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Article Cite This: ACS Omega 2019, 4, 4519−4529
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Dendrimer Donepezil Conjugates for Improved Brain Delivery and Better in Vivo Pharmacokinetics Anurag Kumar Singh,† Avinash Gothwal,† Sarita Rani,† Monika Rana,‡ Anuj K. Sharma,‡ Awesh K. Yadav,§ and Umesh Gupta*,† †
Department of Pharmacy, School of Chemical Sciences and Pharmacy and ‡Department of Chemistry, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, NH-8 Ajmer Jaipur Expressway, Bandarsindri, Ajmer, Rajasthan 305817, India § Bhagyoday Tirth Pharmacy College, Khurai Road, Sagar, Madhya Pradesh 470002, India
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S Supporting Information *
ABSTRACT: In neurodegenerative disorders, crossing the blood-brain barrier to achieve higher brain uptake of drugs has attracted considerable interest of researchers in recent years. The present approach is designed with a hypothesis that polyamidoamine (PAMAM) dendrimer can be suitable for better brain delivery of donepezil (DZ) utilizing the conjugation strategy. Amine-terminated 4.0 G PAMAM dendrimers (utilizing ethylenediamine core) were synthesized and characterized by different spectroscopic methods (1H and 13C NMR, UV−vis, Fourier transform infrared, electrospray ionization mass). The synthesized PAMAM dendrimer was then conjugated with DZ-ester and finally DZ (ester)-PAMAM conjugate (PDZ) was prepared. The chemical shifts of −CHO (δ = 9.92) and O−CH3 (δ = 3.153) in 1H NMR spectrum confirmed the synthesis of PDZ. The percent drug conjugation of DZ was approximately 26%, and 16 DZ molecules were conjugated with each PAMAM molecule. The size and ζ-potential observed of PDZ conjugate were 122 ± 1.88 nm and 0.434 ± 0.322, respectively. In vitro release studies suggested that DZ release was in a sustained fashion until 120 h at physiological pH conditions. The in vitro acetylcholine esterase (AChE) inhibition activity of PDZ formulation was significantly higher (p < 0.05) than that of the DZ alone at 1 μM dose. In the in vivo studies, the brain uptake of PDZ was quite higher than that of DZ following intravenous administration in Sprague-Dawley rats. The plasma drug concentration studies resulted into improved pharmacokinetic parameters. Half-life (t1/2), volume of distribution (Vd), and clearance were found to be 5.75 ± 0.41 h−1, 0.135 ± 0.02 L, and 0.016 ± 0.0021 L/h, respectively, in the case of PDZ formulation and 1.09 ± 0.10 h−1, 0.172 ± 0.016 L, and 0.108 ± 0.014 L/h, respectively, in the case of DZ solution. The improved half-life and 4-fold increase in brain uptake was observed in the case of the dendrimer-conjugated formulation, which suggested that the synthesized conjugates provide significantly higher DZ brain delivery. The prepared PDZ-conjugated formulation improved AChE inhibition in vitro and the brain delivery in vivo. This strategy may be explored further for better delivery of DZ to the brain.
1. INTRODUCTION It is difficult to transport endogenous molecules across the blood-brain barrier (BBB), and drug solubility plays a major role here. The newer drugs (drugs that are under the preclinical phase) are facing challenges due to their insufficient solubility, absorption, distribution, metabolism, excretion, and such unachieved physical properties, which badly affects its pharmacokinetics and therapeutic action as well. This limits the development of new neurotherapeutics. Genes and almost 98% of large protein molecules of molecular weight more than 400−600 Da cannot cross the BBB.1 Lipophilicity of the transported therapeutic candidate proves its efficient passage across the BBB. The inadequate aqueous solubility of most of the active pharmaceutical ingredients (APIs) also presents a blockage for the development of novel therapeutic approaches. Pharmaceutical industries have excluded approximately 40% drug candidates that are subjected to further development into © 2019 American Chemical Society
successful formulation by optimizing their aqueous solubility and systemic bioavailability.1 Donepezil (DZ) is the USFDA approved second-generation acetylcholinesterase inhibitor (AChE) drug used to treat mildto-moderate Alzheimer’s disease (AD) (Figure 1A). CYP 450 isoenzymes 2D6 and 3A4 metabolize DZ into four major metabolites, followed by glucuronidation and finally excretion through urine. The smaller drug molecules that have limited aqueous solubility (0.0045 mg/mL), however, need to be formulated either using a micellar approach or maybe using nanoparticles (NPs) to achieve higher bioavailability. Despite the indisputable use of NPs to improve the brain drug delivery by various routes of administration,2−5 particles beyond 250− Received: December 8, 2018 Accepted: February 14, 2019 Published: March 1, 2019 4519
DOI: 10.1021/acsomega.8b03445 ACS Omega 2019, 4, 4519−4529
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Figure 1. (A) Chemical structure of donepezil (DZ) (IUPAC name: (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine) (B); synthetic scheme of DZ (ester) (1b)−PAMAM 4.0 G conjugate (PDZ).
