Brain Uptake of a Zidovudine Prodrug after Nasal Administration of

Article pubs.acs.org/molecularpharmaceutics

Brain Uptake of a Zidovudine Prodrug after Nasal Administration of Solid Lipid Microparticles Alessandro Dalpiaz,*,† Luca Ferraro,‡ Daniela Perrone,† Eliana Leo,§ Valentina Iannuccelli,§ Barbara Pavan,‡ Guglielmo Paganetto,‡ Sarah Beggiato,∥ and Santo Scalia† †

Department Department § Department ∥ Department ‡

of of of of

Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Medical Sciences, University of Ferrara, Ferrara, Italy

ABSTRACT: Our previous results demonstrated that a prodrug obtained by the conjugation of the antiretroviral drug zidovudine (AZT) with ursodeoxycholic acid (UDCA) represents a potential carrier for AZT in the central nervous system, thus possibly increasing AZT efficiency as an anti-HIV drug. Based on these results and in order to enhance AZT brain targeting, the present study focuses on solid lipid microparticles (SLMs) as a carrier system for the nasal administration of UDCA−AZT prodrug. SLMs were produced by the hot emulsion technique, using tristearin and stearic acid as lipidic carriers, whose mean diameters were 16 and 7 μm, respectively. SLMs were of spherical shape, and their prodrug loading was 0.57 ± 0.03% (w/w, tristearin based) and 1.84 ± 0.02% (w/w, stearic acid based). The tristearin SLMs were able to control the prodrug release, whereas the stearic acid SLMs induced a significant increase of the dissolution rate of the free prodrug. The free prodrug was rapidly hydrolyzed in rat liver homogenates with a half-life of 2.7 ± 0.14 min (process completed within 30 min). The tristearin SLMs markedly enhanced the stability of the prodrug (75% of the prodrug still present after 30 min), whereas the stabilization effect of the stearic acid SLMs was lower (14% of the prodrug still present after 30 min). No AZT and UDCA−AZT were detected in the rat cerebrospinal fluid (CSF) after an intravenous prodrug administration (200 μg). Conversely, the nasal administration of stearic acid based SLMs induced the uptake of the prodrug in the CSF, demonstrating the existence of a direct nose−CNS pathway. In the presence of chitosan, the CSF prodrug uptake increased six times, up to 1.5 μg/mL within 150 min after nasal administration. The loaded SLMs appear therefore as a promising nasal formulation for selective zidovudine brain uptake. KEYWORDS: solid lipid microparticles, zidovudine, ursodeoxycholic acid, prodrug, hydrolysis, liver homogenate, nasal formulation, brain uptake

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INTRODUCTION 3′-Azido-3′-deoxythymidine (zidovudine or AZT, Figure 1) is the first antiretroviral drug approved by FDA for the treatment of acquired immunodeficiency syndrome (AIDS) which is caused by infection with the human immunodeficiency virus (HIV). AZT is currently employed in therapeutic multiple-drug combination protocols.1 Although antiretroviral nucleoside derivatives are largely used in AIDS treatment and their administration dramatically reduces viral loads in HIV patients, a total eradication of the virus from the body cannot be obtained. Indeed, HIV enters the brain2,3 through infected monocytes that differentiate into macrophages and microglia in the brain,4 whereas the antiretroviral drugs are not able to reach these sites.5,6 As a consequence, the central nervous system (CNS) and the macrophages constitute a sanctuary for HIV, from which the periphery can be reinfected and where the drug resistance is induced.7,8 The lack of penetration of antiretroviral © 2014 American Chemical Society

drugs in the HIV sanctuaries is mainly attributed to the expression of active efflux transporters (AET) on the membranes of macrophages9,10 and the cells that constitute the blood−brain (BBB) and blood−cerebrospinal fluid (BCSFB) barriers.11,12 In order to obtain the brain uptake of AZT, the administration of high doses of drug is usually recommended, but their long-term use is impractical, due to hematological intolerance.13 Alternatively, the coadministration of AZT with an inhibitor of AET systems has been suggested in order to increase its uptake in the brain.14 However, this type of inhibitor can induce serious side effects by interfering with the Received: Revised: Accepted: Published: 1550

December 9, 2013 February 20, 2014 April 9, 2014 April 9, 2014 dx.doi.org/10.1021/mp400735c | Mol. Pharmaceutics 2014, 11, 1550−1561

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on the surface.30 Consequently, their constituents are physiologically compatible and biodegradable, providing excellent in vivo tolerability and optimal biocompatibility for the mucosal tissues.30 The obtained SLMs were characterized by evaluating their prodrug loading, morphology, size, and ability to control the UDCA−AZT release. In order to verify the capacity of the SLMs to stabilize the prodrug in physiological environments, rat liver homogenates were employed to compare the hydrolysis rate of UDCA−AZT in the free and encapsulated forms. Finally, in vivo studies were carried out in rats comparing the intravenous infusion of a prodrug solution and the nasal administration of UDCA−AZT loaded SLMs. The AZT and UDCA−AZT levels in blood and CSF were quantified during time after intravenous and nasal administrations. The effect of chitosan in nasal formulations as absorption promoter25 has also been evaluated.

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Figure 1. Chemical formula of zidovidune (AZT), ursodeoxycholic acid (UDCA), and their prodrug UDCA−AZT obtained by ester conjugation.

