Liquid chromatography method for quantifying N-(4-hydroxyphenyl)retinamide and N-(4-methoxyphenyl)retinamide in tissues

A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H(2)O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.     

4-oxo-N-(4-hydroxyphenyl)retinamide: two independent ways to kill cancer cells

Background

The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a polar metabolite of fenretinide (4-HPR) very effective in killing cancer cells of different histotypes, able to inhibit 4-HPR-resistant cell growth and to act synergistically in combination with the parent drug. Unlike 4-HPR and other retinoids, 4-oxo-4-HPR inhibits tubulin polymerization, leading to multipolar spindle formation and mitotic arrest. Here we investigated whether 4-oxo-4-HPR, like 4-HPR, triggered cell death also via reactive oxygen species (ROS) generation and whether its antimicrotubule activity was related to a ROS-dependent mechanism in ovarian (A2780), breast (T47D), cervical (HeLa) and neuroblastoma (SK-N-BE) cancer cell lines.

Methodology/Principal Findings

We provided evidence that 4-oxo-4-HPR, besides acting as an antimicrotubule agent, induced apoptosis through a signaling cascade starting from ROS generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and upregulation of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Through time-course analysis and inhibition of the ROS-related signaling pathway (upstream by vitamin C and downstream by PLAB silencing), we demonstrated that the antimitotic activity of 4-oxo-4-HPR was independent from the oxidative stress induced by the retinoid. In fact, ROS generation occurred earlier than mitotic arrest (within 30 minutes and 2 hours, respectively) and abrogation of the ROS-related signaling pathway did not prevent the 4-oxo-4-HPR-induced mitotic arrest.

Conclusions/Significance

These data indicate that 4-oxo-4-HPR anticancer activity is due to at least two independent mechanisms and provide an explanation of the ability of 4-oxo-4-HPR to be more potent than the parent drug and to be effective also in 4-HPR-resistant cell lines. In addition, the double mechanism of action could allow 4-oxo-4-HPR to efficiently target tumour and to eventually counteract the development of drug resistance.     

Identification of dihydroceramide desaturase as a direct in vitro target for fenretinide

The dihydroceramide desaturase (DES) enzyme is responsible for inserting the 4,5-trans-double bond to the sphingolipid backbone of dihydroceramide. We previously demonstrated that fenretinide (4-HPR) inhibited DES activity in SMS-KCNR neuroblastoma cells. In this study, we investigated whether 4-HPR acted directly on the enzyme in vitro. N-C8:0-d-erythro-dihydroceramide (C(8)-dhCer) was used as a substrate to study the conversion of dihydroceramide into ceramide in vitro using rat liver microsomes, and the formation of tritiated water after the addition of the tritiated substrate was detected and used to measure DES activity. NADH served as a cofactor. The apparent K(m) for C(8)-dhCer and NADH were 1.92 ± 0.36 μm and 43.4 ± 6.47 μm, respectively; and the V(max) was 3.16 ± 0.24 and 4.11 ± 0.18 nmol/min/g protein. Next, the effects of 4-HPR and its metabolites on DES activity were investigated. 4-HPR was found to inhibit DES in a dose-dependent manner. At 20 min, the inhibition was competitive; however, longer incubation times demonstrated the inhibition to be irreversible. Among the major metabolites of 4-HPR, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) showed the highest inhibitory effect with substrate concentration of 0.5 μm, with an IC(50) of 1.68 μm as compared with an IC(50) of 2.32 μm for 4-HPR. N-(4-Methoxyphenyl)retinamide (4-MPR) and 4-Oxo-N-(4-methoxyphenyl)retinamide (4-oxo-4-MPR) had minimal effects on DES activity. A known competitive inhibitor of DES, C(8)-cyclopropenylceramide was used as a positive control. These studies define for the first time a direct in vitro target for 4-HPR and suggest that inhibitors of DES may be used as therapeutic interventions to regulate ceramide desaturation and consequent function.     