reported in the literature.3,6 In a recent study, bortezomib was loaded in a specially tailored acidic pH sensitive dendrimer with a switching drug release property for the management of tumor and was found to be less toxic.12 The same on-off property of dendrimer was exploited to deliver thiolated drugs (cisplatin, doxorubicin, 6-mercaptopurine, etc.) by gold NP encapsulation triggered to glutathione for the management of cancer.13 Stimuli-responsive dendrimers are of great importance in controlled payload release against various stimuli, such as exogenous stimuli (temperature, light, and ultrasound) and endogenous stimuli (redox potentials, enzyme, and acid), which offers sufficient springiness to aid the delivery of various therapeutic agents associated with PK/PD challenges.14 Fluorinated dendrimers are reported to facilitate cellular uptake, serum resistance of the polyplexes of the dendrimer and DNA, and endosomal escape. They also mimic lipid and cationic polymer gene vectors with least cytotoxicity, low charge ratio-assisted efficient transfection, and minimized DNA dosage. The ability of dendrimers to wind or unwind DNA is regulated by it too.15−17 Dendrimer−ligand interactions can well be understood by utilizing NMR by analyzing chemical shift titration, nuclear Overhauser effect measurements, diffusion analysis, relaxation measurements, and saturation transfer difference. These tools can be used in understanding the mechanism of interaction between
300 nm size range exhibit inefficient drug delivery in the brain because of less penetration transport across intracellular and paracellular regions.3 Such shortcomings may be overcome via utilizing nanocarriers that result into favorable drug delivery across BBB. Here, in the present work, we preferred dendrimers because of their attractive size (less than 10 nm), that proved them as important drug carriers for CNS delivery of poor hydrophilic drugs.3,6 Dendrimers bear a core in their distinct nanostructure and are synthesized via a step-by-step synthesis in an interactive manner. The exclusive oversized range and surface properties have piloted to the engineering of “critical nanoscale design parameters” and nanoparticle structure control as an approach for improving pharmacokinetics, pharmacodynamics, and disease-specific peculiar site targeting.7 Our recent report also stated that chitosan-coated polyamidoamine (PAMAM) dendrimer formulation can improve the delivery of temozolamide (TMZ) in the brain. The intraperitoneal delivery of TMZ using chitosan−PAMAM dendrimer-based formulation resulted into a 2-fold increase in the brain bioavailability in vivo.8 Dendrimers are excellent solubilizers, as evidenced by the plethora of reports in the last few decades.9−11 Superficially positively charged dendrimers are believed to aid enhanced transport of drugs to the brain because positively charged NPs in association with mucus have increased cellular uptake as 4520
DOI: 10.1021/acsomega.8b03445 ACS Omega 2019, 4, 4519−4529
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Figure 2. 1H NMR spectra of DZ (ester)−PAMAM conjugate (PDZ).
