Inhalation treatment of cystic fibrosis with lumacaftor and ivacaftor co-delivered by nanostructured lipid carriers.

Cystic fibrosis (CF), a most deadly genetic disorder, is caused by mutations of CF transmembrane receptor (CFTR) - a chloride channel present at the surface of epithelial cells. In general, two steps have to be involved in treatment of the disease: correction of cellular defects and potentiation to further increase channel opening. Consequently, a combinatorial simultaneous treatment with two drugs with different mechanisms of action, lumacaftor and ivacaftor, has been recently proposed. While lumacaftor is used to correct p.Phe508del mutation (the loss of phenylalanine at position 508) and increase the amount of cell surface-localized CFTR protein, ivacaftor serves as a CFTR potentiator that increases the open probability of CFTR channels. Since the main organ that is affected by cystic fibrosis is the lung, the delivery of drugs directly to the lungs by inhalation has a potential to enhance the efficacy of the treatment of CF and limit adverse side effects upon healthy tissues and organs. Based on our extensive experience in inhalation delivering of drugs by different nanocarriers, we selected nanostructured lipid carriers (NLC) for the delivery both drugs directly to the lungs by inhalation and tested NLC loaded with drugs in vitro (normal and CF human bronchial epithelial cells) and in vivo (homozygote/homozygote bi-transgenic mice with CF). The results show that the designed NLCs demonstrated a high drug loading efficiency and were internalized in the cytoplasm of CF cells. It was found that NLC-loaded drugs were able to restore the expression and function of CFTR protein. As a result, the combination of lumacaftor and ivacaftor delivered by lipid nanoparticles directly into the lungs was highly effective in treating lung manifestations of cystic fibrosis.

Characterization and application of a nose-only exposure chamber for inhalation delivery of liposomal drugs and nucleic acids to mice.

BACKGROUND: A small nose-only exposure chamber was evaluated for inhalation delivery of drug carrier systems (DCSs) to mice for the treatment of lung cancer. The chamber then was used for inhalation delivery of an anticancer drug, antisense oligonucleotides (ASO), and small interfering RNA (siRNA) directly to the cancerous lungs of mice. METHODS: The uniformity of particle delivery across the ports of the exposure chamber and stability of the DCS (liposomes) during continuous aerosolization by a Collison nebulizer were examined. The mean produced particle size by number was approximately 130 nm, and the mass median diameter was approximately 270 nm. The system was then used to deliver DCS containing doxorubicin (DOX) and ASO or siRNA targeted to multidrug resistance-associated protein 1 (MRP1) mRNA as suppressors of cancer cell resistance. The retention of the drug in the lungs and the effect on tumor size were compared after inhalation delivery and intravenous injection in a nu/nu mouse model of lung cancer. RESULTS: The aerosol mass across the four inhalation ports had a coefficient of variation of less than 12%, and approximately 1.4% of the nebulized mass was available for inhalation at each port. The mean size of 130 nm of liposomal DCS did not change significantly during continuous 60-min aerosolization. For inhalation delivery of DCS with DOX+ASO/siRNA, the amount of drugs available for inhalation was lower compared with intravenous injection of DOX; however, the observed lung dose and the retention time were significantly higher. The delivery of DOX+ASO/siRNA via inhalation resulted in tumor volume reduction of more than 90%, whereas only about 40% reduction was achieved after intravenous injection of DOX. CONCLUSIONS: The investigated exposure system is suitable for inhalation delivery of complex DCS, and its use to deliver DCS containing anticancer drugs and resistance suppressors via inhalation offered a superior method for lung cancer treatment in mice compared with intravenous injections.

Preliminary evaluation of the genotoxic potential of a hydrophilic polymer with three preservation systems.

This study compared the genotoxic potential of a polymeric associative thickener used in topically applied emulsions preserved with three different preservative systems. The method used for the assessment of genotoxicity is the in vitro micronucleus test [Organization for Economic Cooperation and Development (OECD) guideline number 487]. When changing an additive such as a preservation system in a raw material, it is crucial to re-evaluate its toxicity potential because this change may significantly alter its properties. This study shows that at the levels tested neither of the systems evaluated demonstrated any cytotoxic or genotoxic effects. Skin exposure must take into consideration factors such as duration, skin condition and metabolism, but most importantly concentration. Although preservatives can be toxic at high concentrations, they are usually safe at the concentrations used in cosmetic raw materials and formulations. If used to preserve raw materials, they undergo further dilution when added to the formulation.

