Lipid-coated nanocrystals of paclitaxel as dry powder for inhalation: Characterization, in-vitro performance, and pharmacokinetic assessment.

BACKGROUND: Nanocrystals can be produced as a dry powder for inhalation (DPIs) to deliver high doses of drug to the lungs, owing to their high payload and stability to the shear stress of aerosolization force. Furthermore, lipid-coated nanocrystals can be formulated to improve the drug accumulation and retention in lung. OBJECTIVE: The present work involved the fabrication of paclitaxel nanocrystals using hydrophilic marine biopolymer fucoidan as a stabilizer. Thereafter, fabricated nanocrystals (FPNC) were surface-modified with phospholipid to give lipid-coated nanocrystals (Lipo-NCs). METHODS: The nanocrystals were fabricated by antisolvent crystallization followed by the probe sonication. The lipid coating was achieved by thin film hydration followed ultrasonic dispersion technique. Prepared nanocrystals were lyophilized to obtain a dry powder of FPNC and Lipo-NCs, used later for physicochemical, microscopic, and spectroscopic characterization to confirm the successful formation of desired nanocrystals. In-vitro and in-vivo investigations were also conducted to determine the role of nanocrystal powder in pulmonary drug delivery. RESULTS: Lipo-NCs exhibited slower drug release, excellent flow properties, good aerosolization performance, higher drug distribution, and prolonged retention in the lungs compared to FPNC and pure PTX. CONCLUSION: Lipid-coated nanocrystals can be a novel formulation for the maximum localization of drugs in the lungs, thereby enhancing therapeutic effects and avoiding systemic side effects in lung cancer therapy.

High-effective biosynthesis of baccatin â ¢ by using the alternative acetyl substrate, N-acetyl-d-glucosamine.

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.

Mortality Assessment of Paclitaxel-Coated Balloons: Patient-Level Meta-Analysis of the ILLUMENATE Clinical Program at 3 Years.

BACKGROUND: A recent summary-level meta-analysis comprising randomized, controlled trials (RCTs) of femoropopliteal paclitaxel-coated balloon and stent intervention identified excess late mortality in the paclitaxel-treated patients. METHODS: We evaluated the safety of the Stellarex drug-coated balloon (DCB) for femoropopliteal artery disease with an independently performed meta-analysis of patient-level data from all patients in the Stellarex femoropopliteal clinical program. To compare mortality after DCB or uncoated percutaneous transluminal angioplasty (PTA), we aggregated data from 2 RCTs comprising 419 patients treated with DCB and 170 patients treated with PTA. In an additional analysis, data were aggregated from 6 poolable Stellarex DCB studies (2 RCTs, 3 single-arm studies, and 1 registry). All serious adverse events including deaths were adjudicated by a blinded, third-party, independent Clinical Events Committee. Kaplan-Meier estimates in the RCTs were compared with restricted mean survival time. Predictors of death were assessed with hazard ratios (HRs) and Cox proportional hazards modeling. RESULTS: Baseline characteristics were similar in the patients treated with DCB and PTA in the pooled RCT analysis, with the exception that the DCB cohort was younger (67.4±9.7 versus 69.4±9.4 years, P=0.02), smoked more frequently (86.6% versus 78.8%, P=0.02), and were less often treated for recurrent lesions (8.8% versus 14.7%, P=0.04). In the RCTs, patients treated with DCB had all-cause mortality rates that were not different from those of patients treated with PTA (Kaplan-Meier estimates 1.8±0.7% versus 1.3±0.9%, 6.5±1.2% versus 5.9±1.9%, and 9.3±1.5% versus 9.9±2.4% at 1, 2, and 3 years, respectively, P=0.86). All-cause mortality rates were similar in a 1906-patient pooled nonrandomized DCB data set (Kaplan-Meier estimates of 2.1%, 4.9%, and 7.0% at 1, 2, and 3 years, respectively). Clinical Events Committee-adjudicated causes of death were balanced between the DCB and PTA cohorts. Multivariable Cox modeling identified age (HR, 1.06; 95% CI, 1.04-1.08; P<0.001), diabetes mellitus (HR, 1.42; 95% CI, 1.01-2.00; P=0.04), congestive heart failure (HR, 1.88; 95% CI, 1.12-3.16; P=0.02), and renal insufficiency (HR, 2.00; 95% CI, 1.33-3.01; P<0.001) as predictors of mortality. Paclitaxel exposure was unrelated to mortality (HR, 1.04; 95% CI, 0.98-1.10; P=0.23). CONCLUSIONS: The mortality rates for patients treated with the DCB and uncoated PTA were indistinguishable over 3-year follow-up. Additional patient-level, adequately powered meta-analyses with larger RCT data sets will be needed to confirm the generalizability of these findings. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT02110524, NCT01858363, NCT01858428, NCT03421561, NCT01912937, NCT01927068, and NCT02769273.