glassy in nature. The final confirmation of the synthesized dendrimers was however ascertained by spectroscopic characterization (FT-IR, 1H NMR, and 13C NMR) discussed in the following sections. 2.1.2. FT-IR and NMR spectroscopic analysis. In FT-IR spectra, the characteristic peaks are at 3350.2 cm−1 (N−H stretch of primary amines); 3283.14 cm−1 (quaternary ammonium-ion peak); 3190.0 cm−1 (antisymmetric N−H stretch of substituted primary amine); 1641.3 cm−1 (CO stretch); 2931.67 cm−1 (aliphatic C−H stretch); 1550.32, 1435.63, and 1358.99 cm−1 (N−H bending); and 1115.19 cm−1 (C−C bending). The progress of dendrimer generation was observed from the vibration peak at 1731 cm−1. The absence of this peak in each full-generation amine-terminated dendrimer confirmed completion of the amidation step of the chemical reaction. All remaining FT-IR spectra for 0.0−4.0 G are provided in the Supporting Information (Figure S2b−k). 1 H NMR (500MHz, Bruker, Switzerland) further confirmed the synthesis, PAMAM G 4.0, δH: 3.334−3.392 (−CH2NH2), δH: 2.410, 2.50 (−CO), δH: 3.535 (methanolic −OH), δH: 2.6−3.2 (amide) (Figure S3j). PAMAM synthesis was followed with necessary modifications as per previous reports, and our results of synthesis were in accordance with the literature.20 2.2. Characterization of DZ (ester) (1b). DZ (ester) synthesis was carried out following a two-step process (Figure 1B), involving the Vilsmeier−Haack reaction.21 A mixture of phosphorus trichloride, N,N-dimethylmethanamide, DZ, and MeOH was added, and FT-IR and NMR (Figures 2 and 4) confirmed the conversion of the aldehyde. In FT-IR spectrum, DZ (aldehyde) (1a) revealed a peak at 1730 cm−1 (CO stretch) that confirmed the synthesis of DZ aldehyde. In 1H NMR spectrum, chemical shifts of −CHO (δ = 9.92) and O− CH3 (δ = 3.153) were observed. In the next step of the reaction, a mixture of DZ (aldehydes) (1a), oxone, and MeOH was obtained in a stepwise manner and this product was purified further and characterized by FT-IR, NMR, and electrospray ionization (ESI) mass. The FT-IR spectrum of PDZ showed peaks at 1690 cm−1 (CO stretch) and 1172 cm−1 (−C−O stretch). In NMR spectroscopic studies, chemical shifts of −CH3COO (δ = 3.841−3.764), O−CH3
dendrimer and the ligand, binding parameter calculations, internal/external competitive binding of ligand with dendrimer surfaces, ligand/ligand complex lateralization in the dendrimer, etc.18,19 In the present study, the potential of G 4.0 polyamidoamine dendrimers (PAMAM) with amine surface groups was explored for achieving improved delivery of DZ to the brain following parenteral administration. First, the DZ ester was synthesized, and then this DZ-ester was conjugated with 4.0 G of PAMAM dendrimers. The conjugate synthesis was confirmed by Fourier transform infrared (FT-IR), 1H NMR, and 13C NMR at each step of the reaction. The developed conjugates were evaluated for in vitro AChE activity and in vivo brain uptake studies in the Sprague-Dawley rat model. The overall objective was to establish the proof of the principle that the conjugated DZ is able to penetrate more in the brain and exert the in vitro activity to support Alzheimer-like disorders.
2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of PAMAM Dendrimers up to 4.0 G. 2.1.1. Copper Sulfate Chemical Test and UV−Vis Absorption Analysis. Ethylenediamine (EDA)-cored PAMAM dendrimers were synthesized and characterized. The initial completion of synthesis was ascertained by the purple/blue coloration of copper sulfate in the presence of primary amines of the full-generation PAMAM dendrimers. Complete or full-generation PAMAM dendrimers (1.0, 2.0, 3.0 G and so on) gave a purple color, whereas the half-generation PAMAM dendrimers ended up giving a deep blue color.20 The wavelength shift of conjugates in the UV absorption range indirectly confirmed the synthesis of dendrimer conjugation. (Figure S2a). In the results, the wavelength changed as the dendrimers’ structure changed in different generations. As the PAMAM dendrimer generation increased, the absorption shifted from higher to lower wave numbers. Dendrimers were found to be oily liquids, and the intrinsic viscosity was higher only in the case of full generations not in half-generations. The color observed was deep yellow with a honeylike consistency. The higher generations were 4521
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Figure 3. 13C NMR spectra of DZ (ester)−PAMAM conjugate (PDZ).