Genotoxic potential evaluation of a cosmetic insoluble substance by the micronuclei assay.

An optical brightener (OB) powder (INCI: sodium silicoaluminate (and) glycidoxypropyl trimethyloxysilane/PEI-250 cross fluorescent brightener 230 salt (and) polyvinylalcohol crosspolymer) that is used in cosmetic facial products was tested for its genotoxic potential using the micronuclei test (MNT). It is a solid dry powder with an average size of 5 microns that is insoluble but dispersible in water. This study describes the exposure of cell culture to positive controls with and without enzymatic activation and to the test compound in different concentrations. We evaluated three end points: microscopic observation and quantification of micronuclei formation, and cell viability and proliferation. Both positive controls induced significant changes that were observed under the microscope and quantified. Based on its chemical nature, it was not anticipated that the test substance will degrade under the conditions of the experiments. However, the test is required to make sure that when solublized, impurities that may be present, even at trace levels, will not induce a genotoxic effect. The test compound did not promote micronuclei formation or change the viability or proliferation rate of cells. During this study we faced challenges such as solubilization and correlating viability data to genotoxicity data. These are described in the body of the paper. We believe that with the emergence of the 7(th) European amendment that bans animal testing, sharing these data and the study protocol serves as a key in building the understanding of the utilization of in vitro studies in the safety assessment of cosmetic ingredients.

Migraine in the Andes and headache at sea level.

In Cerro de Pasco (CP), Peru (altitude 4338 m) 24% of men have migraine with aura. We studied 30 men. Twenty CP natives, examined in CP, were rated using a chronic mountain sickness (CMS) score to separate controls (10) from those with CMS (10), a maladaptation syndrome in natives to altitude which includes severe, recurring headache. We collected white cells in CP and, from the same men, within 1 h of arrival in Lima (150 m above sea level). Ten normal US men volunteered white cells for comparison. After RNA extraction we assessed gene expression by reverse transcription-polymerase chain reaction. Low ATP1A1 subunit of the ATPase gene mRNA expression in CP was correlated with headache (P=0.002), acral paraesthesias (P=0.004) and CMS score (P<0.001). ATP1A1 subunit expression was increased in all Andeans in Lima (P<0.001). There were no differences between Andean controls in Lima and US controls. Manipulation of Na+/K+ATPase could offer relief for migraineurs at sea level.

Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. Invariably the side effects impose dose reduction, treatment delay, or discontinuance of therapy. To limit the adverse side effects of cancer chemotherapy on healthy organs, we proposed a drug delivery system (DDS) with specific targeting ligands for cancer cells. The proposed DDS minimizes the uptake of the drug by normal cells and enhances the influx and retention of the drug in cancer cells. This delivery system includes three main components: (i) an apoptosis-inducing agent (anticancer drug), (ii) a targeting moiety-penetration enhancer, and (iii) a carrier. We describe one of the variants of such a system, which utilizes camptothecin as an apoptosis-inducing agent and poly(ethylene glycol) as a carrier. Luteinizing hormone-releasing hormone (LHRH) was used as a targeting moiety (ligand) to LHRH receptors that are overexpressed in the plasma membrane of several types of cancer cells and are not expressed detectably in normal visceral organs. The results showed that the use of LHRH peptide as a targeting moiety in the anticancer DDS substantially enhanced the efficacy of chemotherapy, led to amplified apoptosis induction in the tumor, and minimized the side effects of the anticancer drug on healthy organs. The LHRH receptor targeting DDS did not show in vivo pituitary toxicity and did not significantly influence the time course or the plasma concentration of luteinizing hormone and its physiological effects on the reproductive functions of mice.

Molecular targeting of drug delivery systems to cancer.

