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Abstract

Aliskiren (ALS) is a direct renin inhibitor with low bioavailability and high drug cost. The goal of this study was to increase the bioavailability of ALS through nanoformulation. The optimized formulation was then evaluated in spontaneously hypertensive rats (SHRs). We developed an ALS poly(lactic-co-glycolic) acid nanoparticle (ALS-NP) through the emulsion–diffusion–evaporation method with various solvents, stabilizer concentrations, and centrifugation speeds. SHRs were orally dosed with 30 mg/kg ALS or dose equivalent ALS-NP. Several parameters were assayed in plasma and/or urine at baseline and 24 h post-dose, while pharmacokinetic analysis included serial sampling. The optimum formulation was found with ethyl acetate, a 1.00% w/v didodecyldimethylammonium bromide concentration, and a 10,000 r/min (15,554 g) centrifugation speed. A 168% relative bioavailability was observed as a result of ALS-NP administration along with significant, as determined by Student’s t-test, increases in the maximum ALS plasma concentration (p = 0.0189) and the area under the plasma concentration–time curve from 0 to infinity (p = 0.0095). Conversely, a reduction was found in oral volume of distribution (p = 0.0009) and oral clearance (p = 0.0298). Blood urea nitrogen increased significantly after dosing in both groups (p < 0.0001 and p < 0.0001); however, no statistical difference was found between endpoint levels (p > 0.05) following one-way analysis of variance (ANOVA). Kidney injury molecule-1 increased following ALS dosing (p = 0.0486), while ALS-NP showed a decrease (p = 0.027) which was also significantly decreased compared to ALS-Final (p = 0.0008) when examined using two-way ANOVA. Urinary potassium excretion decreased significantly, as shown by two-way ANOVA, only in the ALS group (p = 0.0274) which was also significantly reduced compared to ALS-NP-Final (p = 0.016). Using the current formulation and at the dosage tested, ALS-NP showed a more favorable pharmacokinetic profile and positive kidney changes compared to ALS in regard to select outcomes. Thus, NP formulation may further improve ALS renoprotection in addition to increasing bioavailabilty.

Keywords

Aliskiren nanoparticles formulation bioavailability kidney

Introduction

One quarter of the world population lives with hypertension, a precursor of more severe cardiovascular disease; therefore, effective regulatory medications have risen in importance.¹ Blood pressure maintenance is typically controlled through the renin–angiotensin–aldosterone system, which prompts renin secretion in response to low plasma volume, decreased renal blood flow, or increased sympathetic central nervous system activity.² In this system, renin enzymatically cleaves angiotensinogen into the inactive peptide angiotensin I. Angiotensin converting enzymes (ACEs) then process angiotensin I into angiotensin II, which interacts with angiotensin II type 1 receptors inducing vasoconstriction and the release of catecholamines. Although ACE inhibitors block the formation of angiotensin II, angiotensin I production continues and may be converted to angiotensin II via other metabolic pathways.³ While multiple inhibition checkpoints are available to prevent angiotensin II formation, inhibition of renin, as the first enzyme in the pathway, is a logical option.⁴ Inhibition at this step may increase circulating renin; however, renin inhibitors block enzyme activity.²

Aliskiren (ALS) is a non-peptide, orally active renin inhibitor discovered using X-ray crystallographic structure analysis and molecular modeling.⁴ In March 2007, ALS became the first in class renin inhibitor to receive approval from the United States Food and Drug Administration (FDA)^5,6 and is effective as a monotherapy or in combination with other antihypertensive drugs, such as hydrochlorothiazide.^3,6 A half-life of approximately 23–36 h provides suitability for once daily ALS dosing, typically 150 or 300 mg in humans. Side effects have included headache, diarrhea, and dizziness, but these were reported in less than 3% of patients.⁷

Although ALS has positive attributes in regard to blood pressure maintenance, such as rapid maximum plasma concentrations (C _max) within 2–4 h, drug bioavailability is notably low (approximately 2.6%) with nearly 91% of the drug excreted unmetabolized in feces.^2
–4,7 Synthetic polymer nanoparticle (NP) formulations are gaining prominence in nanomedicine for the improvement of bioavailability, systemic absorption, and/or minimizing effective dosage.^8,9 Defined by a size range from 10 nm to 1000 nm, NPs have the potential to modify particle size and surface characteristics and thereby alter drug pharmacokinetics and pharmacodynamics.^10
–12 In addition, nanoencapsulation can influence drug stability, specificity, efficacy, and/or tolerability.^13,14 Further advantages include improvement of intracellular penetration and oral bioavailability along with the option of targeting drug delivery.^15,16 With the high current average wholesale cost of ALS, NP formulation could reduce drug costs via increased bioavailability.¹⁷

Particle size plays a role in NP surface property modifiability which may affect degradation time, elimination processes, and intracellular uptake. When the diameter of NPs are reduced below 100 nm, degradation in the mononuclear phagocytic system can be avoided, enabling extended circulation in plasma and increasing the probability of delivery to the site of action.¹⁸ Particle charge also has a significant effect on NP distribution.^9,19 Adherence of cationic NPs to the negatively charged cell membrane essentially increases cellular uptake.