MATERIALS AND METHODS Materials. The prodrug UDCA−AZT was synthesized as previously described.15 AZT, 7-n-propylxanthine (7n-PX) and bovine serum albumin (BSA) were obtained from SigmaAldrich (Milan, Italy). Methanol, acetonitrile, ethyl acetate, and water were high performance liquid chromatography (HPLC) grade from Merck (Darmstadt, Germany). Chitosan hydrochloride (Protasan UP CL 113) was purchased from FMC BioPolymer AS (Drammen, Norway). Monopowder P insufflators were furnished by Valois Dispray (Mezzovico, Switzerland). Male Wistar rats were purchased from Harlan SRC (Milan, Italy). Tristearin, stearic acid, Tween 20, and Tween 60 were supplied by Fluka Chemie (Bucks, Switzerland). All other reagents and solvents were of analytical grade (Sigma-Aldrich). HPLC Analysis. The quantification of the prodrug UDCA− AZT and its hydrolysis product, AZT, was performed by HPLC. The chromatographic apparatus consisted of a modular system (model LC-10 AD VD pump and model SPD-10A VP variable wavelength UV−vis detector; Shimadzu,Kyoto, Japan) and an injection valve with 20 μL sample loop (model 7725; Rheodyne, IDEX, Torrance, CA, USA). Separations were performed at room temperature on a 5 μm Hypersil BDS C-18 column (150 mm × 4.6 mm i.d.; Alltech Italia Srl, Milan, Italy), equipped with a guard column packed with the same Hypersil material. Data acquisition and processing were accomplished with a personal computer using CLASS-VP Software, version 7.2.1 (Shimadzu Italia, Milan, Italy). The detector was set at 260 nm. The mobile phase consisted of a mixture of water and methanol regulated by a gradient profile programmed as follows: isocratic elution with 20% (v/v) MeOH in H2O for 10 min; then a 1 min linear gradient to 75% (v/v) MeOH in H2O ; the mobile phase composition was finally maintained at 75% MeOH for 10 min. After each cycle the column was conditioned with 20% (v/v) MeOH in H2O for 10 min. The flow rate was 1 mL/min. The xanthine derivative 7n-PX was employed as internal standard for the analysis of rat blood and liver homogenate extracts (see below). The retention times for 7n-PX, AZT, and the prodrug UDCA−AZT were 6.5, 8.4, and 19.6 min, respectively. The HPLC assay of UDCA−AZT alone was performed isocratically with 80% (v/v) MeOH in H2O. In this case the retention time of UDCA−AZT was 4.8 min. The chromatographic precision was evaluated by repeated analysis (n = 6) of the same sample solution containing each of the examined compounds at a concentration of 25 μM. Calibration curves of peak areas versus concentration were

critical physiological role of AET systems in protecting the brain.12 Recently, we have demonstrated that the conjugation of AZT with ursodeoxycholic acid (UDCA, Figure 1), a bile acid known for its antiapoptotic properties, allows one to obtain a prodrug (UDCA−AZT, Figure 1) able to elude the AET systems.15 It has been therefore proposed that this prodrug, when taken up in the CNS, should not be extruded in the bloodstream, being able to elude the AET systems. Thus, nasal administration of this prudrug has been suggested as a potential way to induce its permeation in the CNS. Indeed, this administration route represents an interesting strategy to obtain the brain uptake of neuroactive agents.16,17 In particular, the olfactory region is the only site of the body where the CNS is in contact with the external environment. Consequently, a drug able to deposit on the olfactory mucosa can potentially reach the cerebrospinal fluid (CSF), upon diffusion across the mucosa itself. The drug can then diffuse into the interstitial fluid (ISF), from where it can penetrate the brain parenchyma.18,19 Moreover, the drug can be transported into the brain by trigeminal or olfactory nerves that reach the nasal cavity.19−22 These processes can be accompanied by systemic absorption of the drug across the nasal mucosa of the respiratory region.16,17 Several strategies have been employed to improve brain delivery of drug by intranasal administration, such as addition of penetration enhancers, the use of mucoadhesive materials or the preparation of micro- and nanoparticulate formulations.23,24 Among these, microspheres based on chitosan have been recently investigated for more efficient nasal drug delivery strategies and CNS uptake;25−27 moreover, solid lipid nanoparticles have been proposed for the brain targeting of neuroactive agents following nasal administration.28,29 As reported above, the demonstration that UDCA−AZT allows one to elude the AET systems led to the suggest of its nasal administration as a potential way to induce the prodrug permeation in the CNS. However, the poor water solubility of the free prodrug strongly hampers its permeation from the nose to the brain. Thus, in order to study new strategies potentially able to enhance the brain targeting of UDCA−AZT, the present study focuses on solid lipid microparticles (SLMs) as a carrier system for the nasal administration of the prodrug. SLMs consist of a solid lipid matrix based on naturally occurring lipids and stabilized by a layer of surfactant molecules 1551

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generated in the range 0.2 to 50 μM for AZT and UDCA− AZT. For CSF simulation, standard aliquots of balanced solution (PBS Dulbecco’s without calcium and magnesium) in the presence of 0.45 mg/mL BSA were employed.31,32 In this case, the chromatographic precision was evaluated by repeated analysis (n = 6) of the same sample solution containing 1.5 μM UDCA−AZT whose calibration curve of peak areas versus concentration was generated in the range 0.1 to 10 μM. Preparation of UDCA−AZT Loaded Microparticles. SLMs were prepared by adding hot (75−85 °C) deionized water (25 mL) containing 0.7% (w/w) Tween 60 to the melted lipid phase (1.5 g of tristearin or stearic acid) in which UDCA− AZT (40 mg) has been dispersed. The sample was subjected to high-shear mixing (21,500 rpm for 2 min) with an Ultra-Turrax T25 (IKA-Werk, Staufen, Germany) at 75−85 °C, and the obtained emulsion was rapidly cooled at room temperature, under magnetic stirring. The formed suspension was centrifuged (6,000 rpm for 15 min) to recover the SLMs, which were freeze-dried to give water-free microparticles. Unloaded particles were also prepared with the same procedure, by omitting UDCA−AZT. Particle Size Measurement and Morphological Analysis. The shape and morphology of the microparticulate samples and the raw UDCA−AZT powder were analyzed by environmental scanning electron microscope (ESEM) (XL-40 Philips, Amsterdam, The Netherlands) in low vacuum modality. A few drops of the concentrated reconstituted SLM suspension were placed on an aluminum stub (TAAB Laboratories Equipment, Ltd, Berks, U.K.) using a double side sticky tab (TAAB Laboratories Equipment, Ltd) and, after drying, vacuum coated with gold−palladium in an argon atmosphere for 60 s (Sputter Coater Emitech K550, Emitech LTD, Ashford, Kent, U.K.). The average size of the SLMs was obtained from the ESEM micrographs by measuring at least 500 particles for each sample using the ImageJ 1.46r software (Wayne Rasbond, NIH, USA). To obtain the ζ potential values, the DLS (dynamic light scattering) technique was employed using a Zetasizer Nano ZS (Malvern, Worcs, U.K.). Each freeze-dried sample was diluted in deionized water until the appropriate concentration of particles was achieved to avoid multiscattering events. The data are the results of three independent experiments. UDCA−AZT Content in the SLMs. The UDCA−AZT content in the microparticulate powders was determined by the following method. The microparticles (about 13 mg) were accurately weighed using a high precision analytical balance (d = 0.01 mg; Sartorius, model CP 225D, Goettingen, Germany), and dissolved in methanol at 80 °C for 15 min. The samples were then cooled at room temperature, and the final volume of the solution was adjusted at 5 mL. Then, 10 μL of filtered solutions (0.45 μm) was injected into the HPLC system for UDCA−AZT assay. The drug loading and entrapment efficiency were calculated according to the following equations: ⎛ W⎞ drug loading ⎜% ⎟ ⎝ W⎠ mass of drug in microparticles = × 100 mass of microparticles