Figure 4. Comparative FT-IR spectra of donepezil (DZ), PAMAM 4.0 G, DZ (ester) (1b), and DZ (ester) (1b)−PAMAM 4.0 G dendrimer conjugate PDZ.
following a reported protocol.22 The peaks of proton (a) and (e) are always observed clearly without any overlapping. These two peaks were considered for the calculation of the overall conjugation of drugs (i.e., DZ) to PAMAM dendrimers. The percent conjugation of DZ (ester) (1b)−PAMAM 4.0 G dendrimer (PDZ) was found 26%, i.e., approximately 16 molecules were conjugated per molecule of G 4.0 PAMAM dendrimer. The ESI mass of DZ (ester) (1b)−PAMAM 4.0 G dendrimer was 21 531.5723 (Figure S4). The increase in mass after conjugation suggested that 16−18 molecules of DZ (ester) (1b) were attached via direct conjugation through an amide bond (Figure S4). As the PAMAM G 4.0 dendrimer has 64 terminal primary amine groups on the surface and provides a platform for chemical conjugation with other moieties, it is
(δ = 3.153), aliphatic CH3 (δ = 5.249), and aromatic CH3 (δ = 7.568−7.029) were observed. 2.3. Characterization of DZ (Ester) (1b)−PAMAM 4.0 G Dendrimer Conjugates (PDZ). In comparative FT-IR spectrum analysis, the characteristics peaks in the case of PDZ were obtained at 1532.57 cm−1 (N−H bending), 1642.36 cm−1 (CO amide stretch), and 2930.97 cm−1 (N−H stretch) (Figure 4). In 1H NMR spectrum, chemical shifts of δ = 7.696−7.037 (Ar-H), δ = 4.120−3.770 (O−CH3), and δ = 3.502 (aliphatic CH) were detected and PDZ synthesis was further confirmed by 13C NMR spectroscopy (Figures 2 and 3). 2.4. Percent Conjugation. The percent drug conjugation was determined with the help of 1H NMR spectroscopy 4522
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not possible to occupy all 64 amines’ functionality with the DZ ester, may be due to steric hindrance (Figure 4).23 2.5. Particle Size, ζ-Potential, and Polydispersity Index (PDI). The size was measured using the dynamic light scattering technique; the average size and PDI of the PDZ were found to be 122 ± 1.88 nm and 0.434 ± 0.322, respectively. The concentration of the analyzed sample was 1 mg/mL throughout the analysis. The size of the synthesized PDZ was increased several folds with respect to PAMAM G 4.0 dendrimer, which indicates that the conjugation was successful. PDI was 0.358 ± 0.121, which confirms uniform distribution of the molecules in the aqueous environment. Results were in the accordance of previously reported dendrimer−drug conjugation.24 2.6. In Vitro Release Studies. The in vitro drug release studies were performed by membrane dialysis method, in water. PDZ conjugate or formulation was released in a sustained release fashion, whereas pure DZ was released rapidly in phosphate buffer saline (PBS) (pH 7.4) and in acid phthalate buffer (pH 4.0) (Figure 5A,B). Results revealed that 100% DZ was released in phosphate buffer saline pH 7.4, within 6−8 h, whereas the PDZ released till 120 h, showing a sustained drug release effect. Again, in acid phthalate buffer (pH 4.0), the drug was released within 6−8 h whereas PDZ formulation showed the release in a sustained fashion till 120 h. The release studies of the drug were performed at acidic pH conditions also, as the amide bond is cleavable at acidic pH. The pH of a hippocampus is also acidic, which is why the release was examined in acidic conditions. β-amyloid processing is markedly affected by low pH, which could link acidosis to AD, and lactate causes a dose-dependent increase in cellular β-amyloid immunoreactivity in hippocampal neurons.25 In the presence of enzymes, such as amylase, the amide bond will break easily.26 Amide linkages within G 4.0 PAMAM dendrimers are highly stable against hydrolysis at all pH buffers studied; their drug release must rely on enzymatic cleavage. Peptidase enzymes or amylases provide such an opportunity for selectively releasing drugs from polymer conjugates in the lysosomal compartment. Being stable, the amide linkage in