The FDA-approved, biodegradable, linear copolymer poly(lactic-co-glycolic) acid (PLGA) possesses several positive properties as NP components, including commercial availability, exceptional biocompatibility, and low toxicity.^9,20 When this polyester copolymer undergoes hydrolysis, the acidic end groups on each monomer autocatalyze and drug is released.^21,22 Water molecules occupy the newly accessible spaces between monomers and contribute to NP degradation, while slowly releasing the drug through the porous features. Eventually the acidic monomers are consumed via the Krebs cycle with marginal toxicity.^14,21,23 Degradation time is affected by the ratio of lactic acid and glycolic acid with the optimum rate being achieved at a 50:50 ratio as in PLGA.²² Typically, particle size ranges from 50 nm to 500 nm, which allows smaller size diameters to consistently be obtained.¹⁶ Upon successful formulation, pharmacokinetic and pharmacodynamic parameters of novel NPs are typically evaluated preclinically in rats.¹¹

In an effort to reduce the amount of ALS per dose and thereby reduce associated drug costs, our laboratory employed nanoformulation using PLGA. Various parameters, including organic solvent choice and stabilizer concentration, were examined to determine an optimized formula. Pharmacokinetic parameters were assessed in rats to determine relative bioavailability. As ALS has been shown to exhibit renoprotective properties,^24,25 renal parameters were examined for both formulations. This study sought to develop an ALS-NP formulation which would exhibit enhanced bioavailability and comparable renal parameters.

Materials and methods

Chemicals

ALS hemifumarate and verapamil (VER) were purchased from ChemScene, LLC (Monmouth Junction, New Jersey, USA) and Selleck Chemicals (Houston, Texas, USA), respectively. Dichloromethane, didodecyldimethylammonium bromide (DMAB), and PLGA (50:50 copolymer compositions; molecular weight 30,000–60,000 Da) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). O-phosphoric acid (85%), ammonium hydroxide, acetone, ethyl acetate, high-performance liquid chromatography (HPLC) grade water, and acetonitrile were acquired from Fisher Scientific (Fair Lawn, New Jersey, USA). All solvents used for LC-mass spectrometry (MS)/MS analysis were of LC-MS grade from Honeywell-Burdick and Jackson (Muskegon, Michigan, USA).

Formulation of NPs

NP formulations were prepared based on a previously described method²⁶ with some modifications such as using a variety of solvents, stabilizer concentrations, or centrifugation speeds.²⁷

Effect of varying organic solvents

The emulsion–diffusion–evaporation method was employed with slight alterations in this study. In brief, 50 mg of PLGA was completely dissolved in 3 mL of dichloromethane, ethyl acetate, or ethyl acetate/acetone. To the organic solvent, 10 mg of ALS was added under moderate stirring. Simultaneously, 0.25% w/v of DMAB was placed in water (6 mL) and allowed to fully dissolve under moderate stirring. While the aqueous phase was under moderate stirring, organic phase was added dropwise. The suspension was sonicated for 5 min at 20 kHz and then the contents were stirred (30–40 min) under a chemical hood to facilitate solvent evaporation. Upon completion, each solution was centrifuged at 10,000 r/min using a Sorvall Biofuge Stratos centrifuge equipped with a Heraeus fixed angle rotor (#3335) made by Thermo Fisher Scientific Inc. (Waltham, Massachusetts, USA) for 5 min. The supernatant was collected to measure entrapment efficiency.

Effect of varying stabilizer concentration and centrifugation speed

The stabilizer concentrations of DMAB included 0.10%, 0.25%, 0.50%, and 1.00% w/v, while the centrifugation speeds were 10,000 or 12,000 r/min (15,554 or 18,665 g). As each parameter has shown the ability to alter NP characteristics in a previous study,²⁸ these variations allowed for the detection of an optimal NP formulation.

NP characteristics

Formulations were evaluated using various NP criteria: entrapment efficiency, particle size, zeta potential, and polydispersity index (PDI).