Liquisolid system of paclitaxel using modified polysaccharides: In vitro cytotoxicity, apoptosis study, cell cycle analysis, in vitro mitochondrial membrane potential assessment, and pharmacokinetics.

The research was aimed to develop a liquisolid formulation of paclitaxel using novel, highly porous liquisolid carriers (modified polysaccharides) to enhance bioavailability of orally administered paclitaxel. Modified polysaccharides namely co-grinded treated guar gum (C-TGG), co-grinded treated tamarind kernel powder (C-TTKP) and co-grinded treated locust bean gum (C-TLBG) were developed by sequentially subjecting the corresponding polysaccharides to wetting, drying and co-grinding with mannitol (1:1). A total of 12 liquisolid systems of paclitaxel (LSP-1 to LSP-12) were formulated using non-volatile solvent (polysorbate 80/Solutol HS 15®), carrier material (C-TGG/C-TTKP/C-TLBG), and Aerosil® 200 as coating material, and evaluated for pre-compression parameters. The liquisolid systems were directly compressed to produce liquisolid tablets (LTP-1 to LTP-12) and assessed for post compression parameters, cytotoxic/cellular analysis and pharmacokinetics. The modified polysaccharides exhibited narrow symmetrical particle size distribution, high liquid absorption potential, diminutive swelling index, favorable in vitro biodegradability and compression amenability. Among the directly compressed liquisolid tabs, LTP-10 exhibited highest CDR of 98.70 ±â€¯2.68% and permeability of 61.59%. The IC50 of <20 mmol/L indicated remarkable cytotoxic potential on human gastro-enteric tumor cancerous cell lines (NCI-N87). Additionally, LTP-10 exhibited significantly high values for cell death 37.92 and 54.17% (P < 0.01) in early and late apoptosis and mitochondrial membrane potential regain (33%) in comparison to paclitaxel (P < 0.05) and 5-fluorouracil (P < 0.01). Pharmacokinetics revealed Cmax of 536.48 ±â€¯4.63 µg/L at 1.64 ±â€¯0.44 h for LTP-10 indicating enhancement in bioavailability (5.43 fold) of paclitaxel on oral administration.

α-Tocopherol Succinate-Anchored PEGylated Poly(amidoamine) Dendrimer for the Delivery of Paclitaxel: Assessment of in Vitro and in Vivo Therapeutic Efficacy.