(1)

entrapment efficiency (%) mass of drug in microparticles = × 100 starting mass of drug

(2)

All the values obtained are the mean of four independent experiments. In Vitro Dissolution and Release Studies from SLMs. An accurately weighed amount of UDCA−AZT (about 0.3 mg weighed with the analytical balance Sartorius CP 225D) or microparticles containing an equivalent quantity of encapsulated substances were added to 30 mL of a mixture of water and methanol (70:30, v/v). Dissolution and release experiments were performed in the absence and in the presence of Tween 20 (0.5%, w/w) as solubilizing agent.33 In some release experiments, chitosan hydrochloride was added to the release medium (chitosan to microparticles ratio 1:4, w/w), since it was employed as absorption promoter25 for the nasal administration of microparticles (see below). The samples were maintained at 37 °C and stirred mechanically (100 rpm). Aliquots (150 μL) were withdrawn at fixed time intervals, and 10 μL of filtered samples (0.45 μm) was injected into the HPLC system. An equal volume of medium was added after each sampling to maintain sink conditions. All the values obtained were the mean of four independent experiments. Kinetic Analysis in H2O/MeOH. Stock solutions of 10−2 M AZT and UDCA−AZT in DMSO were prepared and stored at −20 °C until their use for kinetic studies. AZT and the prodrug UDCA−AZT were incubated at 37 °C in a mixture of H2O/MeOH (70/30 v/v). The incubation phase (6 mL) was spiked with 18 μL of a 10−2 M stock solution of AZT and/or UDCA−AZT in DMSO resulting in a final concentration of 30 μM. At regular time intervals, 200 μL was withdrawn from the samples and 10 μL aliquots were immediately injected into the HPLC apparatus. All the values were obtained as the mean of four independent experiments. Preparation of Rat Liver Homogenates. The livers of male Wistar rats were immediately isolated after their decapitation, washed with ice cold saline solution, and homogenized in 4 volumes (w/v) of TrisHCl (50 mM, pH 7.4, 4 °C) with a Potter−Elvehjem apparatus (Vetrotecnica, Padova, Italy). The supernatant obtained after centrifugation (2000g for 10 min at 4 °C) was decanted off and stored at −80 °C before its employment for kinetic studies. The total protein concentration in the tissue homogenate was determined using the Lowry procedure34 and resulted as 31.8 ± 1.3 μg of protein/μL. Kinetic Analysis of UDCA−AZT in Rat Liver Homogenates. The free prodrug UDCA−AZT was incubated at 37 °C in 3 mL of rat liver homogenates, resulting in a final concentration of 30 μM, obtained by adding 9 μL of 10−2 M stock solution in DMSO. During the experiment, the samples were shaken continuously and gently in an oscillating water bath. At regular time intervals, 100 μL aliquots of samples were withdrawn and immediately quenched in 200 μL of ethanol (4 °C); 100 μL of internal standard (30 μM 7n-PX) was then added. After centrifugation at 13000g for 10 min, 300 μL aliquots were reduced to dryness under a nitrogen stream and redissolved in 150 μL of water−methanol (70:30 v/v), and, after centrifugation, 10 μL was injected into the HPLC system for AZT and UDCA−AZT assay. The same procedure was adopted for the analysis of samples containing the free prodrug in combination with unloaded SLMs (3.5 mg/mL tristearin based LMs; 1.1 mg/mL stearic acid based SLMs). The only difference was the addition of 50 μL of dichloromethane to the samples before the extraction procedure. All the values were obtained as the mean of three independent incubation experiments. 1552



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collected during the experimental session. Considering that the total CSF volume in adult rats ranges from 300 to 400 μL,36,37 50 μL of CSF should represent about 12% of the original total volume. Four rats were employed for femoral intravenous infusions. CSF samples (10 μL) were immediately injected into HPLC system for UDCA−AZT detection. The blood samples were hemolyzed immediately after their collection with 500 μL of ice cold water, and then 50 μL of 10% sulfosalicylic acid and 100 μL of internal standard (30 μM 7n-PX) were added. The samples were extracted twice with 1 mL of water saturated ethyl acetate, and, after centrifugation, the organic layer was reduced to dryness under a nitrogen stream. Two hundred microliters of a water−methanol mixture (70:30 v/v) was added, and, after centrifugation, 10 μL was injected into the HPLC system for AZT and UDCA−AZT detection. The accuracy of the analytical method was determined by recovery experiments, comparing the peak areas extracted from blood test samples at 4 °C (n = 6) with those obtained by injection of an equivalent concentration of the analytes dissolved in their mobile phase. For all compounds analyzed, the calibration curves were constructed by employing eight different concentrations in whole blood at 4 °C ranging from 0.2 to 50 μM and expressed as peak area ratios of the compounds to the internal standard versus concentration. The in vivo half-life of AZT in the blood was calculated by nonlinear regression (exponential decay) of concentration values in the time range within 3 h after infusion and confirmed by linear regression of the log concentration values versus time. Nasal administration of UDCA−AZT was performed on anesthetized rats laid on their backs, following three procedures. The first one consisted of the introduction of 50 μL of an aqueous suspension of UDCA−AZT (2 mg/mL) in each nostril of rats using a semiautomatic pipet which was attached to a short polyethylene tubing. The tubing was inserted approximately 0.6−0.7 cm into each nostril. After the administration, blood (100 μL) and CSF samples (50 μL) were collected at fixed time points, and they were analyzed with the same procedures described above. Four rats were employed for nasal administration of UDCA−AZT suspension. The second procedure was based on the insufflations of UDCA−AZT-loaded microparticles (stearic acid based) to each nostril of anesthetized rats by single dose Monopowder P insufflators (Valois Dispray SA, Mezzovico, Switzerland). These devices were constituted by a pump, a nasal adapter, and a solid formulation reservoir.38 The insufflators were loaded with about 6 mg of UDCA−AZT-loaded microparticles (corresponding to about 100 μg of UDCA−AZT), and the rats received this amount to each nostril. The amount of powder emitted during administration was obtained by the difference in the insufflator weight before and after each insufflation. After the administration, blood (100 μL) and CSF samples (50 μL) were collected at fixed time points, and they were analyzed with the same procedures described above. Four rats were employed for nasal administration of the microparticulate powder. The third way consisted of the introduction in each rat nostril of 55 μL of an aqueous suspension of stearic acid based microparticles (100 mg/mL) in the presence of chitosan hydrochloride (25 mg/mL), employed as absorption enhancer.25 After the administration, blood (100 μL) and CSF samples (50 μL) were collected at fixed time points, and they were analyzed with the same procedures described above. Four