Entrapment efficiency

For all measurements of entrapment efficiency, a calibration curve was constructed using solutions of known ALS concentrations (10–1000 µg/mL) and the corresponding absorbance values detected using ultraviolet–visible spectroscopy (Eppendorf Biophotometer, Hauppauge, New York, USA) with a wavelength set at 260 nm.

A 1:2 ratio dilution of collected supernatant to HPLC-grade acetonitrile was performed, and the corresponding absorbance values were measured. These values were employed for the calculation of entrapment efficiency as previously described.²⁸

Particle size, zeta potential, and PDI

A NICOMP Particle Sizer (Particle Sizing Systems, Port Richey, Florida, USA) was used to evaluate particle size via dynamic light scattering, while zeta potential was approximated under an electrical field with a foundation of electrophoretic mobility. The PDI of each formulation, as an indication of particle size distribution, was also measured using the NICOMP Particle Sizer.

Animals and drug administration

In order to administer the NP formulation to the animals, a fresh batch of ALS-NP (40 mL, final volume of 35 mL) was prepared by dissolving 350 mg of PLGA polymer and 70 mg of ALS (ALS hemifumarate salt) in organic phase consisting of 21 mL of ethyl acetate. For the aqueous phase, 1% w/v DMAB stabilizer was added to 42 mL of water. Organic phase was added to aqueous phase in a dropwise manner and sonicated for 5 min at 20 kHz. After sonication, solutions were constantly stirred at 750 r/min to facilitate organic phase evaporation. Following evaporation, NP solution was centrifuged at 10,000 r/min for 5 min and supernatant was collected. The parameters of the formulation dosed are presented in Table 1.

Table 1.

Nanoformulation parameters of dosed ALS NPs.

Parameter	Mean	SD
Particle size (nm)	66.73	0.21
Zeta potential (mV)	18.85	0.64
PDI	0.32	0.01
Drug entrapment (%)	81.8	0.40

ALS: aliskiren; NP: nanoparticle; SD: standard deviation; PDI: polydispersity index.

Fourteen male spontaneously hypertensive rats (SHRs; 265.36 ± 8.04 g), approximately 7 weeks old, were randomly assigned to receive either orally gavaged ALS (30 mg/kg) or ALS-NP (equivalent to ALS dose) in accordance with an animal protocol approved by the East Tennessee State University Committee on Animal Care. On day 0, one blood sample (0.5 mL) was taken, via cannulation, for baseline biomarker assay as well as a pre-dose ALS time point along with a 12-h urine collection (baseline). On day 1, rats were administered ALS (n = 7) or ALS-NP (n = 7), then serial blood samples (0.25 mL) were taken and urine (final) was collected (approximately 12 h). On day 2, a 24-h blood sample (0.5 mL) was collected followed by exsanguination under anesthesia.

Sample preparation and LC-MS/MS conditions

The detection method used in this study was based upon a previous method by Burckhardt et al.²⁹ Sample preparation was achieved using 1 mL Isolute supported liquid extraction (SLE+) cartridges from Biotage (Charlotte, North Carolina, USA). The sample analysis occurred on a Shimadzu LCMS-IT-TOF (ion trap-time of flight) system from Shimadzu Scientific (Columbia, Maryland, USA). This system consisted of two LC-AD20 pumps, a SIL-20ACHT autosampler, an in-line degasser (DGU-20A3), and a CTO-20A column oven. Chromatographic separations were carried out using a C18 XBridge column (150 × 4.6 mm, 3.5 µm) from Waters (Milford, Massachusetts, USA).

SLE+ of ALS and the internal standard, VER, utilized a 0.5 M ammonium hydroxide buffer pretreatment and elution with methyl-t-butyl ether. Rat plasma samples of 100 µL were spiked with VER to achieve a final internal standard concentration of 50 ng/mL. Calibration samples, prepared from drug-free rat plasma, were spiked with VER and appropriate amounts of ALS stock solutions to achieve specific concentrations (10, 50, 100, 250, and 500 ng/mL). Prior to LC-MS/MS analysis, all samples were filtered using 0.22 µm centrifuge filters from Sigma-Aldrich (St Louis, Missouri, USA). Chromatographic separation was isocratic, using a mobile phase of 80% acetonitrile/20% aqueous formic acid (0.1%), delivered at a flow rate of 0.400 mL/min. The mass spectrometer was set up in positive electrospray (+ESI) mode with nitrogen as the nebulizing gas (1.5 L/min) and argon as the collision gas. The +ESI source was heated to 200°C. Detection of ALS and VER was achieved using direct MS2 channels (fragment ions) of precursors m/z 552.3683 (ALS) and m/z 455.3041 (VER). Identity of analyte and internal standard was confirmed in each chromatogram via mass transitions of m/z 552 → m/z 534 and m/z 552 → m/z 436 for ALS and m/z 455 → m/z 303 and m/z 455 → m/z 165 for VER.