This study involves development of a dendrimer-based nanoconstruct by conjugating α-tocopheryl succinate (α-TOS) and polyethylene glycol (PEG) on a poly(amidoamine) dendrimer (G4 PAMAM) to improve intracellular delivery of a poorly water-soluble chemotherapeutic drug, paclitaxel (PTX). The conjugates were characterized by NMR, and PTX-loaded nanocarriers (G4-TOS-PEG-PTX) were evaluated for hydrodynamic diameter, polydispersity index (PDI), zeta potential, percentage encapsulation efficiency (%EE), and percentage drug loading (%DL). A hemolysis study was performed, which indicated that the synthesized dendrimer conjugates were not toxic to red blood cells; hence, they were biocompatible. A cellular uptake study in (B16F10 and MDA MB231) monolayer cells and 3D spheroids showed that α-TOS conjugation improved the time dependent uptake of nanosized dendrimer conjugates. The cell viability assay revealed that G4-TOS-PEG-PTX enhanced the cytotoxicity of PTX as compared to free PTX and PTX-loaded G4-PEG (G4-PEG-PTX) at tested concentrations. Correspondingly, the α-TOS-anchored dendrimer induced more apoptosis as compared to free PTX and G4-PEG-PTX. Moreover, the fluorescently labeled G4-TOS-PEG penetrated deeper into MDA MB231 3D spheroids as visualized by confocal microscopy. G4-TOS-PEG-PTX showed significant growth inhibition in 3D spheroids as compared to free PTX and G4-PEG-PTX. Further, the in vivo efficacy study using B16F10 xenografted C57Bl6/J mice indicated that the G4-TOS-PEG localized in tumor sections. G4-TOS-PEG-PTX reduced the tumor growth significantly compared to free PTX and G4-PEG-PTX. G4-TOS-PEG-PTX had more apoptotic potential in tumor sections as analyzed by TUNEL assay. Hence, the newly developed dendrimer conjugate, G4-TOS-PEG, has the potential to target loaded drug to the tumor, and G4-TOS-PEG-PTX has the potential to be utilized successfully in cancer treatment.

Production of LMWH-conjugated core/shell hydrogels encapsulating paclitaxel for transdermal delivery: In vitro and in vivo assessment.

Topical applications that reduce systemic toxic effects while increasing therapeutic efficacy are a promising alternative strategy. The aim of this study was to provide an enhanced transdermal delivery of low molecular weight heparin (LMWH) through the stratum corneum by using cationic carrier as a novel permeation enhancer. Recent studies have shown that heparin-conjugated biomaterials can be effective in inhibiting tumor growth during cancer treatment due to their high ability to bind growth factors. Paclitaxel (PCL) was co-encapsulated into the same cationic carrier for the purpose of improving of therapeutic efficacy for a combined cancer treatment with LMWH. In vitro and in vivo studies showed that the LMWH and PCL release was significantly affected by polymer molecular weight and block composition. Skin penetration tests have indicated that larger amounts of LMWH were absorbed from LMWH-gel conjugate through SC, than aqueous formula. However, it was found that the plasma transition of LMWH released from gel conjugate was lower compared to the plasma concentration of LMWH released from aqueous solution. It is recommended that PCL-loaded LMWH-conjugated core/shell hydrogels can be used as promising drug release systems for transdermal applications that can improve therapeutic efficacy and reduce side effects in a combined cancer treatment.

Nanocarrier-Based Combination Chemotherapy for Resistant Tumor: Development, Characterization, and Ex Vivo Cytotoxicity Assessment.

A folic acid-conjugated paclitaxel (PTX)-doxorubicin (DOX)-loaded nanostructured lipid carrier(s) (FA-PTX-DOX NLCs) were prepared by using emulsion-evaporation method and extensively characterized for particle size, polydispersity index, zeta potential, and % entrapment efficiency which were found to be 196 ± 2.5 nm, 0.214 ± 0.04, +23.4 ± 0.3 mV and 88.3 ± 0.2% (PTX), and 89.6 ± 0.5% (DOX) respectively. In vitro drug release study of optimized formulation was carried out using dialysis tube method. FA-conjugated PTX-DOX-loaded NLCs showed 75.6 and 78.4% (cumulative drug release) of PTX and DOX respectively in 72 h in PBS (pH 7.4)/methanol (7:3), while in the case of FA-conjugated PTX-DOX-loaded NLCs, cumulative drug release recorded was 80.4 and 82.8% of PTX and DOX respectively in 72 h in PBS (pH 4.0)/methanol (7:3). Further, the formulation(s) were evaluated for ex vivo cytotoxicity study. The cytotoxicity assay in doxorubicin-resistant human breast cancer MCF-7/ADR cell lines revealed lowest GI50 value of FA-D-P NLCs which was 1.04 ± 0.012 µg/ml, followed by D-P NLCs and D-P solution with GI50 values of 3.12 ± 0.023 and 3.89 ± 0.007 µg/ml, respectively. Findings indicated that the folic acid-conjugated PTX and DOX co-loaded NLCs exhibited lower GI50 values as compared to unconjugated PTX and DOX co-loaded NLCs; thus, they have relatively potential anticancer efficacy against resistant tumor.