Hydrolysis of 4-HPR to atRA occurs in vivo but is not required for retinamide-induced apoptosis

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.     

Boc-lysinated-betulonic acid: a potent, anti-prostate cancer agent

Betulonic acid, derived from betulinol, a pentacyclic styrene, has shown a highly specific anti-prostate cancer activity in in vitro cell cultures. However, due to the lack of solubility of betulonic acid in aqueous medium, its potent anti-cancer activity in vivo has not been determined to the fullest extent. The present study describes the chemical synthesis of hydrophilic Boc-lysinated-betulonic acid, which has improved its solubility in an aqueous biocompatible solvent. Evaluation in cytotoxicity assays, Boc-lysinated-betulonic acid dissolved in phosphate-buffered saline (PBS) containing 22% ethanol and 4% human serum albumin, has shown 95.7% inhibition of LNCaP prostate cancer cells in culture after 72 h incubation at a concentration of 100 microM, but with little effect on normally proliferating fibroblast cells. In the in vivo assay, male athymic mice transplanted with human prostate LNCaP xenografts were injected with Boc-lysinated-betulonic acid intraperitoneally at a dose of 30 mg/kg daily for 17 days. The treated mice exhibited 92% inhibition of tumor growth as compared to controls. Histological sections of the tumors showed that Boc-lysinated-betulonic acid arrested mitosis and induced apoptosis, which was confirmed by TUNEL assay, Yo-Pro-1 staining, and the release of cleaved caspase-3 from the ex vivo in tumor culture. These studies, for the first time, demonstrate that a non-toxic hydrophilic lysinated derivative of betulonic acid and its solubility in a biocompatible aqueous medium has enhanced the bioavailability of the drug and has thus unleashed its full anti-prostate cancer activity.     

Synthesis and in vitro/in vivo evaluation of novel oral N-alkyl- and N,N-dialkyl-carbamate esters of entacapone

Entacapone has a relatively low oral bioavailability which may, in part, be due to its low aqueous solubility at low pH and/or its hydrophilic character at neutral pH. Various novel N-alkyl and N,N-dialkyl carbamate esters of entacapone were synthesized as possible prodrugs of entacapone in order to increase its aqueous solubility at an acidic pH and to increase its lipophilicity at neutral pH. Oral bioavailability of entacapone and selected carbamate esters were investigated in rats. Both N-alkyl and N,N-dialkyl carbamate esters were relatively stable against chemical hydrolysis at pH 7.4 (t1/2 = 14.9-20.7 h), but hydrolyzed rapidly (t1/2 = 0.8-2.7 h) in human serum. However, in contrast to N-alkyl carbamates, N,N-dialkyl carbamates did not release entacapone in in vitro enzymatic hydrolysis (human serum) studies. N-Alkyl carbamates, 2a-c, showed increased aqueous solubility at pH 7.4, of which 2a and 2c also show increased aqueous solubility at pH 5.0, compared to entacapone. In addition to increased aqueous solubility, 2c showed increased lipophilicity at pH 7.4. However, two N-alkyl carbamates of entacapone did not increase the oral bioavailability of the parent drug in rats. Thus, it can be concluded that the relatively low lipophilicity of entacapone is not the cause of its low bioavailability.     

Chitosan-based delivery systems for curcumin: A review of pharmacodynamic and pharmacokinetic aspects

Effective drug delivery is one of the most important issues associated with the administration of therapeutic agents that have low oral bioavailability. Curcumin is an active ingredient in the turmeric plant, which has low oral bioavailability due to its poor aqueous solubility. One strategy that has been considered for enhancing the aqueous solubility, and, thus, its oral bioavailability, is the use of chitosan as a carrier for curcumin. Chitosan is a biodegradable and biocompatible polymer that is relatively water-soluble. Therefore, various studies have sought to improve the aqueous solubility of chitosan. The use of different pharmaceutical excipients and formulation strategies has the potential to improve aqueous solubility, formulation processing, and the overall delivery of hydrophobic drugs. This review focuses on various methods utilized for chitosan-based delivery of curcumin.     