Real sample peak area ratios were compared against linear regression equations of daily calibration curves to calculate ALS concentrations in these samples (coefficient of variation of <5%).

Pharmacokinetic parameters

Pharmacokinetic sampling occurred at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-dose. Pharmacokinetic parameters, including half-life (t _1/2), C _max, time to reach C _max (t _max), the area under the plasma concentration–time curve from 0 to infinity (AUC_0-∞), apparent volume of distribution (V _d/F), and oral clearance (CL_oral), were calculated using non-compartmental modeling with WinNonlin v 6.3 software (Pharsight, California, USA).

Electrolyte analysis

An EasyLyte electrolyte analyzer (Medica Corporation, Bedford, Massachusetts, USA) was utilized to assay sodium (Na) and potassium (K) concentration in both plasma and urine. An equation incorporating ion concentration (C) in millimoles per liter, urine volume (V) in milliliters, duration of collection (T) in minutes, and animal body weight (W) in grams was used to determine ion excretion rate.

Ion excretion rate (µmol/min / 100 g) = \frac{C \times V \times 100}{T \times W}

Kidney injury molecule-1 assay

Kidney injury molecule-1 (KIM-1) concentration was determined in urine using a Kamiya Biomedical Company enzyme-linked immunosorbent assay following manufacturer instructions (Seattle, Washington, USA). Briefly, urine samples were removed from −80°C, brought to room temperature, and diluted as 1:3 provided diluent prior to analysis. One hundred microliters of rat KIM-1 calibrator (0.1–10 ng/mL) or sample were added to respective wells in duplicate. The covered plate was then incubated at room temperature for 2 h on an orbital shaker. Following four washes with wash solution, detection antibody (100 µL) was added to each well and then shaken for 20 min protected from light. Then horseradish peroxidase-streptavidin (100 µL) was added following four more washes. After a subsequent shaking, light-protected incubation (20 min), the plates were washed four times. One hundred milliliters of tetramethylbenzidine substrate solution were added and then incubated for 10 min under the prior incubation conditions. Finally, 100 µL of stop solution was added and the plate was read at 450 nm. A four-parameter logistics curve fit was established using MyAssays software (Sussex, England).

Blood urea nitrogen assay

A blood urea nitrogen (BUN) enzymatic assay kit (Bioo Scientific Corporation, Austin, Texas, USA) was used to analyze baseline and post-dose plasma. Samples and kit reagents were brought to room temperature and then 5 µL of standard (0–200 ppm) or sample was added to respective wells in duplicate. One hundred and fifty microliters of urease mixture were transferred into the wells and gently mixed. Following a 15-min room temperature incubation, alkaline hypochlorite (150 µL) was added. The plate was read at 620 nm after a 10-min incubation. The slope and intercept of the standard curve data trend line was used to determine BUN concentration.

Creatinine assay

Plasma and urine samples were analyzed for creatinine concentration using a creatinine colorimetric/fluorometric assay kit (BioVision, Milpitas, California, USA). Fifty microliters of plasma, 5 µL dilute urine (prior to analysis urine was diluted 1 µL urine to 24 µL creatinine assay buffer), or 0–10 µL creatinine standard (1 nmol/µL) was added to the microplate wells. The urine and standard volumes were adjusted to 50 µL with creatinine assay buffer and then 50 µL reaction mix was added to each well. Following a 1-h incubation at 37°C, the plate was read at 570 nm. The respective concentrations were determined using a standard curve.

Data treatment and statistical analysis

In the formulation optimization stage, each experiment was carried out in triplicate. Variation in centrifugation speeds and pharmacokinetic parameters was compared with the use of a Student’s t-test. For weight parameters, plasma electrolytes, BUN levels, and solvent effect, one-way analysis of variance (ANOVA) was used to compare groups. The urinary electrolyte excretion rates and KIM-1 concentration comparisons among the groups were made by two-way ANOVA using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, North Carolina, USA). Data are presented as mean ± standard deviation with significance set at p < 0.05. SPSS version 23 (IBM, Armonk, New York, USA) was used to identify outliers.

Results

Effect of varying organic solvents

Different organic solvents were examined to optimize results in terms of drug entrapment efficiency.