dendrimeric conjugates provides the advantage of improved pharmacokinetics by prolonging the duration in blood circulation. Generally, amide linkage is cleaved by proteases, such as serine proteases, cysteine, and zinc-dependent endopeptidases.27 The above-mentioned results indicated that the drug was stabilized probably due to conjugation in PDZ formulation. In the release profile, it can be observed that 80% of the drug was released within 120 h. The conjugation might be the reason for the sustained release pattern. PDZ is being given as a formulation and not as a pure drug; hence, it is a nanomaterial and showed some advantages over pure drug. In AD pathophysiology, it was observed that the neurodegeneration leads to an acidic environment; the linkage between dendrimer and DZ is acid labile. This factor was taken into consideration at the time of the conjugation scheme preparation. Therefore, the in vitro release was performed in acidic pH condition. The void space of PAMAM dendrimers is highly hydrophobic, which restricts the aqueous phase intake within the cavity. Hence, dendrimer is highly stable against the hydrolysis at all pH environments. Therefore, the acidic pH affects only the drug release and not the architecture of the dendrimer. 2.7. Hemolytic Studies. The percent hemolysis for each sample was compared to that of distilled water, which was
Figure 5. (A) Comparative release pattern of DZ-1 and PDZ-1 and DZ-2 and PDZ-2 in phosphate buffer saline (pH 7.4) and acid phthalate buffer (pH 4.0), respectively; DZdonepezil and PDZ (ester) (1b)−PAMAM 4.0 G conjugate. (B): Release pattern of PDZ formulation in acidic pH (PDZ-1) and phosphate buffer saline pH 7.4 (PDZ-2), respectively, up to 4 h (C); Hemolytic toxicity of drug and different formulations. (Values represent mean ± SD; n = 3).
considered as 100% hemolytic. The percent hemolysis of red blood cells (RBCs) with PDZ (7.45%; n = 3) was comparatively higher than that with DZ (Figure 5C). Percent hemolytic toxicities of PAMAM, DZ, and PDZ were observed to be 45.63 ± 0.02, 4.50 ± 0.10, and 7.45 ± 0.25% (Figure 5C), respectively (p < 0.05). The DZ and PDZ formulation showed 10 times and 6 times less hemolytic toxicity than that of PAMAM, respectively. From the results, we can conclude that the pure drug DZ and PDZ formulation exhibited similar hemolytic toxicity profiles in results. Therefore, the prepared conjugates PDZ with very less hemolytic toxicity may be used for drug delivery in the AD. The high hemotoxicity of PAMAM is due to a cationic charge of itself,23 whereas PDZ reflects very less hemotoxicity; this may be due to the reduced cationic charge/ζ-potential of the PDZ. Amine-terminated PAMAM dendrimer showed higher hemolysis compared with surface-modified PAMAM dendrimer. 4523
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2.8. Amyloid Inhibition (Lysozyme Fibrillation and Fluorescence Measurement). Different cells, such as epithelial cells, macrophages, and astrocytes, secrete lysozyme in the body. Lysozyme is found abundantly in body fluids and tissues, like spleen, liver, milk, and cerebral spinal fluid (CSF).28 Lysozyme has anti-inflammatory and antioxidant properties,29 but its amount is higher in CSF. Although it is more relevant to use model peptides of AD like Aβ40 or Aβ42 for amyloid aggregation studies, these are difficult to obtain and aggregation varies for different batches. Recent studies suggest that chicken egg white lysozyme (CEWL) has become an appealing model to study the protein misfolding and amyloid fibril formation mechanism.30 CEWL was used to study the effect of DZ and PDZ on fibrillation, since the studies reported here are only preliminary. Interestingly, DZ and PDZ inhibit CEWL aggregation by approximately 42 and 35%, respectively (Figure 6A, B). Therefore, results recommended that both DZ and PDZ formulation exhibited significant amyloid inhibition to fibrillation compared with free CEWL (p < 0.05).
