Immunotoxic effects of cyclophosphamide and cyclosporine in the dog.

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation.

Systemic exposure to parabens: pharmacokinetics, tissue distribution, excretion balance and plasma metabolites of [14C]-methyl-, propyl- and butylparaben in rats after oral, topical or subcutaneous administration.

Parabens (PB) are preservatives used in food, drugs and personal care products preventing microbial and fungal contamination. We investigated ADME profiles of [14C]-methyl-, propyl- or butylparaben (MP, PP, BP) following single oral, dermal or subcutaneous (BP) doses at 100 mg/kg to Sprague-Dawley rats. Plasma Cmax and AUC values after oral or subcutaneous doses were 4- to 10-fold higher relative to respective values after dermal administration. tmax ranged from 0.5, 2 or 8 h after oral, subcutaneous or dermal administration, respectively. MP produced higher blood Cmax and AUC levels relative to those after PP or BP. Following oral or subcutaneous administration, urinary excretion was predominant (>70%, mainly during the first 24 h), less than 4% were eliminated in the feces, 2% were retained in the tissues and carcasses. Following dermal application, >50% of the dose was unabsorbed, 14-27% or <2% were respectively excreted in the urine or feces, respectively. Overall, parabens were well absorbed after oral and subcutaneous, and partially absorbed after dermal administration. All administration routes produced a single peak in the plasma, corresponding to that of para-hydroxybenzoic acid (PHBA) suggesting that PB produce no significant systemic exposure of mammalian organisms after oral, topical or subcutaneous administration.

Improved anti-tumoral capacity of mixed and pure anti-oestrogens in breast cancer cell xenografts after their administration by entrapment in colloidal nanosystems.

Anti-oestrogens (AEs) are currently used for treating hormone-dependent breast cancers. They specifically bind to oestrogen receptors (ERs) and inhibit their transactivation capacity. However, ERs are present in various other tissues in which AEs may have either a beneficial or detrimental action. AE administration via systems targeting breast tumours may be an important therapeutic improvement. Thus, several biodegradable drug delivery systems containing either "mixed" (4-hydroxytamoxifen - 4-HT) or "pure" (RU 58668 - RU) AEs were prepared. Liposomes and nanospheres (NS, composed of non-toxic and biodegradable lipids and poly(d,l-lactic acid) incorporated up to 1 and 0.5 mM AE, respectively. Nanocapsules (NCs) in which an oily core solubilises the AE incorporated no more than 0.02 mM of the drug. PEG-functionalised nanoparticles survived longer in plasma and had better controlled release of the drug. The small size of the vectors (100-250 nm) was compatible with their extravasation through the discontinuous endothelium of tumour vasculature, allowing their accumulation in MCF-7 cell xenografts and leading to a prolonged exposure of the tumour to AEs. In these tumours and in MCF-7/ras xenografts, RU-NS and RU-NC (6.5mg/kg/week and 0.27 mg/kg/week, respectively, doses at which free RU had a very weak effect), both inhibited tumour growth. Entrapped RU significantly induced involution of tumours and strongly induced apoptosis in tumour cells, concomitantly with inhibiting tumour angiogenesis. 4-HT-nanoparticles also arrest oestradiol-induced tumour growth, inducing apoptosis and inhibiting angiogenesis. However, unlike RU-nanoparticles, they did not promote ERalpha subtype loss in tumour cells. Subcutaneous administration of both RU- and 4-HT-NS in MCF-7 xenografts strongly arrested tumour growth for prolonged periods and RUNS decreased the number of tumour epithelial cells. Analysis of the proteins involved in cell cycle proliferation and apoptosis confirmed that RU-nanoparticles were more efficient than 4-HT-nanoparticles. Their lack of toxicity and high anti-tumour potency that affects only tumour cells in the xenograft models mean these AE-loaded colloidal systems are a breakthrough in hormone-dependent breast cancer treatment.

Innovative drug delivery nanosystems improve the anti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiple myeloma.

Anticancer drug efficiency is governed by its bioavailability. In order to increase this parameter, we synthesized several injectable and biodegradable systems based on incorporation of anti-estrogens (AEs) in nanoparticles (NPs) and liposomes were synthesized. Both nanospheres (NS) and nanocapsules (NCs, polymers with an oily core in which AEs were solubilized) incorporated high amounts of 4-hydroxy-tamoxifen (4-HT) or RU 58668 (RU). Physico-chemical and biological parameters of these delivery systems, and coupling of polyethylene-glycol chains on the NP surface revealed to enhance the anti-tumoral activity of trapped AEs in a breast cancer MCF-7 cell xenograft model and to induce apoptosis. These features correlated with an augmentation of p21(Waf-1/Cip1) and of p27(Kip1) and a concomitant decrease of cyclin D1 and E in tumor extracts. Liposomes containing various ratios of lipids enhanced the apoptotic activity of RU in several multiple myeloma (MM) cell lines tested by flow cytometry. MM cell lines expressed both estrogen receptor alpha and beta subtypes except Karpas 620. Karpas 620 cells which did not respond to AEs became responsive following ER cDNA transfection. A new MM xenograft model was generated after s.c. injection of RPMI 8226 cells in nude mice. RU-loaded liposomes, administered i.v. in this model, at a dose of 12mgRU/kg/week, induced the arrest of tumor growth contrary to free RU or to empty liposomes. Thus, the drug delivery of anti-estrogens enhances their ability to arrest the growth of tumors which express estrogen receptors and are of particular interest for estrogen-dependent breast cancer treatment. In addition it represents a new potent therapeutic approach for multiple myeloma.

Drug delivery systems for oestrogenic hormones and antagonists: the need for selective targeting in estradiol-dependent cancers.