Entrapment efficiency

For each known concentration of ALS formulation, the absorbance value was measured in triplicate, and the average value was plotted against the known concentration of ALS solution. With an R ² value of 0.99716, this calibration curve was employed for all entrapment efficiency measurements. The ability to sufficiently dissolve hydrophilic ALS, as well as produce a desired entrapment efficiency, was imperative for organic solvent selection. Three solvents, dichloromethane, ethyl acetate, and ethyl acetate/acetone (50:50), were considered based on their availability and polarity range. As shown in Table 2, entrapment efficiency was highest (82.68 ± 1.18%) for PLGA-NPs that were formulated employing ethyl acetate as the organic solvent, 0.25% w/v DMAB, and 10,000 r/min centrifugation speed.

Table 2.

Effect of various organic solvents at 0.25% w/v DMAB and 10,000 r/min on ALS entrapment efficiency.

	Entrapment efficiency (%)
Organic solvent	Mean	SD
Dichloromethane	72.75^a	0.43
Ethyl acetate	82.68	1.18
Ethyl acetate/acetone	59.09^a	1.26

DMAB: didodecyldimethylammonium bromide; ALS: aliskiren; SD: standard deviation.

^a p < 0.05: significantly less than ethyl acetate.

Effects of varying stabilizer concentration and centrifugation speed

The use of increasing concentrations of stabilizer along with variation in centrifugation speed allowed for the optimization of NP formulation.

Particle size

When DMAB stabilizer concentration or centrifugation speed was varied, the first characteristic analyzed was particle size (Figure 1). Centrifugation speed alteration produced significantly different particle sizes for the PLGA-NP formulations containing 0.10%, 0.50%, and 1.00% stabilizer concentrations. The smallest acquired particle size (67.27 ± 0.87 nm) was found with the use of 0.50% w/v DMAB stabilizer and a centrifugation speed of 12,000 r/min.

Figure 1.

Effect of DMAB concentration at 10,000 or 12,000 r/min on NP particle size. DMAB: didodecyldimethylammonium bromide; NP: nanoparticle; p < 0.05: significantly different from 10,000 rpm.

Zeta potential

For zeta potential, variation in centrifugation speed produced a significant change within the formulations employing 0.50% and 1.00% stabilizer concentration (Figure 2). When analyzed, the highest zeta potential (18.73 ± 0.03 mV) was seen with 1.00% w/v DMAB stabilizer and 10,000 r/min centrifugation speed.

Figure 2.

Effect of DMAB concentration at 10,000 or 12,000 r/min on NP zeta potential. DMAB: didodecyldimethylammonium bromide; NP: nanoparticle; p < 0.05: significantly different from 10,000 rpm.

Polydispersity index

In this study, the best PDI (0.15 ± 0.03) value was observed when employing 0.25% w/v DMAB stabilizer and 10,000 r/min centrifugation speed (Figure 3). However, the chosen formulation of 1.00% w/v DMAB stabilizer at 10,000 r/min centrifugation speed displayed a value of 0.32 ± 0.01 for PDI. Though a high value, the formulation demonstrated sufficiently consistent dispersion of PLGA-NPs in the final formulation. A significant change was observed in PDI only in the formulation using 0.50% stabilizer concentration.

Figure 3.

Effect of DMAB concentration at 10,000 or 12,000 r/min on NP PDI. DMAB: didodecyldimethylammonium bromide; NP: nanoparticle; PDI: polydispersity index; p < 0.05: significantly different from 10,000 rpm.

Entrapment efficiency

Entrapment efficiency was significantly different between centrifugation speed in all formulations using 0.10%, 0.25%, 0.50%, and 1.00% stabilizer concentrations. Although the highest entrapment efficiency (82.68 ± 1.18%) was achieved when 0.25% w/v DMAB stabilizer and 10,000 r/min centrifugation speed were used (Figure 4), we chose the formulation of 1.00% w/v DMAB stabilizer at 10,000 r/min centrifugation speed displaying 71.62 ± 0.11% for entrapment efficiency, due to sufficient encapsulation of ALS, optimized particle size, and zeta potential values.

Figure 4.

Effect of DMAB concentration at 10,000 or 12,000 r/min on NP entrapment efficiency. DMAB: didodecyldimethylammonium bromide; NP: nanoparticle; p < 0.05: significantly different from 10,000 rpm.

Pharmacokinetic parameters

Mass chromatograms (Figure 5) associated with the m/z 552 → m/z 436 and m/z 455 → m/z 303 transitions for ALS and VER, respectively, were integrated using the instrumental LC-MS Solutions software from Shimadzu, and peak area ratios (analyte: internal standard) were used as the dependent variable in the generation of calibration curves.

Figure 5.

Mass chromatogram of ALS and VER. ALS: aliskiren; VER: verapamil.