This review presents molecular targeting approaches in anticancer drug delivery systems (DDS) and identifies new developments in these systems. Targeting approaches include passive targeting (enhanced permeability and retention effect), targeting specific tumor conditions, topical delivery and active targeting, namely, targeting organs, cells, intracellular organelles and molecules, sandwich targeting, promoter targeting, indirect targeting and targeting by external stimuli. A novel advanced proapoptotic anticancer DDS that utilizes several molecular targets will be considered. Experimental data suggest that this DDS can simultaneously: (1) induce cell death, (2) prevent adverse effects on healthy tissues; (3) suppress and prevent multidrug resistance; and (4) inhibit cellular antiapoptotic defense.

Molecular targeting of drug delivery systems to ovarian cancer by BH3 and LHRH peptides.

Novel targeted proapoptotic anticancer drug delivery systems were developed and evaluated. Poly(ethyleneglycol) (PEG) conjugates were used as carriers. Camptothecin (CPT) was used as an anticancer agent-apoptosis inductor. Two types of molecular targets were investigated: (1) an extracellular membrane receptor specific to ovarian cancer and (2) intracellular controlling mechanisms of apoptosis. Synthetic peptides similar to luteinizing hormone-releasing hormone (LHRH) and BCL-2 homology 3 (BH3) peptide were used as a targeting moiety and a suppressor of cellular antiapoptotic defense, respectively. Three different conjugates (CPT-PEG, CPT-PEG-BH3 and CPT-PEG-LHRH) were synthesized and examined in A2780 human ovarian cancer cells. Cytotoxicity, expression of genes encoding BCL-2, BCL-XL, SMAC, APAF-1 proteins and caspases 3 and 9, the activity of caspases 3 and 9 and apoptosis induction were studied. Taken together the results indicate much higher cytotoxicity and apoptosis-inducing activity of PEG-CPT conjugates when compared to free CPT. Moreover, the effects of targeted CPT-PEG-BH3 and CPT-PEG-LHRH conjugates were more pronounced than the non-targeted PEG-CPT conjugate. The results confirmed the feasibility of this new two-tier molecular targeting strategy for enhancing the efficacy of cancer chemotherapy.

Enhancing the efficacy of chemotherapeutic drugs by the suppression of antiapoptotic cellular defense.

The study was aimed at evaluating the combination of a traditional anticancer drug doxorubicin (DOX) with a suppressor of antiapoptotic cellular defense--synthetic peptide corresponding to the minimal sequence of BCL-2 homology 3 (BH3) domain. BH3 peptide was delivered into cells by fusion with a peptide corresponding to the Antennapedia (Ant) internalization sequence. The cytotoxicity of DOX, Ant-BH3 and Ant-BH3 mixed in with DOX, mitochondrial transmembrane potential, expression of genes encoding pro- and antiapoptotic members of BCL-2 protein family and caspases, caspases activity, apoptosis induction were assessed in human ovarian carcinoma cells. It was found that the combination in one drug formulation of DOX and Ant-BH3 produced two main effects: (1) enhancing the apoptosis induction by an anticancer drug, and (2) preventing the development of antiapoptotic cellular drug resistance. The results confirmed that anticancer drug-BH3 combination might form the basis for a new advanced anticancer proapoptotic drug delivery systems.

Selected contribution: Lung hypoxia: antioxidant and antiapoptotic effects of liposomal alpha-tocopherol.

The aim of this study is to examine the antioxidant and antiapoptotic activity of liposomal alpha-tocopherol (LAT) in anesthetized rats exposed to severe hypoxia. It was shown that intratracheal application of LAT normalized lung phospholipid composition and inhibited lipid peroxidation in lung tissues, which in turn decreased lung edema and damage and improved breathing pattern, oxygen diffusion, and lung gas exchange. LAT also limited the overexpression of genes encoding hypoxia inducible factor-1alpha and both studied forms of phospholipase A(2), and it increased the power of cellular antioxidant and antiapoptotic defense by overexpressing genes encoding Mn- and Cu-Zn-cofactored superoxide dismutases, Bcl-2, and heat shock 70 proteins. The overexpression of studied caspases and their activity were downregulated, which significantly (1.6-2 times) limited apoptosis in lung cells. Finally, all these positive changes decreased mortality during hypoxia from approximately 60% in untreated animals to approximately 30% in the group of rats treated with LAT. The data obtained indicate that LAT may be useful for the correction of hypoxic lung injury.

Water soluble polymers in tumor targeted delivery.