Stability studies on the microencapsulated UDCA−AZT were performed by spiking the rat liver homogenate (3 mL) at 37 °C with loaded SLMs, resulting in a final concentration of about 30 μM for UDCA−AZT. Because the loaded SLMs were suspended in the medium, it was difficult to withdraw several homogeneous samples, and consequently stability was assessed by a single sampling after 30 min incubation. Dichloromethane (1 mL) was added, and 250 μL was withdrawn from the samples and quenched in 400 μL of ice-cold ethanol added with 200 μL of 30 μM 7n-PX as internal standard. After 10 min of centrifugation at 13000g, 600 μL aliquots were reduced to dryness under a nitrogen stream. The obtained residue was redissolved in 300 μL of the water−methanol mixture (70:30 v/v) and, after centrifugation, 10 μL was injected into the HPLC system for AZT and UDCA−AZT detection. All the values obtained are the mean of four independent experiments. A preliminary analysis performed on blank rat liver homogenate samples showed that its components did not interfere with the AZT, UDCA−AZT, and 7n-PX retention times. The accuracy of the method was determined by recovery experiments, comparing the peak areas of AZT and UDCA− AZT extracted from the rat liver homogenate samples at 4 °C (n = 6) with those obtained by injection of an equivalent concentration of the analytes dissolved in their mobile phase. For all compounds analyzed, the calibration curves were constructed by employing eight different standard solutions in rat liver homogenates at 4 °C, ranging from 0.5 to 50 μM and plotted as analyte to internal standard peak area ratios versus concentration. In Vivo UDCA−AZT Administration and Quantification. Male Wistar rats (200−250 g) anesthetized during the experimental period received a femoral intravenous infusion of 0.1 mg/mL UDCA−AZT dissolved in a medium constituted by 20% (v/v) DMSO and 80% (v/v) physiologic solution, with a rate of 0.2 mL/min for 10 min. At the end of infusion and at fixed time points, blood samples (100 μL) were collected and CSF samples (50 μL) were withdrawn by the cysternal puncture method described by van den Berg et al.35 Briefly, before the experiments the rats were anesthetized and fixed in a stereotaxic apparatus, after shaving of the skin overlying the neck. A needle connected to a syringe by means of polyethylene tubing and filled with sterile filtered water was attached to a holder of the stereotaxic frame. Following the appropriate stereotaxic coordinates,35 the needle was brought into position to carry out the puncture. Before puncturing, an air bubble was drawn into the needle with the syringe at the other end of the collection tubing. For puncturing, the needle was gently moved through the skin and muscles toward the cisterna magna. During the needle placement, the syringe plunger was pulled back to create negative pressure; thereafter, the needle advancement was continued until the air bubble moved into the tubing followed by CSF. Then, the syringe and the tubing were disconnected from the CSF collection system and a Hamilton syringe was attached to the CSF collection tubing and used for CSF withdrawal. After sampling, the Hamilton syringe was disconnected and the tubing was closed with a clamp. Subsequent samples were taken by removing the clamp from the tubing, followed by attachment of the Hamilton syringe. This procedure requires a single needle stick and allows the collection of serial (40−50 μL) CSF samples which are virtually blood-free. A total volume of about 150 μL of CSF was 1553



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Table 1. Size, Physical-Chemical, and Loading Parameters of SLMs Obtained through Hot Emulsion Techniquea sample tristearin unloaded loaded stearic acid unloaded loaded a

mean diameter (μm)

potential ζ (mV)

UDCA−AZT content (%)

encapsulation effic (%)

9.29 ± 5.23 16.66 ± 3.69

−28.0 ± 6.9 −15.7 ± 4.7

0.57 ± 0.06

25.89 ± 2.70

5.87 ± 2.00 6.77 ± 2.67

−51.4 ± 9.3 −44.3 ± 6.6

1.84 ± 0.04

76.34 ± 1.66

Data are reported as the mean ± SD of four independent experiments.

rats were employed for nasal administration of the suspension of microparticles in the presence of chitosan hydrochloride. All in vivo experiments were performed in accordance with the guidelines issued by the Italian Ministry of Health (D.L. 116/92 and D.L. 111/94-B), the Declaration of Helsinki, and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Bethesda, Maryland). The protocol of all the in vivo experiments has been approved by Local Ethics Committee (University of Ferrara, Ferrara, Italy). Any effort has been done to reduce the number of the animals and their suffering. The area under concentration curves of UDCA−AZT in the CSF (AUC, μg mL−1 min) were calculated by the trapezoidal method. All the calculations were performed by using the computer program Graph Pad Prism (GraphPad Software Incorporated, La Jolla, CA, USA).

