The Impact of Systemic Lupus Erythematosus on the Clinical Phenotype of Antiphospholipid Antibody-Positive Patients: Results From the AntiPhospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Clinical Database and Repository.

OBJECTIVE: Although systemic lupus erythematosus (SLE) is the most common autoimmune disease associated with antiphospholipid antibodies (aPL), limited data exist regarding the impact of SLE on the clinical phenotype of aPL-positive patients. The primary objective of this study was to compare the clinical, laboratory, and treatment characteristics of aPL-positive patients with SLE with those of aPL-positive patients without SLE. METHODS: A secure web-based data capture system was used to store patient demographic characteristics and aPL-related clinical and laboratory characteristics. Inclusion criteria included positive aPL according to the updated Sapporo classification criteria. Antiphospholipid antibody-positive patients fulfilling the American College of Rheumatology criteria for the classification of SLE ("aPL with SLE") and those with no other autoimmune diseases ("aPL only") were included in the analysis. RESULTS: Six hundred seventy-two aPL-positive patients were recruited from 24 international centers; 426 of these patients did not have other autoimmune disease, and 197 had SLE. The frequency of thrombocytopenia, hemolytic anemia, low complement levels, and IgA anti-ß2 -glycoprotein I (anti-ß2 GPI) antibodies was higher in the aPL-positive patients with SLE, whereas the frequency of cognitive dysfunction and IgG anti-ß2 GPI antibodies was higher in the aPL-only group. The frequency of arterial and venous thromboses (including recurrent) as well as pregnancy morbidity was similar in the 2 groups. The prevalence of cardiovascular disease risk factors at the time of entry into the registry entry did not differ between the 2 groups, with the exception of current smoking, which was more frequent in aPL-positive patients with SLE. CONCLUSION: Although the frequencies of thrombosis and pregnancy morbidity are similar in aPL-positive patients with and those without SLE, the diagnosis of SLE in patients with persistently positive aPL is associated with an increased frequency of thrombocytopenia, hemolytic anemia, low complement levels, and positive IgA anti-ß2 GPI antibodies.

Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.

OBJECTIVES: A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells. MATERIALS AND METHODS: Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on ß-tubulin organization was carried out by confocal microscopy. RESULTS: The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the ß-tubulin microtubular mesh with subsequent cell death. CONCLUSIONS: Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.

Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of beta2-glycoprotein I.

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N-terminal domain (DmI) of beta2-glycoprotein I (beta2GpI). To possibly block anti-beta2GpI Abs activity, we synthesized the entire DmI comprising residues 1-64 of beta2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N-DmI) was purified by RP-HPLC, and its chemical identity and correct disulfide pairing (Cys4-Cys47 and Cys32-Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far- and near-UV CD and fluorescence spectroscopy, provided strong evidence for the native-like structure of DmI, which is also quite resistant to both Gdn-HCl and thermal denaturation. However, the thermodynamic stability of N-DmI at 37 degrees C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N-DmI in direct binding experiments. From ELISA competition experiments with plate-immobilized beta2GpI, a mean IC(50) value of 8.8 microM could be estimated for N-DmI, similar to that of the full-length protein, IC(50)(beta2GpI) = 6.4 microM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-beta2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N-terminally biotin-(PEG)(2)-derivative of N-DmI (Biotin-N-DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin-N-DmI loaded onto a streptavidin-coated plate selectively recognized aAbs from APS patients.

Biokinetic and dosimetric studies of 188Re-hyaluronic acid: a new radiopharmaceutical for treatment of hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and has very limited therapeutic options. Recently, it has been found that hyaluronic acid (HA) shows selective binding to CD44 receptors expressed in most cancer histotypes. Since the trend in cancer treatment is the use of targeted radionuclide therapy, the aim of this research was to label HA with rhenium-188 and to evaluate its potential use as a hepatocarcinoma therapeutic radiopharmaceutical. METHODS: (188)Re-HA was prepared by a direct labelling method to produce a ReO(O-COO)(2)-type coordination complex. (188)Re-HA protein binding and its stability in saline, phosphate buffer, human serum and cysteine solutions were determined. Biokinetic and dosimetric data were estimated in healthy mice (n=60) using the Medical Internal Radiation Dose methodology and mouse model beta-absorbed fractions. To evaluate liver toxicity, alanine aminotranferase (AST) and aspartate aminotranferase (ALT) levels in mice were assessed and the liver maximum tolerated dose (MTD) of (188)Re-HA was determined. RESULTS: A stable complex of (188)Re-HA was obtained with high radiochemical purity (>90%) and low serum protein binding (2%). Biokinetic studies showed a rapid blood clearance (T(1/2)alpha=21 min). Four hours after administration, (188)Re-HA was almost totally removed from the blood by the liver due to the selective uptake via HA-specific receptors (73.47+/-5.11% of the injected dose). The liver MTD in mice was approximately 40 Gy after 7.4 MBq of (188)Re-HA injection. CONCLUSIONS: (188)Re-HA complex showed good stability, pharmacokinetic and dosimetric characteristics that confirm its potential as a new agent for HCC radiation therapy.

Biodistribution imaging of a paclitaxel-hyaluronan bioconjugate.