Assessment of novel core-shell Fe3O4@poly l­DOPA nanoparticles for targeted Taxol® delivery to breast tumor in a mouse model.

Drug delivery systems using nanoparticles can deliver to tumor cells without affecting normal cells. In this study, a novel well dispersed magnetic nano drug was synthesized. Thus, a selective drug delivery system was designed for potential cancer treatment. A new nanocomposite, poly 3,4­dihydroxy­l­phenylalanine/Fe3O4 (l­DOPA/Fe3O4), was synthesized and used for targeted Taxol® delivery to breast tumor in inbreed Balb/c mice model with or without magnetic field. Fe and Taxol® concentrations were measured by flame atomic absorption spectrometry and high-performance liquid chromatography, respectively. Antitumor effectiveness was investigated in terms of tumor growth features. In the presence of magnetic field, Taxol® was significantly deposited in tumor tissue in Taxol-nanocomposite-treated group. In addition, the Taxol®-nanocomposite-treated group with magnetic field showed higher antitumor efficacy than the commercial Taxol and Taxol-nanocomposite without magnetic field. The magnetic nanocomposite is promising for targeted Taxol® delivery to breast tumor in a mouse model yielding high performance.

Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers.

An approach to characterizing P-glycoprotein (Pgp) interaction potential for sparingly water-soluble compounds was developed using bidirectional transport kinetics in MDR1-MDCK cell monolayers. Paclitaxel, solubilized in a dilute polysorbate 80 (PS80) micellar solution, was used as a practical example. Although the passage of paclitaxel across the cell monolayer was initially governed by the thermodynamic activity of the micelle-solubilized drug solution, Pgp inhibition was sustained by the thermodynamic activity (i.e., critical micelle concentration) of the PS80 micellar solution bathing the apical (ap) membrane. The mechanistic understanding of the experimental strategies and treatment of data was supported by a biophysical model expressed in the form of transport events occurring at the ap and basolateral (bl) membranes in series whereas the vectorial directions of the transcellular kinetics were accommodated. The derived equations permitted the stepwise quantitative delineation of the Pgp efflux activity (inhibited and uninhibited by PS80) and the passive permeability coefficient of the ap membrane, the passive permeability at the bl membrane and, finally, the distinct coupling of these with efflux pump activity to identify the rate-determining steps and mechanisms. The Jmax/KM(∗) for paclitaxel was in the order of 10(-4) cm/s and the ap- and bl-membrane passive permeability coefficients were asymmetric, with bl-membrane permeability significantly greater than ap.

Low cost microfluidic cell culture array using normally closed valves for cytotoxicity assay.

A reusable low cost microfluidic cell culture array device (MCCAD) integrated with a six output concentration gradient generator (cGG) and 4×6 arrays of microchamber elements, addressed by a series of row and columnar pneumatically actuated normally closed (NC) microvalves was fabricated for cell-based screening of chemotherapeutic compounds. The poly(dimethylsiloxane) (PDMS) device consists of three layers: fluidic, control and membrane which are held by surface contact and made leak-proof by clamping pressure. The NC valves are actuated by a thick PDMS membrane that was created by a novel method based on the self-assembly of PDMS pre-polymer molecules over a denser calcium chloride solution. The membrane actuated the valves reliably and particulates such as alumina particles (3 µm) and MCF-7 cells (20-24 µm) (2×10(5) cells/mL) were flowed through the valves without causing blockage or leakage and consequently avoiding contamination of the different cell culture elements. The MCCAD was cast and assembled in a standard laboratory without specialist equipment and demonstrated for performing quantitative cell-based cytotoxicity assays of pyocyanine on human breast cancer (MCF-7) cells and assessed for toxic effect on human hepatocyte carcinoma (HepG2) cells as an indicator for liver injury. Then, the MCCAD was demonstrated for sequential drug combinatorial screening involving gradient generation of paclitaxel doses followed by treatment with aspirin doses on the viability of MCF-7 cells. The interaction between paclitaxel and aspirin was evaluated by using the Bliss independence predictive model and results showed reasonable agreement with the model. A robust, portable, easily fabricated and low cost device is therefore shown to conveniently carry out culturing of multiple cell lines for high throughput screening of anti-cancer compounds using minimal reagents.

Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups.

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers--dipiperidine trismethylene, piperazine, and aminomethylpiperidine--were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2'-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (~6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.

Dissecting paclitaxel-microtubule association: quantitative assessment of the 2'-OH group.

Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to ß-tubulin in assembled microtubules. The 2'-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated. The contribution of the 2'-OH group to the affinity and tubulin-assembly efficacy of PTX has been evaluated through quantitative analysis of PTX derivatives possessing side chain deletions: 2'-deoxy-PTX, N-debenzoyl-2'-deoxy-PTX, and baccatin III. The affinity of 2'-deoxy-PTX for stabilized microtubules was more than 100-fold lower than that of PTX and only ~3-fold greater than the microtubule affinity of baccatin III. No microtubule binding activity was detected for the analogue N-debenzoyl-2'-deoxy-PTX. The tubulin-assembly efficacy of each ligand was consistent with the microtubule binding affinity, as was the trend in cytotoxicities. Molecular dynamics simulations revealed that the 2'-OH group of PTX can form a persistent hydrogen bond with D26 within the microtubule binding site. The absence of this interaction between 2'-deoxy-PTX and the receptor can account for the difference in binding free energy. Computational analyses also provide a possible explanation for why N-debenzoyl-2'-deoxy-PTX is inactive, in spite of the fact that it is essentially a substituted baccatin III. We propose that the hydrogen bonding interaction between the 2'-OH group and D26 is the most important stabilizing interaction that PTX forms with tubulin in the region of the C-13 side chain. We further hypothesize that the substituents at the 3'-position function to orient the 2'-OH group for a productive hydrogen bonding interaction with the protein.

Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Mechanistic population pharmacokinetics of total and unbound paclitaxel for a new nanodroplet formulation versus Taxol in cancer patients.

PURPOSE: Our objectives were (1) to compare the disposition and in vivo release of paclitaxel between a tocopherol-based Cremophor-free formulation (Tocosol Paclitaxel) and Cremophor EL-formulated paclitaxel (Taxol) in human subjects, and (2) to develop a mechanistic model for unbound and total paclitaxel pharmacokinetics. METHODS: A total of 35 patients (average +/- SD age: 59 +/-13 years) with advanced non-hematological malignancies were studied in a randomized two-way crossover trial. Patients received 175 mg/m(2) paclitaxel as 15 min (Tocosol Paclitaxel) or 3 h (Taxol) intravenous infusion in each study period. Paclitaxel concentrations were determined by LC-MS/MS in plasma ultrafiltrate and whole blood. NONMEM VI was used for population pharmacokinetics. RESULTS: A linear disposition model with three compartments for unbound paclitaxel and a one-compartment model for Cremophor were applied. Total clearance of unbound paclitaxel was 845 L/h (variability: 25% CV). The prolonged release with Tocosol Paclitaxel was explained by the limited solubility of unbound paclitaxel of 405 ng/mL (estimated) in plasma. The 15 min Tocosol Paclitaxel infusion yielded a mean time to 90% cumulative input of 1.14 +/- 0.16 h. Tocosol Paclitaxel was estimated to release 9.8% of the dose directly into the deep peripheral compartment. The model accounted for the presence of drug-containing nanodroplets in blood. CONCLUSIONS: Population pharmacokinetic analysis indicated linear disposition and a potentially higher bioavailability of unbound paclitaxel following Tocosol Paclitaxel administration due to direct release at the target site. The prolonged release of Tocosol Paclitaxel supports 15 min paclitaxel infusions. This mechanistic model may be important for development of prolonged release formulations that distribute in and from the systemic circulation.

Kinetics of human peptidylarginine deiminase 2 (hPAD2)--reduction of Ca2+ dependence by phospholipids and assessment of proposed inhibition by paclitaxel side chains.