Oral formulation of a novel antiviral agent,PG301029, in a mixture of Gelucire 44/14 and DMA (2∶1, wt/wt)

To develop an oral formulation for PG301029, a novel potent agent for the treatment of Hepatitis C virus infection, that not only has very low aqueous solubility but also degrades rapidly in water. The solubility of PG301029 was determined in water, various aqueous media, and several neat organic solvents. The stability of PG301029 was monitored at room temperature in buffess for 4 days, and in several neat organic solvents for up to 8 mo. Drug concentrations were measured by high-performance liquid chromatography (HPLC). Based on solubility and stability data, Gelucire 44/14 and DMA (N,N-dimethylacetamide) at a weight ratio of 2 to 1 were chosen as the formulation vehicle. After the vehicle was prepared, it was maintained in liquid form at ∼40°C until the PG301029 was dissolved. The final formulation product was a semisolid at room temperature. The bioavailability of the formulation was tested on 4 female BALB/c mice. PG301029 is insoluble in all tested aqueous media, while its solubility is promising in DMA. This compound is unstable in aqueous media and some organic solvents; however, it is stable in DMA. This proposed formulation is able to hold up to 10 mg/mL of drug and is stable at 4°C. The shelf life for this formulation stored at 4°C is extrapolated to be greater than 4 years. This formulation dramatically increases the bioavailability of PG301029. This nonaqueous formulation solves the stability, solubility, and bioavailability problems for PG301029. This semisolid formulation can easily be incorporated into soft elastic capsules.     

Characterization and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of the Complexes of Posaconazole with β- and 2,6-di-<Emphasis Type="Italic">O</Emphasis>-methyl-β-cyclodextrin

Posaconazole is a triazole antifungal drug that with extremely poor aqueous solubility. Up to now, this drug can be administered via intravenous injection and oral suspension. However, its oral bioavailability is greatly limited by the dissolution rate of the drug. This study aimed to improve water solubility and dissolution of posaconazole through characterizing the inclusion complexes of posaconazole with β-cyclodextrin (β-CD) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD). Phase solubility studies were performed to calculate the stability constants in solution. The results of FT-IR, PXRD, ¹H and ROESY 2D NMR, and DSC all verified the formation of the complexes in solid state. The complexes showed remarkably improved water solubility and dissolution rate than pure posaconazole. Especially, the aqueous solubility of the DM-β-CD complex is nine times higher than that of the β-CD complex. Preliminary in vitro antifungal susceptibility tests showed that the two inclusion complexes maintained high antifungal activities. These results indicated that the DM-β-CD complexes have great potential for application in the delivery of poorly water-soluble antifungal agents, such as posaconazole.     

Albumin-insulin conjugate releasing insulin slowly under physiological conditions: a new concept for long-acting insulin

The covalent linkage of peptides or protein drugs to human serum albumin (HSA) greatly prolongs their lifetime in vivo but is pharmacologically irrelevant when it irreversibly inactivates them. We retain drug bioactivity by synthesizing a heterobifunctional reagent (MAL-Fmoc-OSu, 9-hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide) that generates HSA-Fmoc-insulin on covalent conjugation to the amino group of insulin and the Cys-34 side chain of HSA. HSA-Fmoc-insulin is water-soluble and, upon incubation in aqueous buffers reflecting normal human serum conditions, slowly, spontaneously, and homogeneously hydrolyzes to release unmodified insulin with a t 1/2 of 25 +/- 2 h. A single subcutaneous or intraperitoneal administration of HSA-Fmoc-insulin to diabetic rodents lowers circulating glucose levels for about 4 times longer than an equipotent dose of Zn2+-free insulin. Following subcutaneous administration, onset of the glucose-lowering effect is delayed 0.5-1 h and persists for 12 h. Thus, we present a prototype insulin formulation possessing three desirable parameters: high aqueous solubility, delayed action following subcutaneous administration, and prolonged therapeutic effect.     

