK characterization of DFO-NPs shows this novel nanochelator holds promise for further preclinical development. Consistent with prior results, DFO-NPs are rapidly eliminated from the body, which suggests they can quickly remove chelated iron and therefore generate sink conditions for iron mobilization from tissue stores into systemic circulation. Moreover, sc. administration substantially alters the PK of DFO-NPs, sustaining the serum concentration within a much narrower range of concentrations than iv. administration, which reduces off-target tissue interactions and C_max-driven toxicity. Importantly, sc. administration increases the extent of exposure in concentration ranges expected to give therapeutic efficacy (e.g., 5–40 μM DFO equivalents) based on published steady state plasma concentrations of DFO obtained using typical therapeutic regimens (i.e., 40 mg/kg infused over 8 h) in clinical settings [32,33]. Ultimately, additional PK analysis is required to better understand the absorption kinetics and therapeutic implications of DFO-NPs administered subcutaneously. It will also be necessary to conduct additional PK analyses in iron overload animals to characterize the impact of any hemodynamic or other physiological changes in severe iron overload conditions on PK parameters and to enable PK/PD modeling of DFO-NPs.

Conclusion

The present study evaluated the absorption, distribution and elimination of DFO-NPs after iv. and sc. injection at therapeutically relevant doses in Sprague–Dawley rats. DFO-NPs exhibited a biphasic concentration-time profile after iv. administration, with a terminal half-life of 2–3 h, dose-dependent clearance, minimal tissue distribution other than the kidney and exclusive renal clearance subject to a possible capacity limited reabsorption mechanism. DFO-NPs administered sc. exhibited absorption-rate-limited kinetics, with a prolonged apparent half-life of 5–10 h, and dose-dependent bioavailability. These results demonstrate that DFO-NPs exhibit nonlinear pharmacokinetics in both sc. absorption and renal elimination and sc. administration substantially improves the duration of exposure without changing the selective renal excretion, thereby making it a clinically viable administration route.

Summary points.

• The dose- and administration route-dependent pharmacokinetics of a renally selective nanochelator (DFO-NP) were characterized in this study.

• Noncompartmental pharmacokinetic (PK) analysis indicates that DFO-NPs follow nonlinear pharmacokinetics after intravenous administration, with increasing t_½, increasing CL, increasing V_ss and decreasing dose-normalized AUC_0-∞ with increasing dose.

• Collectively, the nonlinear intravenous PK and dose-dependent urine excretion and kidney distribution suggest that DFO-NPs undergo a capacity-limited reabsorption process during renal elimination, most likely involving the proximal tubule.

• Noncompartmental PK analysis quantitatively demonstrates the benefits of the subcutaneous administration route, as highlighted by the favorable bioavailability (>47%) and increased MRT (9.4–15.7 h) relative to intravenous administration.

• When administered via subcutaneous injection, DFO-NPs showed absorption rate-limited kinetics, which improved the duration of exposure at therapeutically relevant concentrations while maintaining selective renal excretion without the need for reformulation.