Figure 7. Percent AChE activities with inhibitors (DZ and PDZ) obtained from the absorbance at 405 nm. One-way analysis of variance (ANOVA) using Newman−Keuls multiple comparison test. p < 0.0001, extremely significant; # means no significance.
DZ at 1 μM concentration showed approximately 84 and 28% AChE inhibition, respectively, which was significantly higher (p < 0.05). When used at a concentration of 10 μM, PDZ and DZ showed approximately 95 and 84% AChE inhibition, respectively. PDZ formulation displayed improved and significantly higher (p < 0.05) activity as compared to that of DZ. The results indicated that PDZ showed improved inhibitory activity in amyloid formation and effective AChE inhibition activity than those in DZ. The encouraging in vitro results obtained for AChE inhibition and amyloid formation also support in vivo studies of the prepared formulations. 2.10. Stability Studies. In the stability study, the morphological characteristics of prepared formulations were observed for 1−2 months and it was assured that the size remained the same in this time period. The formulations were found to be the most stable in the dark at room temperature (Table 1). Minimum drug leakages were found at room temperature as compared to 0 and 50 °C for 7 weeks, may be due to a conformational change of the dendritic structure that lead to a decreased drug enclosing cavity. This study confirmed that the PDZ formulation was more stable. The drug content was quantified in PDZ as 91.6 ± 7.32%, as per the pharmacopoeial limit 100 ± 10%.31 2.11. In Vivo Studies. 2.11.1. Brain Distribution and Pharmacokinetic Studies. Plasma drug level studies of PDZ formulation were performed in Sprague-Dawley rats to determine the feasibility of delivering DZ through intravenous (IV) route. It is observed from Figure 8A that PDZ formulation contributed remarkably toward DZ bioavailability in comparison to DZ solution as a positive (control) (Figure 8A). Pharmacokinetic parameters, such as bioavailability (AUC0‑∞) of PDZ (32.65 + 3.73 ng h/mL), were almost 4fold higher (p ≤ 0.05) than that of the DZ alone (Table 2). Half-life (t1/2), volume of distribution (Vd), and clearance (Cl) were found to be 5.75 ± 0.41 h−1, 0.135 ± 0.02 L, and 0.016 ± 0.0021 L/h, respectively, in the case of PDZ formulation and 1.09 ± 0.10 h−1, 0.172 ± 0.016 L, and 0.108 ± 0.014 L/h, respectively, in the case of DZ solution (Table 2). This may be attributed to the conjugation of DZ to PAMAM, which led to enhanced penetration of the drug into the brain. The biodistribution pattern of PDZ formulations and DZ following IV administration revealed a higher concentration of DZ in the brain (Figure 8B) from the PDZ formulation as compared with pure DZ. The DZ concentration of PDZ in tissues/organs was significantly higher than that of free DZ. The formulations
Figure 6. (A) Normalized ThT fluorescence intensities for CEWL fibrils without inhibitors and with inhibitors of DZ and PDZ. The values represent mean ± SD (n = 3). (DZdonepezil; PDZ; DZ (ester) (1b)−PAMAM 4.0 G conjugate); (B) ThT fluorescence spectrum depicting the inhibitory effect of DZ and PDZ on CEWL.
2.9. In Vitro Anticholinesterase Assay. The AChE inhibitory properties of DZ and PDZ were assessed against AChE from Electrophorus electricus (EeAChE) using the wellestablished Ellman method to determine the AChE activities at selected concentrations (Figure 7). It was observed that the developed formulation of DZ (ester) (1b)−PAMAM 4.0 G dendrimer (PDZ) was able to inhibit AChE activity. PDZ and 4524
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Table 1. Stability Studies of Prepared PDZ up to 7 Weeks of Batch Loadinga dark
light
parameters (after 7 weeks)
formulation
0 °C
RT
50 °C
0 °C
RT
50 °C
turbidity precipitation crystallization color change change in consistency
DZ (ester) (1b)−PAMAM 4.0 G dendrimer (PDZ)
+ + + < ++
-
++ ++ + +