Figure 6 shows the plasma concentration–time curve obtained from each formulation, while Table 3 denotes individual parameters. The t _1/2 (p = 0.0517) and t _max (p = 0.0961) were not significantly altered; however, C _max in the ALS-NP group (448.53 ± 49.07 ng/mL) was elevated compared to control (288.60 ± 148.07 ng/mL; p = 0.0189). ALS-NP also presented with a 168% relative bioavailability compared to ALS with respective AUC_0-∞ values of 2592.82 ± 600.51 h.ng/mL and 1538.40 ± 678.17 h.ng/mL (p = 0.0095). The V _d/F of ALS-NP (128.56 ± 43.67 L/kg) was significantly reduced (p = 0.0009) compared to ALS (540.33 ± 245.57 L/kg). A significant reduction (p = 0.0298) was also detected in CL_oral (ALS-NP, 12.26 ± 3.59 L/h/kg vs. ALS, 23.44 ± 11.45 L/h/kg).

Figure 6.

Plasma concentration–time curves following a single oral dose of ALS (30 mg/kg) and equivalent ALS-NP in rats. ALS: aliskiren; ALS-NP: aliskiren-loaded nanoparticle.

Table 3.

The pharmacokinetic parameters of ALS following a single oral dose of ALS (30 mg/kg) or a PLGA NP equivalent in rats.^a

Formulation	N	t _1/2 (h)	C _max (ng/mL)	t _max (h)	AUC_0-∞ (ng.h/mL)	CL_oral (L/h/kg)	V _d/F (L/kg)
ALS	7	18.39 ± 12.08	288.60 ± 148.08	1.07 ± 0.45	1538.40 ± 678.17	23.44 ± 11.45	540.33 ± 245.57
ALS-NP	7	8.01 ± 3.96	448.53 ± 49.07^b	0.71 ± 0.27	2592.82 ± 600.51^b	12.26 ± 3.59^b	128.56 ± 43.67^b

ALS: aliskiren; ALS-NP: aliskiren-loaded nanoparticles; PLGA: poly(lactic-co-glycolic) acid.

^a Values expressed as mean ± standard deviation.

^b p < 0.05: significantly different from ALS.

Electrolyte analysis

As ANOVA detected significance among the groups (p = 0.0149), urinary K excretion (Figure 7) was decreased significantly only in the ALS group (ALS-B vs. ALS-F, 0.16 ± 0.02 vs. 0.09 ± 0.03 µmol/min/100 g; p = 0.0274) which was also significantly reduced compared to ALS-NP-F (ALS-F vs. ALS-NP-F, 0.09 ± 0.03 vs. 0.19 ± 0.13 µmol/min/100 g; p = 0.016). Na excretion rate (ANOVA; p = 0.4346), seen in Figure 8, along with plasma electrolyte levels (K; p = 0.1957 & Na; p = 0.9524), seen in Table 4, were unchanged.

Figure 7.

Effect of formulation on urinary K excretion rate in rats. K: potassium; ALS: aliskiren. *p > 0.05: significantly different from baseline. ^# p < 0.05: significantly different from ALS-Final.

Figure 8.

Effect of formulation on urinary Na excretion rate in rats. Na: sodium. p > 0.05: values were not significantly different.

Table 4.

Plasma electrolyte concentrations on day 2.^a

Group	Na (mM)	K (mM)
ALS	137.56 ± 1.77	4.19 ± 0.07
ALS-NP	137.62 ± 1.72	4.90 ± 1.17

ALS: aliskiren; ALS-NP: aliskiren-loaded nanoparticles; Na: sodium; K: potassium.

^a p > 0.05: values were not significantly different.

Urinary KIM-1 assay

ANOVA testing showed a significant alternation between groups (p = 0.0078). As shown in Figure 9, KIM-1 excretion increased following ALS dosing (ALS-B vs. ALS-F, 0.11 ± 0.03 vs. 0.17 ± 0.05 ng/mL; p = 0.0486), while ALS-NP showed a decrease (ALS-NP-B vs. ALS-NP-F, 0.13 ± 0.05 vs. 0.07 ± 0.04 ng/mL; p = 0.027) which was also significantly decreased compared to ALS-F (p = 0.0008).

Figure 9.

Effect of formulation on urinary KIM-1 in rats. KIM-1: kidney injury molecule-1; ALS: aliskiren; ALS-NP: aliskiren-loaded nanoparticle. *p > 0.05: significantly different from baseline. ^# p < 0.05: significantly different from ALS-NP-Final.