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.     

Use of Polyvinyl Alcohol as a Solubility-Enhancing Polymer for Poorly Water Soluble Drug Delivery (Part 1)

Polyvinyl alcohol (PVAL) has not been investigated in a binary formulation as a concentration-enhancing polymer owing to its high melting point/high viscosity and poor organic solubility. Due to the unique attributes of the KinetiSol® dispersing (KSD) technology, PVAL has been enabled for this application and it is the aim of this paper to investigate various grades for improvement of the solubility and bioavailability of poorly water soluble active pharmaceutical ingredients. Solid amorphous dispersions were created with the model drug, itraconazole (ITZ), at a selected drug loading of 20%. Polymer grades were chosen with variation in molecular weight and degree of hydroxylation to determine the effects on performance. Differential scanning calorimetry, powder X-ray diffraction, polarized light microscopy, size exclusion chromatography, and dissolution testing were used to characterize the amorphous dispersions. An in vivo pharmacokinetic study in rats was also conducted to compare the selected formulation to current market formulations of ITZ. The 4-88 grade of PVAL was determined to be effective at enhancing solubility and bioavailability of itraconazole.     

The biopharmaceutical classification system-experimental model of prediction of drug bioavailability

The Biopharmaceutical Classification System (BCS) is based on solubility tests, correlating for certain drugs with their bioavailability in human body. It is widely used in design and development of innovation drugs, new dosage forms (permeability amplifiers), in clinical pharmacology (drug-drug, drug-food interaction) and also by regulation agencies of several countries as the scientific approach, for testing of waiver on bioavailability. The review considers modern concepts and theoretical bases for prediction of bioavailability according to BCS. It gives characteristics of fundamental parameters of the system: absorption number, solubility number and ratio of dose to the soluble part of the drug. Possible versions of BCS modification for its subsequent optimization are discussed.     

Pharmacokinetics of saquinavir after intravenous and oral dosing of saquinavir: hydroxybutenyl-beta-cyclodextrin formulations

The current research evaluated the ability of hydroxybutenyl-beta-cyclodextrin (HBenBCD) to enhance saquinavir in vitro solubility and in vivo oral bioavailability; both the base and mesylate salt forms of saquinavir were investigated. HBenBCD was effective and significantly improved saquinavir solubility in aqueous media. In the presence of 10 wt % HBenBCD, saquinavir base solubility in water was increased to ca. 5.5 +/- 0.4 mg/mL and represents a 27-fold increase from that observed in water (207 +/- 5 microg/mL) in the absence of HBenBCD. Saquinavir-HBenBCD formulations were found to have rapid dissolution over a wide pH range (1.2-6.8), and saquinavir solubility in these media was maintained throughout the experiments. When saquinavir-HBenBCD formulations were administered to Wistar-Hannover rats, saquinavir was rapidly absorbed and rapidly eliminated. Rapid saquinavir elimination was particularly pronounced when saquinavir-HBenBCD formulations were given as an oral aqueous gavage. Saquinavir oral bioavailability in rats obtained from saquinavir mesylate capsules (2.0% +/- 0.7%) was increased (9 +/- 4)-fold (18.6% +/- 7.3%) when dosed with saquinavir base-HBenBCD capsules. Clearly, HBenBCD can significantly improve the solubility and oral bioavailability of saquinavir; however, further formulation studies are required to optimize saquinavir oral delivery using this technology.     