INTRODUCTION: Gamma-ray detectors represent sensitive and noninvasive instruments to evaluate in vivo the metabolic trapping of radiopharmaceuticals. This study aimed to assess the imaging biodistribution of a [(99m)Tc]-radiolabelled new prototype bioconjugate composed of paclitaxel linked to hyaluronan (ONCOFID-P). METHODS: A small gamma camera providing high-resolution images was employed. Imaging of biodistribution following intravenous, intraperitoneal, intravesical and oral administration was carried out for a 2-h period in anesthetized mice receiving [(99m)Tc]ONCOFID-P. At the end of the observation time, radioactivity in organs was directly measured. As a control, groups of mice were treated with free [(3)H]paclitaxel given according to the same administration routes, and organ biodistribution of the drug was assessed after 2 h. RESULTS: Intravenous inoculation of [(99m)Tc]ONCOFID-P was followed by a rapid and strong liver uptake. In fact, almost 80% of the imaging signal was detected in this organ 10 min after injection and such value remained constant thereafter, thus indicating that the bioconjugate given through the intravenous route could be well suited to targeting primary or metastatic liver neoplasias. Imaging of the bladder, abdomen and gastrointestinal tract after local administration disclosed that the radiolabelled compound remained confined to the cavities, suggesting a potential regional application for transitional bladder cell carcinomas, ovarian cancers and gastric tumors, respectively. Free [(3)H]paclitaxel biodistribution profoundly differed from that of [(99m)Tc]ONCOFID-P. CONCLUSIONS: Conjugation of drugs with polymers results in new chemical entities characterized by a modified biodistribution pattern. Therefore, preclinical studies based on imaging analysis of such new compounds can suggest novel therapeutic applications.

A paclitaxel-hyaluronan bioconjugate targeting ovarian cancer affords a potent in vivo therapeutic activity.

PURPOSE: This study was designed to evaluate the pharmacologic and biological properties of a paclitaxel-hyaluronan bioconjugate (ONCOFID-P) against IGROV-1 and OVCAR-3 human ovarian cancer xenografts following i.p. administration. EXPERIMENTAL DESIGN: In vitro tumor sensitivity to ONCOFID-P was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas bioconjugate interaction with cells was studied cytofluorimetrically and by confocal microscopy. In vivo toxicity was assessed by a single-dose maximum-tolerated dose, peripheral blood cell count determination and by histologic analysis. Biodistribution of the compound was evaluated with a small animal-dedicated scintigraphy gamma camera following injection of 99mTc-labeled ONCOFID-P. Pharmacokinetic analysis was also carried out. Female severe combined immunodeficiency mice implanted with ovarian cancer cells underwent treatment with ONCOFID-P or free paclitaxel starting from day 7 or 14 after tumor injection, and survivals were compared. RESULTS: ONCOFID-P interacted with CD44, entered cells through a receptor-mediated mechanism, and exerted a concentration-dependent inhibitory effect against tumor cell growth. After i.p. administration, the bioconjugate distributed quite uniformly within the peritoneal cavity, was well-tolerated, and was not associated with local histologic toxicity. Pharmacokinetic studies revealed that blood levels of bioconjugate-derived paclitaxel were much higher and persisted longer than those obtained with the unconjugated free drug. Intraperitoneal treatment of tumor-bearing mice with the bioconjugate revealed that ONCOFID-P exerted a relevant increase in therapeutic activity compared with free drug. CONCLUSIONS: ONCOFID-P significantly improved results obtained with conventional paclitaxel, in terms of in vivo tolerability and therapeutic efficacy; these data strongly support its development for locoregional treatment of ovarian cancer.

Novel types of carborane-carrier hyaluronan derivatives via "click chemistry".

Two new HA derivatives bearing carborane rings were synthesized by click chemistry. The optimal conditions were assessed for the preparation of biocompatible boron carriers, potentially suitable for application in BNCT and capable of targeting the CD44 antigen. The new polymeric samples were characterized by means of NMR-spectroscopy techniques that gave degrees of 17 and 8% for HAAACB and HapACB, respectively. Both HAAACB and HApACB turned out to be nontoxic for colorectal, ovarian and bladder tumor cell lines, to disclose a specific interaction with the CD44 antigen as the native hyaluronan moiety, and to deliver boron-atom concentrations largely sufficient for BNCT therapy when accumulated in cancer cells.

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy.

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.

HYTAD1-p20: a new paclitaxel-hyaluronic acid hydrosoluble bioconjugate for treatment of superficial bladder cancer.

OBJECTIVE: To report the development of a new water-soluble paclitaxel-hyaluronic acid bioconjugate, HYTAD1-p20, for intravesical treatment of superficial bladder cancer. MATERIALS AND METHODS: HYTAD1-p20 was synthesized by carboxyl esterification of hyaluronic acid with paclitaxel, and its physicochemical and biologic properties were characterized. RESULTS: Paclitaxel loading was optimized at 20% w/w; this procedure increased by 500-fold the paclitaxel concentration in the resulting water-soluble biomaterial. In vitro, HYTAD1-p20 exerted a much higher dose-dependent inhibitory effect against RT-4 and RT-112/84 bladder carcinoma cell growth than that of free drug, and directly interacted with CD44 expressed by bladder tumor cells. In vivo, results of pharmacokinetic studies performed in mice after bladder catheterization and intravesical instillation of HYTAD1-p20 disclosed that drug leakage was negligible during a 2-hour analysis. Histologic examination of drug-instilled bladders revealed that HYTAD1-p20 was extremely well tolerated, while paclitaxel alone produced mucosal disruption and submucosal infiltration of inflammatory cells. Treatment of severe combined immunodeficient mice bearing subcutaneous RT-112/84 tumors with maximum tolerated doses of bioconjugate or paclitaxel showed that HYTAD1-p20 exerted a therapeutic activity comparable to that of free drug. CONCLUSIONS: These data suggest that HYTAD1-p20 significantly improved results obtained with conventional paclitaxel in terms of hydrosolubility, in vitro activity against human bladder cancer cells, and in vivo biocompatibility. This bioconjugate is a potentially useful treatment for superficial urothelial malignancy.