BUN assay

Significant (ANOVA; p < 0.0001) BUN differences were found among groups. Plasma BUN (Figure 10) increased significantly after dosing in both groups (ALS-NP-B vs. ALS-NP-F, 12.34 ± 0.91 vs. 15.14 ± 0.70 mg/dL; p < 0.0001 and ALS-B vs. ALS-F, 13.17 ± 0.27 vs. 15.60 ± 1.27 mg/dL; p < 0.0001); however, no statistical difference was found between endpoint levels. It seems that nanoformulation had no effect on the plasma BUN response.

Figure 10.

Effect of formulation on plasma BUN in rats. BUN: blood urea nitrogen. *p < 0.05: significantly different from baseline.

Creatinine assay

Following group testing via ANOVA, creatinine levels were found to be significantly (p < 0.0001) changed. Plasma creatinine levels (Figure 11) were elevated following both dosage forms (ALS-B vs. ALS-F, 0.87 ± 0.50 vs. 1.50 ± 0.21 mg/dL; p < 0.0001 and ALS-NP-B vs. ALS-NP-F, 0.96 ± 0.31 vs. 1.63 ± 0.17 mg/dL; p < 0.0001) with no significant change between endpoint values. Urinary creatinine, Figure 12, did not significantly change (ANOVA; p = 0.2657). The results in our study show that both ALS formulations significantly increase plasma creatinine; however, the increase is comparable between groups which may indicate no NP influence on this outcome.

Figure 11.

Effect of formulation on plasma creatinine in rats. *p < 0.05: significantly different from baseline.

Figure 12.

Effect of formulation on urinary creatinine excretion rate in rats. p > 0.05: values were not significantly different.

Discussion

Different organic solvents were examined to optimize results in terms of drug entrapment efficiency. The role of the organic solvent is to provide adequate solubility for the drug, while ultimately promoting drug entrapment. Of the solvents considered (dichloromethane, ethyl acetate, and ethyl acetate/acetone), entrapment efficiency was highest (82.68 ± 1.18%) for PLGA-NPs that were formulated employing ethyl acetate as the organic solvent, 0.25% w/v DMAB, and 10,000 r/min centrifugation speed. Ethyl acetate is the most polar of the solvents considered, making it a suitable match for the ALS. As shown in Table 2, dichloromethane facilitated adequate entrapment (72.75 ± 0.43%). The ethyl acetate/acetone mixture produced the lowest overall entrapment (59.09 ± 1.26%). The ethyl acetate formulation exhibited the most ideal characteristics including small particle size and high entrapment efficiency. These conditions are more suited to the in vivo conditions of the vascular and lymphatic systems, therefore decreasing the chance of clearance from the circulatory system.³⁰ Particles that measure less than 100 nm are small enough to permeate submucosal membranes, whereas particles greater than 100 nm remain in the epithelial lining and do not reach the target delivery site.¹⁴ In turn, cellular uptake is affected as the number of NPs which reach the cell are minimized. The decreased particle size is related to an increased surface area which allows for more interaction with the solvent.²⁷ The low entrapment efficiency with ethyl acetate/acetone and dichloromethane indicate that, of those organic solvents tested, ethyl acetate was the best choice for this PLGA-NP formulation in regard to adequate entrapment.

For a drug delivery system to be considered successful, a high loading capacity is essential to curtail the overall dosage requirement. The larger entrapment efficiency value indicated that there was a greater amount of ALS inside the NP per total amount of ALS used for NP production. Further, the amount of drug bound to the NP was greatly influenced by the copolymer PLGA and the chemical structure of ALS.³¹ It is possible that entrapment efficency was overestimated as a result of free drug within the NP containing supernatant.

The use of increasing concentrations of stabilizer along with variation in centrifugation speed allowed for the optimization of NP formulation. Zeta potential and PDI are both parameters that guide NP formulation optimization. A more positive zeta potential value, around 20 mV, is desirable so that the NPs may adhere to the negatively charged cell membrane.¹⁶ Higher bioavailability will be achieved if the PLGA-NP is capable of permeating the cell membrane and transported to the primary endosomes within the cell.¹⁴ From there, the PLGA-NPs can be separated, and selected NPs are recycled to the cell exterior to maintain a required concentration gradient of NPs. Without this balance, exocytosis of the NPs will be activated. Inside the cell, the remaining encapsulated PLGA-NPs migrate to the cytoplasm where controlled release of ALS occurs. Charge was highly considered when choosing an optimal formulation due to the vital role this property plays in cellular uptake and drug release.