Multiple sclerosis is a complex human neurodegenerative disease, characterized by the active destruction of the insulating myelin sheath around the axons in the central nervous system. The physical deterioration of myelin is mediated by hyperdeimination of myelin basic and other proteins, catalysed by the Ca2+ -dependent enzyme peptidylarginine deiminase 2 (PAD2). Thus, inhibition of PAD2 may be of value in treatment of this disease. Here, we have first characterized the in vitro kinetic properties of the human peptidylarginine deiminase isoform 2 (hPAD2). Phosphatidylserine and phosphatidylcholine reduced its Ca2+ dependence by almost twofold. Second, we have explored the putative inhibitory action of the methyl ester side chain of paclitaxel (TSME), which shares structural features with a synthetic PAD substrate, viz., the benzoyl-L-arginine ethyl ester (BAEE). Using the known crystallographic structure of the homologous enzyme hPAD4 and in silico molecular docking, we have shown that TSME interacted strongly with the catalytic site, albeit with a 100-fold lower affinity than BAEE. Despite paclitaxel having previously been shown to inhibit hPAD2 in vitro, the side chain of paclitaxel alone did not inhibit this enzyme's activity.

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis.

The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts alpha-phenylalanine to beta-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield beta-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both beta-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of beta-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of beta-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.

O-N intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides.

N-Ointramolecular acyl migration in Ser- or Thr-containing peptides is a well-known side reaction in peptide chemistry. It results in the mutual conversion of ester and amide bonds. Our medicinal chemistry study focused on the fact that the O-acyl product can be readily converted to the original N-acyl form under neutral or slightly basic conditions in an aqueous buffer and the liberated ionized amino group enhances the water solubility of O-acyl products. Because of this, we have developed a novel class of "O-N intramolecular acyl migration"-type water-soluble prodrugs of HIV-1 protease inhibitors. These prodrugs released the parent drugs via a simple chemical mechanism with no side reaction. In this study, we applied this strategy to important cancer chemotherapeutic agents, paclitaxel and its derivatives, to develop water-soluble taxoid prodrugs, and found that these prodrugs, 2'-O-isoform of taxoids, showed promising results with higher water solubility and proper kinetics in their parent drug formation by a simple pH-dependent chemical mechanism with O-N intramolecular acyl migration. These results suggest that this strategy would be useful in toxicology and medical economics. After the successful application of O-N intramolecular acyl migration in medicinal chemistry, this concept was recently used in peptide chemistry for the synthesis of "difficult sequence-containing peptides." The strategy was based on hydrophilic O-acyl isopeptide synthesis followed by the O-N intramolecular acyl migration reaction, leading to the desired peptide. In a model study with small, difficult sequence-containing peptides, synthesized "O-acyl isopeptides" not only improved the solubility in various media and efficiently performed the high performance liquid chromatography purification, but also altered the nature of the difficult sequence during SPPS, resulting in the efficient synthesis of O-acyl isopeptides with no complications. The subsequent O-N intramolecular acyl migration of purified O-acyl isopeptides afforded the desired peptides as precipitates with high yield and purity. Further study of the synthesis of a larger difficult sequence-containing peptide, Alzheimer's disease-related peptide (A beta 1-42), surprisingly showed that only one insertion of the O-acyl group drastically improved the unfavorable nature of the difficult sequence in A beta 1-42, and achieved efficient synthesis of 26-O-acyl isoA beta 1-42 and subsequent complete conversion to A beta 1-42 via the O-N intramolecular acyl migration reaction of 26-O-acyl isoA beta 1-42. This suggests that our new method based on O-N intramolecular acyl migration is an important method for the synthesis of difficult sequence-containing bioactive peptides.

A novel approach of water-soluble paclitaxel prodrug with no auxiliary and no byproduct: design and synthesis of isotaxel.

A novel water-soluble paclitaxel prodrug, isotaxel 2, that realizes a higher water-solubility and the formation of paclitaxel through a simple pH-dependent chemical mechanism via the O-N acyl migration was synthesized and showed promising results in water-solubility and kinetics. This prodrug, a 2'-O-benzoyl isoform of paclitaxel, has no additional functional auxiliaries released during conversion to paclitaxel, which would be a great advantage in toxicology and medical economics.