The pleiotropic activity of oestrogens and their mechanism of action via their binding to the two oestrogen receptors alpha (ER alpha) and beta (ER beta) subtypes in the different tissues where oestrogens exert their action have been briefly described. The fate of these compounds trapped into different galenic forms is discussed with regard to their therapeutic applications. Firstly, the advantages and disadvantages of the different forms (pills, i.v. forms and transdermal patches) used in contraception are compared. Secondly, the therapeutic use of formulated oestrogens for the post-menopausal hormone replacement therapy (HRT) is analysed through the various results obtained in different trials. The link between HRT and the risks of breast cancer and cardiovascular disease is underlined. Finally, comparing the activity of selective oestrogen receptor modulators such as tamoxifen and pure anti-oestrogens such as RU58668 and ICI182780, we analysed the reasons leading to the need for a tumor targeting of the latters, but not of the former for the treatment of oestrogen-dependent breast cancer. Different injectable and biodegradable formulations, that lead to a remarkable anti-tumor efficiency in xenografts, have been recently developed and we believe that they may represent promising new administration ways of added therapeutic values for anti-oestrogens. Such devices could be extended to the delivery of other anti-cancer drugs with more aggressive activities than anti-oestrogens.

Pure antiestrogen RU 58668-loaded nanospheres: morphology, cell activity and toxicity studies.

Nanospheres (NS) formulated using biodegradable and biocompatible polymers, poly(D,L-lactide-co-glycolide) (PLGA), poly(D,L-lactide) (PLA) and poly(epsilon-caprolactone) (PCL), loaded with the pure anti-estrogen RU 58668 (RU), a promising estrogen-dependent anticancer agent, have been prepared. They all possess a small size compatible with an intratumoral extravasation behavior and their pegylation reduce significantly their zeta potential. Characterization by freeze fracture electron microscopy have shown that NS are spheric particles with a size ranging between 30 and 50nm and a tendency to agglomerate which is reduced by polyethylene glycol (PEG) grafting. PEG-grafted NS are all non-toxic as revealed by cell viability assay. A specific cellular model has been used to evaluate not only the release extent of the drug but also its biological activity. All formulations tested showed that they release slowly RU as measured by the delayed ability of RU to inhibit estrogen-induced transcription in human breast cancer cells and that they possess only a small amount of surface adsorbed RU.

In vitro and in vivo biologic evaluation of long-circulating biodegradable drug carriers loaded with the pure antiestrogen RU 58668.

We have developed a parenteral delivery system for the administration of the highly promising pure antiestrogen RU 58668 (RU). Two types of nanoparticles (NP) made of biodegradable copolymers and coated with polyethylene-glycol (PEG) chains were prepared: nanospheres (NS) (diameter, approximately 110 nm) and nanocapsules (NC) with an oily core (diameter, approximately 250 nm). The amount of RU incorporated into NS and NC was approximately 33 vs. approximately 5 microg RU/mg of polymer, respectively. Coating with PEG chains prolonged the antiestrogenic potency of RU, as shown by a prolonged antiuterotrophic activity of encapsulated RU into PEG-poly(D,L lactic acid) (PLA) NS, as compared to that of conventional nonpegylated NS. In mice bearing MCF-7 estrogen-dependent tumors, free RU injected at 4.3 mg/kg/week by i.v. route slightly decreased the estradiol-promoted (0.5 mg/kg/week) tumor growth while RU-loaded PEG-PLA NS injected at the same dose strongly reduced it. Analysis of cell cycle parameters in tumors treated with RU indicated that RU-loaded PEG-PLA NS injected at 4.3 mg/kg/week in MCF-7 tumors decreased cyclin D(1) and cyclin E simultaneously, and increased p27. The antitumoral activity of RU encapsulated within pegylated NC was stronger than that of RU entrapped with pegylated NS loaded at an equivalent dose. Indeed, the former decreased the tumor size in nude mice transplanted with the estrogen receptor-positive but estrogen-independent MCF-7/Ras breast cancer cells at a concentration 2.5 times lower than that of the latter (0.4 mg/kg/week compared to 1 mg/kg/week). Empty PEG-PLA NS and NC were devoid of antiuterotrophic and antitumoral activities. Altogether, these results suggest that the incorporation of the pure antiestrogen RU into long-circulating NP could represent a novel antiestrogen drug delivery system for the parenteral route.

Polyester-poly(ethylene glycol) nanoparticles loaded with the pure antiestrogen RU 58668: physicochemical and opsonization properties.

PURPOSE: The pure antiestrogen RU58668 (RU) was encapsulated within nanospheres (NS) and nanocapsules (NC) prepared from different polyester copolymers with poly(ethylene glycol) (PEG) chains. The influence of their physicochemical properties on drug release in vitro and their susceptibility to opsonization were evaluated. METHODS: RU-loaded PEG-bearing nanoparticles (NP) prepared by interfacial deposition of preformed polymer were characterized (size, zeta potential, percentage encapsulation and loading). In vitro release kinetics were studied in the presence of 10% fetal calf serum (FCS). Their opsonization in mouse serum was evaluated by silver staining of SDS-PAGE and Western blotting of desorbed proteins. RESULTS: The NS were smaller than NC and had a zeta potential close to zero and a higher percentage of loading. RU release from NS in vitro was reduced as compared with the dissolution profile of free RU in a serum-containing medium. Decreased opsonin adsorption at the surface of pegylated NS was observed. CONCLUSION: Small nanoparticulate systems containing a high load of pure antiestrogen, showing reduced drug release, have been developed. Among the six nanosphere preparations containing RU, two show a size below 200 nm, and two others undergo reduced protein adsorption in the presence of serum, compatible with increased persistence in the blood.