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RESULTS Characterization of the SLMs: Size, Morphology, ζ Potential, and UDCA−AZT Loading. SLMs loaded with the prodrug UDCA−AZT were developed through a hot emulsion technique using tristearin or stearic acid as lipid material, since they represent commonly used excipients in SLMs,30 and Tween 60 as a pharmaceutically acceptable emulsifier. The mean diameter and ζ potential of both unloaded and loaded SLMs are reported in Table 1, together with the UDCA−AZT loading and encapsulation efficiency values, obtained by HPLC analysis. The mean diameters of the tristearin based microparticles were about 9 and 16 μm for unloaded and loaded SLMs, respectively, whereas in the case of unloaded and loaded stearic acid based microparticles the mean diameters were about 6 and 7 μm, respectively. Figures 2 and 3 report representative ESEM images of the loaded and unloaded microparticles based on tristearin and stearic acid, respectively. ESEM pictures of tristearin based SLMs (Figure 2) showed particles characterized by a very uniformly round morphology and a smooth surface. By contrast, the analysis of the stearic acid based SLMs (Figure 3) revealed a smooth surface with some broken or poorly formed particles, especially for the loaded microparticles. Figure 4 reports the scanning electron micrograph of the raw UDCA− AZT powder. As it can be observed, the prodrug crystals appeared irregular in shape with a size larger than SLMs and in a range from 50 to 300 μm. The ζ potential of the tristearin based microparticles (Table 1) showed negative values, ranging from about −28 mV for unloaed SLMs to −16 mV for particles loaded with UDCA-ZT. The stearic acid based microparticles showed increased negative values with respect to the tristearin based SLMs, ranging from about −51 mV for unloaded SLMs to −44 mV for the prodrug

Figure 2. Environmental scanning electron microscopy (ESEM) micrographs of unloaded (A) and UDCA−AZT loaded (B) SLMs based on tristearin.

loaded particles (Table 1), indicating a probable screening effect of UDCA−AZT on the ζ potential of the SLMs. The loading values were obtained by HPLC analysis, as previously described.15The chromatographic precision for UDCA−AZT dissolved in methanol was represented by the relative standard deviation (RSD) value of 0.96%. The calibration curve of the prodrug was linear over the range of 0.5−50 μM (n = 8, r > 0.998, P < 0.0001). The amount of encapsulated UDCA−AZT in tristearin based microparticles was found to be 0.57 ± 0.06% (w/w), which 1554



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Figure 4. Environmental scanning electron microscopy (ESEM) micrographs of raw UDCA−AZT powder.

UDCA−AZT was not degraded during incubation at 37 °C for 8 h in the H2O/MeOH 70:30 (v/v) dissolution medium. Figure 5 reports the release profiles of UDCA−AZT from the loaded SLMs. The release patterns are compared with the

Figure 3. Environmental scanning electron microscopy (ESEM) micrographs of unloaded (A) and UDCA−AZT loaded (B) SLMs based on stearic acid.

corresponded to an encapsulation efficiency of 25.9 ± 2.7%, whereas the amount of the prodrug encapsulated in stearic acid based microparticles was found to be 1.84 ± 0.04% (w/w), which corresponded to an encapsulation efficiency of 76.3 ± 1.6% (Table 1). In Vitro UDCA−AZT Dissolution and Release from SLMs. The dissolution and release studies of UDCA−AZT were performed in a H2O/MeOH (70:30, v/v) mixture. The employment of methanol as cosolvent was necessary to increase the low solubility of the prodrug in water, thus ensuring sink conditions. In particular, the solubility value of UDCA−AZT in water was 0.0030 ± 0.0001 mg/mL (4.75 ± 0.19 × 10−6 M), whereas in the water−methanol (70:30, v/v) mixture, it increased to 0.058 ± 0.002 mg/mL (9.0 ± 0.3 × 10−5 M).15 Dissolution and release data were obtained by HPLC analysis. The chromatographic precision for UDCA−AZT dissolved in a H2O/MeOH mixture 70:30 (v/v) was represented by the relative standard deviation (RSD) value of 0.95%. The calibration curve of the prodrug was linear over the range of 0.5−50 μM (n = 8, r > 0.996, P < 0.0001). The prodrug

Figure 5. (A) In vitro release of UDCA−AZT from SLMs based on tristearin or stearic acid. The release profiles are compared with those of the raw UDCA−AZT powder dissolution during time. (B) Dissolution and release profiles in the presence of 0.5% Tween 20, chosen as solubilizing agent, or chitosan hydrochloride (chitosanmicroparticles 1:4 w/w), employed as absorption promoter for nasal administration of microparticles. Results are the mean of four independent experiments.

dissolution of the raw powder of the prodrug. All dissolution and release data were obtained in the absence (Figure 5A) and in the presence (Figure 5B) of 0.5% Tween 20, chosen as solubilizing agent,33 or chitosan hydrochloride (chitosanmicroparticles 1:4 w/w), which was employed as absorption 1555



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promoter25 for nasal administration of the UDCA−AZT microparticles (see below). As illustrated in Figure 5A, the dissolution rate of UDCA− AZT in the absence of Tween 20 appeared to be very low. Indeed, after 5 h of incubation less than the 25% of the total raw powder amount was solubilized. Under the same conditions, the release rate of UDCA−AZT from the SLMs was significantly higher than its dissolution rate and distinct variations in UDCA−AZT release were observed between the microparticles based on the different lipids (Figure 5A). In particular, the tristearin based sample showed a release pattern characterized by a burst effect of about 40% of the incorporated UDCA−AZT, followed by a relatively slow release, with about 50% of encapsulated prodrug released within 5 h. It is interesting to observe that the stearic acid based SLMs, despite the relatively high encapsulation efficiency, showed an UDCA− AZT release pattern characterized by a burst effect of about 80%, followed by a relatively fast release that was completed within 1 h. These data indicate poor modulation of UDCA− AZT release by the stearic based SLMs, which, on the other hand, achieved a marked enhancement of the prodrug dissolution rate (Figure 5A). In order to study the potential influence of the surfactants on the dissolution and release of UDCA−AZT, release experiments were also performed in the presence of 0.5% Tween 20, chosen as solubilizing agent. It is interesting to observe that the addition of this surfactant to the incubation medium produced a marked increase of the dissolution rate of the prodrug, which was completely dissolved within 1.5 h (Figure 5B). However, the presence of Tween 20 did not induce comparable changes on the release rate of UDCA−AZT from the SLMs. In particular, no significant variations were observed for the stearic acid based microparticle samples. Indeed, also in the presence of the solubilizing agent, the release of UDCA− AZT from stearic acid SLMs was faster than the dissolution of the nonencapsulated prodrug (Figure 5B). Moreover, the addition of Tween 20 to the release medium did not influence the initial portion of the release curves from the tristearin based microparticles; however the amount of prodrug released within the monitored time period (5 h) increased from about 50% to 74% (Figure 5B). It is important to underline that, in the presence of Tween 20, the release rate of UDCA−AZT from the tristearin based SLMs was lower than the dissolution rate of the free prodrug. Therefore, in the presence of this surfactant, the tristearin based microparticles were able to control the release of UDCA−AZT. Finally, the data reported in Figure 5B indicate that the presence of chitosan hydrochloride in the release medium did not induce significant changes on the release profiles of UDCA−AZT from the SLMs. Hydrolysis Studies of Free and Encapsulated UDCA− AZT in Rat Liver Homogenates. In order to evaluate the ability of the SLMs to protect and stabilize the prodrug in physiologic environments, we have compared the hydrolysis rate of free and encapsulated UDCA−AZT in rat liver homogenates. Rat liver homogenates were selected for the study, because in this biological medium the hydrolysis rate of the prodrug is relatively fast, showing a half-life of 2.70 ± 0.14 min, with the process completed within 30 min.15 The hydrolysis has been evaluated via HPLC analysis of extracted samples. The average recovery ± SD of UDCA−AZT and AZT from rat liver homogenates was 83.2 ± 2.9% and 85.5% ± 3.4% respectively. The concentrations of these compounds were