Design of a liquid nano-sized drug delivery system with enhanced solubility of rivaroxaban for venous thromboembolism management in paediatric patients and emergency cases

Abstract

The increasing incidence of venous thromboembolism (VTE) in paediatric population has stimulated the development of liquid anticoagulant formulations. Thus our goal is to formulate a liquid formulation of poorly-water soluble anticoagulant, rivaroxaban (RIVA), for paediatric use and to assess the possibility of its intravenous administration in emergencies. Self-nanoemulsifying drug delivery systems (SNEDDSs) were developed and characterized. SNEDDS constituents were estimated from the saturated solubility study followed by plotting the corresponding ternary phase diagrams to determine the best self-emulsified systems. Thermodynamic stability, emulsification, dispersibility, robustness to dilution tests, in vitro dissolution, particle size, and zeta potential were executed to optimize the formulations. The optimized formulation, that composed of Capryol 90:Tween 20:PEG 300 (5:45:50), increased RIVA solubility (285.7-fold than water), it formed nanoemulsion with a particle size of 16.15?nm, PDI of 0.25 and zeta potential of ?21.8. It released 100.83?±?2.78% of RIVA after 5?min. SNEDDS was robust to dilution with oral and parenteral fluids and showed safety to human RBCs. SNEDDS showed enhanced bioavailability after oral and intravenous administration than the oral drug suspension (by 1.25 and 1.26-fold, respectively). Moreover, it exhibited enhanced anticoagulant efficacy in the prevention and treatment of carrageenan-induced thrombosis rat model.     

Progesterone inhibits the growth of human neuroblastoma: in vitro and in vivo evidence

We investigated the antitumorogenic effects of progesterone (P4) in a human neuroblastoma (SK-N-AS) cell line in vitro and in a mouse xenograft model of neuroblastoma. The safety of P4 was tested in rat primary cortical neurons and human foreskin fibroblasts (HFF-1). At high doses, P4 significantly (P < 0.05) decreased SK-N-AS cell viability in vitro, and this effect was not blocked either by 5α-reductase inhibitor, finasteride or the P4 receptor antagonist RU486. Even at very high doses, P4 did not induce any cell death in healthy primary cortical neurons or HFF-1. The bioavailability of P4 24 h after the last injection in the serum of treated animals was significantly (P < 0.05) higher (10-33 μg/mL) than in untreated animals. In nude mice, P4 (50 and 100 mg/kg) inhibited neuroblastoma growth by ~50% over 8 d of treatment. No drug toxicity was observed in the mice, as measured by body weight and activity. P4 suppressed the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-9, MMP-2), which are involved in tumor vascular development. High-dose P4 inhibited tumor growth by suppressing cell proliferation and inducing apoptosis, as evidenced by the expression of proliferating cell nuclear antigen and cleaved caspase-3. P4 significantly increased the expression of P4 receptor isoform-A and suppressed phospho-Akt (Ser437) expression. In conclusion, at high doses, P4 effectively inhibits the growth of solid neuroblastoma tumor and has high bioavailability, selective toxicity and a high margin of safety, making it a possible candidate for further study as a potential clinical treatment of neuroblastoma.     

Molecular Interaction Studies of Amorphous Solid Dispersions of the Antimelanoma Agent Betulinic Acid

Betulinic acid (BA), a novel natural product with antimelanoma activity, has poor aqueous solubility (<0.1 μg/mL) and therefore exhibits poor bioavailability. The purpose of this study was to explore the feasibility of preparing BA solid dispersions (BA-SDs) with hydrophilic polymers to enhance the aqueous solubility of BA. Melt-quenched solid dispersions (MQ-SDs) of BA were prepared at various ratios with the hydrophilic polymers including Soluplus, HPMCAS-HF, Kollidon VA64, Kollidon K90, and Eudragit RLPO. BA was found to be miscible in all polymers at a 1:4 (w/w) ratio by modulated differential scanning calorimetry (MDSC). BA/Soluplus MQ-SD exhibited the highest solubility in simulated body fluids followed by BA/Kollidon VA64 MQ-SD. The MQ-SDs of BA/Soluplus, BA/HPMCAS-HF, and BA/Kollidon VA64 were found to be amorphous as indicated by X-ray powder diffraction (XRPD) studies. Fourier transform infra-red (FT-IR) studies indicated molecular interactions between BA and Soluplus. Our preliminary screening of polymers indicates that Soluplus and Kollidon VA64 exhibit the greatest potential to form BA-SDs.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0220-x) contains supplementary material, which is available to authorized users.KEY WORDS: betulinic acid, DSC, FT-IR, HPLC, SEM, solid dispersions, solubility, XRPD     