The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained. Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.

Preliminary evaluation of caspases-dependent apoptosis signaling pathways of free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

The purpose of the study was to examine the role of caspases in signaling pathways of apoptosis induced by free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) in human ovarian carcinoma cells. Sensitive A2780 and DOX resistant A2780/AD cells were exposed to different doses of drugs within 12, 18, 24 and 36 h. Caspase activity, expression of genes encoding human caspases 1-10, Apaf-1 and bcl-2 proteins and apoptosis were studied. In sensitive cells both free and P(GFLG)-DOX activated caspases 3, 7 and 9. In addition, P(GFLG)-DOX activated caspases 6 and 8. In resistant cells apoptosis induced by free DOX depended on the activation of caspases 2, 7 and 9, while caspase 3 was not involved; this explains the low degree of apoptosis induced by free DOX in resistant cells. P(GFLG)-DOX triggered the additional caspases 3, 6 and 8. A more pronounced degree of caspase activation and apoptosis after the action of P(GFLG)-DOX depended on the inhibition of bcl-2-encoded cellular defensive mechanisms and a more significant activation of Apaf-1. It was concluded that HPMA copolymer-bound DOX induced additional caspase-dependent apoptosis signaling pathways and the degree of the induction was higher, which led to more pronounced apoptosis when compared to free DOX.

Time- and concentration-dependent apoptosis and necrosis induced by free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

A2780 sensitive and A2780/AD doxorubicin (DOX) resistant human ovarian carcinoma cells were exposed to different concentrations (0.25, 0.5, 1, 5 and 10xIC(50)) of free and HPMA copolymer-bound DOX for 12, 24, 36, 48, 60 and 72 h. Apoptosis and necrosis were evaluated using the FITC-conjugated annexin V and propidium iodide staining. The data obtained showed that the induction of apoptosis and necrosis by both free DOX and HPMA copolymer-bound DOX were time- and concentration-dependent. The data also showed significant differences between the drugs. It was found that: (i) under the action of HPMA copolymer-bound doxorubicin the alterations in the plasma membrane permeability preceded disturbances in cellular metabolism; (ii) HPMA copolymer-bound doxorubicin kills the cells mainly by necrosis; (iii) HPMA copolymer-bound doxorubicin is a more effective anticancer drug than free doxorubicin.

The influence of cytotoxicity of macromolecules and of VEGF gene modulated vascular permeability on the enhanced permeability and retention effect in resistant solid tumors.

PURPOSE: To study the influence of cytotoxicity of macromolecules, VEGF gene expression, and vascular permeability on the enhanced permeability and retention (EPR) effect. METHODS: Mice bearing xenografts of A2780 multidrug resistant human ovarian carcinoma were treated by free doxorubicin (DOX) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound DOX (P(GFLG)-DOX), Texas Red (P-TR), and FITC (P-FITC). Antitumor activity, drug distribution in tumor, vascular permeability, VEGF gene expression, and DNA fragmentation were studied. RESULTS: The accumulation of free DOX led to the VEGF gene overexpression and increased the vascular permeability, which in turn enhanced the drug accumulation in the same location. This positive feedback loop led to a highly inhomogeneous distribution of the drug within the tumor. In contrast, P(GFLG)-DOX down-regulated the VEGF gene and decreased vascular permeability. This negative feedback seemed to prevent additional drug accumulation in dead necrotic tissue, resulting in a more uniform drug distribution and enhanced the antitumor activity P(GFLG)-DOX. CONCLUSIONS: The EPR effect significantly differed for macromolecules containing DOX when compared to macromolecules without drug. The cytotoxicity of P(GFLG)-DOX amplified the EPR effect, led to a more homogenous distribution of the drug, increased the average drug concentration in tumor and augmented its efficacy.

HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action.

The design, synthesis and properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers as carriers of anticancer drugs are reviewed. Macromolecular therapeutics based on HPMA copolymers are biocompatible, preferentially accumulate in tumors, and possess a higher anticancer efficacy than low molecular weight drugs. Novel designs of HPMA copolymer carriers resulted in long-circulating conjugates and gene and oligonucleotide delivery systems. HPMA copolymer based macromolecular therapeutics were active against numerous cancer models and are in clinical trials. The data obtained indicated that macromolecular therapeutics activated different signaling pathways and possessed a different mechanism of action than free drugs. This bodes well for the success of future research aimed at identification of new intracellular molecular targets as a basis for the design of the second generation of macromolecular therapeutics.

Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma.

Anticancer activity and main mechanisms of action of free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) were studied in solid tumor mice models of DOX sensitive and resistant human ovarian carcinoma. Free DOX was effective only in sensitive tumors decreasing the tumor size about three times, whereas P(GFLG)-DOX decreased the tumor size 28 and 18 times in the sensitive and resistant tumors. An enhanced accumulation of P(GFLG)-DOX in the tumor was observed, whereas only low concentrations of DOX were detected in other organs following P(GFLG)-DOX administration. This effect was dependent on the high permeability of blood vessels in untreated tumors. After treatment with P(GFLG)-DOX the permeability decreased concomitantly with the downregulation of VEGF gene expression. P(GFLG)-DOX effectively killed both types of tumors inducing apoptosis and necrosis through the activation of p53, Apaf-1, caspase 9, c-fos, or c-jun pathways, and the downregulation of the bcl-2 gene. HPMA copolymer-bound DOX preserved its activity inside cells, inhibited detoxification and defensive mechanisms encoded by GST-pi, BUDP, and HSP-70 genes, and limited DNA repair, replication, and biosynthesis by downregulation of Topo-IIalpha,beta, and TK1 genes. P(GFLG)-DOX also produced tumor tissue hypoxia and significantly activated lipid peroxidation in tumors. No damage to other organs after exposure to P(GFLG)-DOX was detectable. On the other hand, free DOX activated lipid peroxidation and led to tissue hypoxia in many organs. All data relevant to the mechanism of anticancer action of P(GFLG)-DOX indicated a higher antitumor activity and lower systemic toxicity of HPMA copolymer-bound DOX when compared with free DOX.

Synthesis of starlike N-(2-hydroxypropyl)methacrylamide copolymers: potential drug carriers.

Starlike HPMA copolymers were synthesized by conjugating semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide] macromolecules (ST-PHPMA, arm) with PAMAM dendrimers (core: G2, G3, G4). ST-PHPMA was synthesized by chain transfer free radical polymerization, and the terminal -COOH was activated with N-hydroxysuccinimide. Doxorubicin (DOX) was introduced into the starlike HPMA copolymer to evaluate its potential as a drug delivery system. The polymers were characterized with SEC, NMR, and UV. Cytotoxicity of the DOX containing starlike HPMA copolymer was determined on an A2780 human ovarian carcinoma cell line and compared with DOX-containing linear HPMA copolymers. The rate of in vitro DOX release from polymer--DOX conjugates in the presence of cathepsin B (CP-B, lysosomal cysteine proteinase) was determined and correlated with cytotoxicity results.

HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line.

The effect of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Adriamycin-OV-TLl6 antibody conjugate [P(GFLG)-ADR-Ab] on OVCAR-3 human ovarian carcinoma cells was studied. A nontargeted HPMA copolymer-ADR conjugate (P(GFLG)-ADR) and free ADR were the controls. The IC(50) doses were 0.65, 3.0, and 65 microM for free ADR, targeted P(GFLG)-ADR-Ab conjugate, and nontargeted P(GFLG)-ADR conjugate, respectively. These differences reflect the different mechanisms of cell entry of the compounds evaluated. Free ADR and HPMA copolymer-ADR conjugates had different impacts on the expression of MDR1, MRP, c-fos, c-jun, and bcl-2 genes which encode the P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP) efflux pumps, and play an important role in cell death signaling pathways (c-fos, c-jun, and bcl-2). Whereas high doses of free ADR induced MDR1 gene expression, HPMA copolymer-bound ADR appeared to be without effect. On the contrary, expression of the MRP gene was not influenced by free ADR, whereas HPMA copolymer-ADR conjugates seemed to suppress the gene expression in a concentration-dependent manner. There were differences in the expression of c-fos, c-jun, and bcl-2 genes after the incubation of OVCAR-3 cells with free and HPMA copolymer-bound ADR indicating differences in activation of cell death signaling pathways.