referred to as peak area ratio with respect to their internal standard 7n-PX. The precision of the method based on peak area ratio was represented by RSD values ranging between 1.1% and 1.3%. The calibration curves referred to AZT and its prodrug UDCA−AZT incubated in rat liver homogenates were linear over the range 0.5−50 μM (n = 8, r > 0.989, P < 0.0001). First, we have verified that the presence of unloaded SLMs in rat liver homogenates did not significantly influence the hydrolysis rate of UDCA−AZT. In particular, the half-lives related to the prodrug hydrolysis were 2.63 ± 0.12, 2.77 ± 0.13, and 2.73 ± 0.14 min in the absence and in the presence of unloaded tristearin or stearic acid based SLMs, respectively in rat liver homogenates. The amounts of AZT and UDCA−AZT detected after 30 min of incubation of free or encapsulated prodrug in rat liver homogenates are reported in Figure 6. All the values are

Figure 6. Degradation in rat liver homogenates of free or encapsulated UDCA−AZT in SLMs. All UDCA−AZT and AZT values are reported as the percentage of the overall amount of incubated prodrug. Data are reported as the mean ± SD of four independent experiments.

reported as percentage of the overall amount of incubated prodrug. It can be observed that after the monitored time period the free prodrug appeared totally hydrolyzed; in fact only AZT was present and no UDCA−AZT was detected. On the other hand, following 30 min incubation of the tristearin based SLMs in rat liver homogenates, 75% of the prodrug was still present. Indeed, much lower amounts of UDCA−AZT were hydrolyzed with respect to the free prodrug, as confirmed by the release of only 28% of AZT. The stearic acid based SLMs also contributed to inhibit the prodrug complete hydrolysis, even though their efficacy appeared much lower than that of the microparticles based on tristearin. In particular, the amount of unchanged UDCA−AZT was about 14%, with a corresponding release of AZT of about 80%. These results indicate that the ability of the SLMs to stabilize the prodrug in rat liver homogenates is related to their capacity to control the release of encapsulated UDCA−AZT (Figure 5). In Vivo UDCA−AZT Administration. Taking into account that the SLMs based on stearic acid were characterized not only by a satisfactory encapsulation efficiency but also by their ability to induce a fast dissolution of UDCA−AZT in physiological environments, we have tested these microparticles for nasal administration of the prodrug, in order to verify its potential uptake in the central nervous system. The nasal administration of UDCA−AZT was performed with the employment of three 1556



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No AZT or UDCA−AZT amounts were detected in the CSF within 180 min after intravenous administration of UDCA− AZT. Nasal Administration of UDCA−AZT. The nasal administration of pure UDCA−AZT as powder was not performed, owing to the very low dose required (about 200 μg in rats). Therefore, as control, we employed a water suspension of the raw drug. No significant amounts of AZT or UDCA−AZT were observed in blood or in CSF, respectively, within 180 min after the administration (data not shown). On the contrary, the nasal administration of the powder constituted by the loaded SLMs based on stearic acid (6 mg, about 100 μg of UDCA−AZT for each nostril) produced detectable amounts of UDCA−AZT in the CSF of the rats, as reported in Figure 8. In particular, the UDCA−AZT

different formulations: (i) a suspension in water of the raw powder, (ii) the microparticulate powder of loaded SLMs based on stearic acid, and (iii) a water suspension of the loaded microparticles in the presence of chitosan hydrochloride, chosen as absorption promoter. After administration, blood and CSF samples were withdrawn in order to analyze via HPLC their AZT or UDCA−AZT concentrations during time. The results have been compared with those obtained by the intravenous infusion of UDCA−AZT to rats. The chromatographic precision for AZT and UDCA−AZT dissolved in a balanced solution in the presence of 0.45 mg/mL BSA (simulating the CSF) was represented by the RSD value ranging from 0.94 and 0.95%. The calibration curves of AZT and its prodrug were linear over the range of 0.1−10 μM, corresponding to 0.027−2.67 μg/mL for AZT and 0.064−6.42 μg/mL for UDCA−AZT (n = 8, r > 0.992, P < 0.0001). The average recovery ± SD of AZT from rat blood was 70.4 ± 3.8%. The concentrations of this compound were therefore referred to as peak area ratio with respect to the internal standard 7n-PX. The precision of the method based on peak area ratio was represented by RSD values of 1.2% . The calibration curves referred to AZT and its prodrug incubated in rat blood was linear over the range 0.2−20 μM corresponding to 0.053 to 5.34 μg/mL (n = 8, r = 0.991, P < 0.0001). Intravenous Administration of UDCA−AZT. The analysis of rat blood samples following the intravenous infusion of UDCA−AZT indicated that the prodrug was undetectable. This result is in agreement with the very fast hydrolysis of UDCA−AZT in rat blood at 37 °C.15As a result of this fast hydrolysis, important amounts of AZT were detected following the intravenous administration of the prodrug, as evidenced in Figure 7. In particular, after infusion of 0.200 mg of UDCA−

Figure 8. UDCA−AZT concentrations (μg/mL) detected in the CSF after nasal administration of loaded SLMs based on stearic acid as powder itself or in suspension in the presence of chitosan hydrochloride (25 mg/mL). Each dose contained 200 μg of UDCA−AZT. Data are expressed as the mean ± SD of four independent experiments.