Novel self-emulsifying formulation of quercetin for improved in vivo antioxidant potential: Implications for drug-induced cardiotoxicity and nephrotoxicity

Quercetin (QT) was formulated into a novel self-emulsifying drug delivery system (SEDDS) to improve its oral bioavailability and antioxidant potential compared to free drug. Capmul MCM was selected as the oily phase on the basis of optimum solubility of QT in oil. Tween 20 and ethanol were selected as surfactant and cosurfactant from a large pool of excipients, depending upon their spontaneous self-emulsifying ability with the selected oily phase. Pseudoternary-phase diagrams were constructed to identify the efficient self-emulsification regions in various dilution media, viz., water, pH 1.2, and pH 6.8. The ratio of 40:40:20 w/w, Capmul MCM:QT (19:1)/Tween 20/ethanol was optimized based on its ability to form a spontaneous submicrometer emulsion in simulated gastrointestinal fluids. DPPH scavenging assay showed comparable antioxidant activity of QT-SEDDS to free QT. QT-SEDDS was robust in terms of stability against short-term excursion of freeze/thaw cycles and accelerated stability for 6 months as per International Conference on Harmonisation guidelines. A fluorescent dye-loaded SEDDS formulation showed rapid internalization within 1 h of incubation with Caco-2 cells as evident by confocal laser scanning microscopy. QT-SEDDS showed a significant increase in cellular uptake by 23.75-fold in comparison with free QT cultured with Caco-2 cells. The SEDDS demonstrated ~5-fold enhancement in oral bioavailability compared to free QT suspension. The in vitro–in vivo relation between in vitro Caco-2 cell uptake and in vivo pharmacokinetics of QT-SEDDS showed a correlation coefficient of ~0.9961, as evident from a Levy plot. Finally, QT-SEDDS showed a significantly higher in vivo antioxidant potential compared to free QT when evaluated as a function of ability to combat doxorubicin- and cyclosporin A-induced cardiotoxicity and nephrotoxicity, respectively.     

Design of novel hexahydropyrazinoquinolines as potent and selective dopamine D3 receptor ligands with improved solubility

We have recently reported hexahydropyrazinoquinolines as a new class of dopamine 3 (D(3)) receptor ligands with high-affinity to the D(3) receptor and excellent selectivity over the closely related D(1)-like and D(2)-like receptors. However, our previously reported most potent and selective D(3) ligands have poor aqueous solubility, which greatly hinders our in vivo studies aimed at evaluation of their therapeutic potential in animal models. In this study, we wish to report the design, synthesis, and evaluation of a series of new hexahydropyrazinoquinolines as D(3) ligands with improved solubility. Among them, compound 4g has a K(i) value of 9.7 nM for the D(3) receptor and displays a selectivity of >5000 and 466 times over the D(1)-like and D(2)-like receptors, respectively. Importantly, the hydrochloride salt form of compound 4g has a good aqueous solubility (>50 mg/mL) and represents a promising D(3) ligand for further in vivo evaluations of its therapeutic potential for the treatment of drug abuse, restless legs syndrome, schizophrenia, Parkinson's disease, and depression.     

Synthetic studies on selective adenosine A2A receptor antagonists. Part II: Synthesis and structure–activity relationships of novel benzofuran derivatives

Based on the previously reported lead compound, a series of benzofuran derivatives were prepared to study their antagonistic activities to A_2A receptor. The replacement of the phenyl group at the 4-position with a heterocyclic ring improved the PK profile and aqueous solubility. From these studies, we discovered a potent new A_2A antagonist, 12a, which has both a good oral bioavailability and in vivo efficacy on motor disability in MPTP-treated common marmosets.