Once our ALS-NP formulation was optimized, and fresh NPs were prepared, we conducted a oral dose pharmacokinetic study using SHRs. In another study in which SHRs were dosed with a single oral administration of 30 mg/kg in 5% dextrose solution, pharmacokinetic sampling yielded an AUC_0-∞ of 3.06 ± 1.8 h.µmol/L (approximately 1688.38 ± 993.17 h.ng/mL) which agrees with the ALS formulation in our study.³² The mean t _1/2 (1.9 ± 0.4 h) in the study conducted by Wood et al.³² was much lower than either t _1/2 in our study; however, this may be a function of the extended sampling scheme of our study.

A reduction in electrolyte excretion is typically a negative effect as subsequent fluid retention will likely occur leading to high blood volume. In the case of K excretion, the NP formulation appears to show an improvement compared to the methylcellulose form; however, the plasma concentration of K did not show significant variation. When these findings are coupled with a high variation in urinary Na excretion and a nonsignificant plasma Na change, the changes observed may be attributable to an increase in food intake over the examined window.

The results of the urinary KIM-1 assay support the use of this ALS-NP through a reduction in this biomarker of kidney injury. However, the change seen with ALS may change over time. Following 28 days of BID 10 mg/kg ALS oral dosing in C57BL/6-(hREN)/(hAGT) double transgenic mice with concurrent coadministered subcutaneous cyclosporine A (20 mg/kg), urinary KIM-1 (approximately 3.25 ng/mL) was not significantly changed (p > 0.05) compared to the control group.³³ In our study, we may have only observed a transient increase in KIM-1 through NP usage.

Our data show no effect on the plasma BUN response using the nanoformulation. This change may also be time-dependent as the Saraswat et al. study showed that BUN (22.33 ± 0.56 mg/dL) was not significantly changed (p > 0.05) compared to the control group.³³

In another study, Sprague-Dawley rats treated with daily intramusculuar gentamicin (100 mg/kg) and ALS (25 mg/kg) via osmotic pump for 14 days, plasma creatinine concentration was reduced compared to gentamicin alone.³⁴ Further, in five out of six nephrectomized rats treated with oral 10 mg/kg ALS over 4 weeks, plasma creatinine was significantly reduced (p < 0.05) compared to nephrectomized controls.³⁵ The change we saw may be due to either our larger dose or short duration.

Conclusion

Of the various parameters assayed, the optimum formulation of ALS-NPs was achieved using ethyl acetate as the organic solvent, 1.00% DMAB stabilizer, and a centrifugation speed of 10,000 r/min. For all possible alterations, these conditions offered the highest zeta potential, reasonable particle size below 100 nm, agreeable entrapment efficiency, and a uniform distribution of NPs in the solution. At the dosage tested, ALS-NP showed favorable kidney changes compared to ALS in regard to KIM-1 and K excretion rate. Additionally, the other parameters tested exhibited equivalent outcomes to the ALS formulation. As patient compliance may be more favorable due to an estimated reduction of cost for the antihypertensive drug, this ALS-NP formulation may be the starting point for improving ALS dosing.

Footnotes

Authors’ note

Portions of this article were published in thesis form in fulfillment of the requirements for the Undergraduate Honors Program for Student Jessica Coleman.

Acknowledgment

The authors would like to thank Gregory Hanley, Dustin L Cooper, Fessou Lawson-Hellu, Hannah V Oakes, Angela V Hanley, Kenny W Bullins, Brian G Evanshen, and Judy A Whitmore for their technical assistance.

Author contributions

Derek E Murrell and Jessica M Coleman contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The funding for this project was made possible through an East Tennessee State University (ETSU) Research and Development Committee Small Grant and an ETSU Honors College Student-Faculty Collaborative Grant. Also, this research was supported in part by the National Institutes of Health grant (C06RR0306551).

ORCID iD

Sam Harirforoosh

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Formulation and in vivo evaluation of aliskiren-loaded poly(lactic- co -glycolic) acid nanoparticles

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Formulation of NPs

Effect of varying organic solvents

Effect of varying stabilizer concentration and centrifugation speed

NP characteristics

Entrapment efficiency

Particle size, zeta potential, and PDI

Animals and drug administration

Sample preparation and LC-MS/MS conditions

Pharmacokinetic parameters

Electrolyte analysis

Kidney injury molecule-1 assay

Blood urea nitrogen assay

Creatinine assay

Data treatment and statistical analysis

Results

Effect of varying organic solvents

Entrapment efficiency

Effects of varying stabilizer concentration and centrifugation speed

Particle size

Zeta potential

Polydispersity index

Entrapment efficiency

Pharmacokinetic parameters

Electrolyte analysis

Urinary KIM-1 assay

BUN assay

Creatinine assay

Discussion

Conclusion

Footnotes

Authors’ note

Acknowledgment

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References