concentrations in the CSF were 0.236 ± 0.034 μg/mL and 0.394 ± 0.022 μg/mL at 30 and 60 min after nasal administration, respectively. The UDCA−AZT concentration then decreased, and after 90 min the prodrug was not detectable. No AZT amounts were detected in the bloodstream and in CSF within 180 min after nasal administration of the powder constituted by the loaded SLMs based on stearic acid. The same amount of loaded SLMs based on stearic acid was nasally administered to rats as suspension in water (100 mg/ mL) in the presence of chitosan hydrochloride (25 mg/mL), chosen as absorption enhancer.25 It is interesting to observe in Figure 8 that this formulation induced a delay of UDCA−AZT appearance in the CSF with respect to the administration of the powder itself, but higher amounts were detected within 120 min after administration. In particular, at 60, 90, and 120 min following the administration of the suspension the concentrations of UDCA−AZT detected in the CSF were 0.90 ± 0.08 μg/mL, 1.35 ± 0.11 μg/mL, and 1.45 ± 0.09 μg/mL, respectively. The UDCA−AZT then decreased to 0.090 ± 0.004 μg/mL at 150 min. No AZT amounts were detected in the bloodstream and in CSF within 180 min after the nasal administration of the SLMs suspension in the presence of chitosan hydrochloride. The area under concentration curve values obtained for UDCA−AZT in the CSF, following the nasal administration of the SLM powder and SLM suspension in the presence of chitosan (Figure 8), were 19.05 ± 1.75 μg mL−1 min (AUC

Figure 7. Elimination profile of AZT after 0.200 mg infusion of UDCA−AZT to rats. Data are expressed as the mean ± SD of four independent experiments. The elimination followed an apparent first order kinetic, confirmed by the semilogarithmic plot reported in the inset (n = 8, r = 0.997, P < 0.0001). The half-life of AZT was calculated to be 57.2 ± 3.4 min.

AZT to rats, the AZT concentration in the bloodstream was 4.10 ± 0.32 μg/mL and decreased during time with an apparent first order kinetic confirmed by the linearity of the semilogarithmic plot reported in the inset of Figure 7 (n = 8, r = 0.997, P < 0.0001), and a half-life of 57.2 ± 3.4 min. 1557



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powder) and 112.5 ± 3.53 μg mL−1 min (AUC suspension), respectively. The ratio between AUC suspension and AUC powder was 5.9, indicating that the nasal formulation constituted by SLM suspension in the presence of chitosan allowed an uptake of UDCA−AZT in the CSF about six times higher than that obtained by the administration of the powder itself.

(approximately = 0.11%) of the drug. The properties of the SLMs appeared strongly different on the basis of the lipids chosen for their formulation. Indeed, tristearin SLMs appeared as spheres with smooth surface, showing an encapsulation efficiency of about 25% and the ability to control the release of the prodrug; on the other hand, stearic acid microparticles appeared fractured and irregularly shaped, showing an encapsulation efficiency of about 75% and poor modulation of the prodrug release. The release rate of UDCA−AZT from both tristearin and stearic acid based SLMs was higher than the dissolution rate of raw UDCA−AZT. The reduced size of the SLMs as compared to the raw UDCA−AZT would contribute to the observed higher release rate due to the increase in specific surface area. The observed effect could also be traced to improved wetting and/or solubilization of the prodrug by the SLM formulation,48,49 due to the presence of the surfactant used as emulsifier agent for the preparation of the SLMs. Indeed, in the presence of Tween 20 the dissolution rate of the prodrug increased sensibly and became higher than the release rate of UDCA−AZT from tristearin based microparticles, which, therefore, appeared able to control the release of the prodrug. Surfactants like Tween are used in the dissolution media to give a better correlation with in vivo conditions.50 Interestingly, in the presence of Tween 20, the release rate of UDCA−AZT from stearic acid based microparticles was higher than the dissolution rate of the prodrug, evidencing this type of SLMs to sensibly promote the UDCA−AZT dissolution rate. The large quantity of rapidly released prodrug indicates that the higher fraction of UDCA−AZT was present on the surface of the SLMs, rather than entrapped in the lipid particle matrix. On the other hand, the ability of the tristearin based microparticles to control the release of the prodrug evidenced its “core” distribution.51 The observed differences in the release patterns of the SLMs could be utilized to deliver the drug at different rates, producing both rapid and prolonged therapeutic effects. The stability studies of free and encapsulated UDCA−AZT in rat liver homogenates evidenced the ability of tristearin based microparticles to enhance the stability of the prodrug, whereas this effect was poorly shown by stearic acid based microparticles, in accordance with the results obtained by release studies. Because of the high encapsulation efficiency of stearic acid based SLMs and their ability to strongly promote the dissolution rate of UDCA−AZT, they were selected as solid formulation for nasal administration of the prodrug, and their potential ability to promote its brain uptake was evaluated. Following insufflation in the rat nostrils of UDCA−AZT SLMs, the AZT and prodrug concentrations in the bloodstream and CSF were measured and compared with those attained by nasal and intravenous administration of nonencapsulated UDCA−AZT. In particular, raw UDCA−AZT was nasally administered as water suspension, because the chosen dose (200 μg) was too low to be insufflated. Intravenous prodrug administration evidenced the extremely rapid UDCA−AZT hydrolysis in rat blood, in accordance with our previous in vitro data.15 Indeed, only AZT amounts were detected, showing concentration values decreasing during time according to an apparent first order kinetic, with a half-life of about 1 h. No AZT and UDCA−AZT amounts were detected in the CSF of rats after intravenous administration, confirming the inability of this drug to permeate in the CNS. On the other hand, it has been reported that, following intravenous administration to rats of higher doses of AZT, the drug was

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DISCUSSION The penetration of antiretroviral drugs in the CNS represents currently a strategic challenge in order to eradicate the HIV reservoirs in the body, the brain being one of the most important sanctuaries of the virus.7,8 HIV needs to be suppressed not only in the brain tissue but also in the CSF and connected spaces, in particular the subarachnoid ones. These spaces contain indeed macrophages that harbor the virus during the early stages of infection constituting, therefore, the only sites of HIV replication in the brain. Drugs able to penetrate in the ventricular CSF can have access to subarachnoid spaces,39 where the action of antiretroviral agents should be of fundamental importance against the HIV stages of CNS infection.40 Unfortunately, the antiretroviral agents are substrates of the AET systems that hamper their penetration in the CNS from the bloodstream, where they are rejected in case of brain uptake.12 Taking into account these aspects, it appears important not only to target antiretroviral agents in the CSF but also to induce their ability to elude the AET systems, allowing therefore their brain permanence. Very recently we have demonstrated that the conjugation of AZT with a bile acid (UDCA) allows one to obtain a prodrug (UDCA−AZT) able to elude the AET system.15 This prodrug appears therefore a good candidate for nasal administration in order to obtain its uptake and permanence in the CNS. Indeed, the prodrug deposition on olfactory mucosa may induce its delivery in the CSF upon diffusion across the mucosa itself;18,19 moreover, when taken up in the CSF, UDCA−AZT should not be extruded in the bloodstream, being able to elude the AET systems and acting, therefore, as a potential carrier for AZT in the CNS. The poor water solubility of UDCA−AZT15 requires formulations able to enhance the prodrug absorption from the nasal route. As a consequence, in order to verify the possibility to target UDCA−AZT in the CSF, we have prepared new solid nasal formulations based on solid lipid microparticles (SLMs) that provide optimal biocompatibility for the mucosal tissues30 being constituted by naturally occurring lipids. Tristearin and stearic acid were selected as lipid materials for the preparation of UDCA−AZT loaded LMs, since they have been reported to be more suitable than other lipid excipients (e.g., mono- and diglycerides) for the entrapment of very lipophilic drugs, such as the studied prodrug.41 Moreover, the micrometric sizes offer the advantage to induce the deposition of particles on nasal mucosa after administration of solid formulations25,26 and, differently from nanoparticulate systems, hamper their inhalation42 and absorption in the bloodstream or in the CNS, avoiding the induction of unwanted effects by accumulation in vital organs.43,44 The SLMs based on tristearin and stearic acid were obtained in the absence of organic solvents, an advantage that contributes to confer on them high biocompatibility.45,46 Even if zidovudine has been classified as a heat-sensitive drug, its degradation kinetic parameters47 allow us to demonstrate that the incubation at 80 °C for 10 min (the maximum time requested for SLM formulation) induces negligible degradation 1558



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detected in CSF,14 a phenomenon probably due to the saturation of AET systems. Indeed, the AZT amounts detected in CSF increased when AZT was intravenously coadministered with probenecid, an inhibitor of AET systems.14 It is however important to remark that this type of administration, even if efficacious in inducing the AZT penetration in the brain, is known to cause both hematological intolerance and toxicity in the brain, due to the high AZT doses13 and the activity of AET inhibitors,12 respectively. As reported in this study, the nasal administration to rats of raw UDCA−AZT did not produced any detectable levels of both UDCA−AZT and AZT in the bloodstream and CSF. This is probably due to very poor water solubility of the prodrug.15 On the other hand, the nasal administration of stearic acid based SLMs induced the uptake of the prodrug in the CSF, up to a concentration of 0.4 μg/mL within 90 min after administration. These results indicate the ability of the stearic acid based SLMs to induce the delivery in nasal mucosa of prodrug amounts ready to be absorbed in the CSF, probably by promoting the UDCA−AZT dissolution. The ζ potential of the formulated SLMs was negative, with the absolute higher values (about −44 mV) attained to stearic acid microparticles. Unfortunately, the mucosal membranes are characterized by the presence of negatively charged species that interfere by electrostatic repulsions with adhesion for the stearic acid based SLMs. It is known that chitosan shows good mucoadhesive properties25 due to the positive charges of its amino groups.52 Chitosan is also known to transiently open the tight junctions in the epithelial membranes,53,54 and for this reason it has been employed as absorption promoter in nasal formulations25,55 Accordingly, an optimized nasal formulation was obtained by suspending the stearic acid based SLMs in a water dispersion of chitosan. This formulation, after nasal administration to rats, produced a remarkable increase in UDCA−AZT concentrations detected in the CSF, up to 1.5 μg/mL within 150 min after administration. Therefore, the presence of chitosan induced a significantly higher UDCA−AZT uptake in the CSF (the relative bioavailability was about six times greater than that achieved by the solid formulation based on the stearic acid SLMs alone). The presence of chitosan in the nasal cavity induced also a delayed and prolonged uptake of UDCA−AZT in CSF with respect to the solid formulation (Figure 8). Interestingly, no AZT amounts were detected either in CSF or in the bloodstream, after nasal administration of the microparticulate formulations. This result emphasizes the ability of stearic acid based SLMs to promote a selective uptake of UDCA−AZT in CSF, demonstrating the existence of a direct nose−CNS pathway for this prodrug. Upon uptake, UDCA−AZT appeared to prolong its permanence in the CSF, in accordance with its ability to elude the AET systems.

istration in solubilized form or as raw powder to obtain its permeation across nasal mucosa. On the contrary, in the present study we have demonstrated that the encapsulation of UDCA−AZT in stearic acid SLMs induced a significant increase of the UDCA−AZT dissolution rate in aqueous solvent. Therefore, we have considered this formulation as eligible for nasal administration of the prodrug. By an in vivo study in rats, we have then demonstrated that the loaded SLMs were effectively able to induce the permeation of the prodrug from nose to CSF, where UDCA−AZT has been detected at concentration values ranging from 0.2 to 1.4 μg/mL ranging from 30 to 120 min. Importantly, the observed absence of UDCA−AZT and AZT in the bloodstream, following the nasal administration of the loaded SLMs, suggests the potential ability of this formulation to allow drug central effects, without peripheral undesired phenomena.

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AUTHOR INFORMATION

Corresponding Author

*Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara 19, I-44121 Ferrara, Italy. Phone: +39-0532-455274. Fax: +39-0532455953. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN 2012). The authors thank Andrea Margutti (Department of Life Sciences and Biotechnology, University of Ferrara, Italy) for technical assistance. Special thanks should be given to Roberta Mazzorana Reolon (ECO.RA.V. S.p.A. Longarone, Belluno Italy) for her significant support on this project.

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REFERENCES

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CONCLUSIONS Taking into account that UDCA−AZT is able to elude the AET systems,15 we have hypothesized that this prodrug should be retained in the CNS, differently from AZT, which is known to be extruded in the bloodstream.56 Thus, it became necessary to develop an UDCA−AZT formulation able to induce the uptake of the prodrug in the CNS. In this regard, we proposed the nasal administration, this route being known to induce the uptake in the brain of the substances nasally administered. However, the very poor solubility and dissolution rate of UDCA−AZT in aqueous solvent did not allow its admin1559



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Brain Uptake of a Zidovudine Prodrug after Nasal Administration of

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