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.

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.

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.

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 bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-adriamycin (ADR) conjugate containing lysosomally degradable oligopeptide (GFLG) side chains terminated in ADR was synthesized. The effect of free and HPMA copolymer-bound ADR on the viability of A2780 sensitive and A2780/AD multidrug resistant human ovarian carcinoma cells was studied in vitro. As expected, the IC50 dose for the HPMA copolymer-ADR conjugate was higher than for free ADR reflecting the difference in the mechanism of cell entry. The resistant A2780/AD cells demonstrated about 40-times higher resistance to free ADR than the sensitive A2780 cells. On the contrary, there was only a small difference in cytotoxicity of the HPMA copolymer-ADR conjugate toward sensitive A2780 or MDR resistant A2780/AD cells. The IC50 value for A2780/AD was only about 20% higher than the value for sensitive A2780 cells. These data seem to indicate that the HPMA copolymer-ADR conjugate may, at least partially, avoid the ATP driven P-glycoprotein (Pgp) efflux pump. The analysis of the expression of the MDR1 gene which encodes the Pgp, has shown that free ADR in high doses stimulated MDR1 gene expression in sensitive A2780 cells. At the same time both free and HPMA copolymer-ADR conjugate partially inhibited the expression of the MDR1 and beta 2 m genes in multidrug resistant A2780/AD cells.

Familial hypercholesterolemia kindred in Utah with novel C54S mutations of the LDL receptor gene.

In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To determine the genetic etiology of the lipoprotein abnormalities, we screened DNA samples for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a T-to-A transversion at nucleotide position 223, causing substitution of Ser for Cys at codon 54 in exon 3 of the receptor gene. This amino acid replacement would disrupt one of the disulfide bonds necessary for maintenance of the secondary structure of the repeat at the N-terminal of the receptor, prevent correct folding of the receptor, and result in defective intracellular transport of the receptor.

Superresistance against hypoxia after preliminary adaptation to repeated stress.

The study investigated the influence of adaptation to stress on resistance to hypoxia. After rats were adaptated to moderate restraint stress, they were anesthetized and exposed to 6% O2. Adaptation increased tidal volume by 2.6-fold, lung and alveolar ventilation by 1.6- and 1.8-fold, respectively, and O2 consumption by 1.6-fold; limited lactate accumulation in the liver by 2-fold, in the heart by 34%, in the lung by 36%, and in the blood by 36%; and elevated pH. At the same time, preliminary adaptation to stress inhibited the hypoxic activation of lipolysis and peroxidation in all tissues. The concentration of lipid peroxides decreased after adaptation by 1.3- to 1.5-fold in different organs, whereas the content of free fatty acids diminished by 1.7- to 2.3-fold. Finally, after adaptation, mortality decreased under severe hypoxia by 6.5-fold. Thus, the data suggest that the cross-protective effect of adaptation was achieved by the economization of respiration and circulation, by marked augmentation in the ability of tissue to utilize blood O2, and by the limitation of processes that are able to damage tissue membranes, namely, acidosis, lipolysis, and lipid peroxidation.

[Adaptation to stress can enhance animal resistance to sublethal hypoxia to a greater extent than adaptation to hypoxia]. / Adaptatsiia k stressu mozhet povyshat' rezistentnost' zhivotnykh k subletal'noi gipoksii v bol'shei mere, chem adaptatsii k gipoksii.

Wistar male rats were adapted to intermittent hypoxia in an altitude chamber (group 1) and to short-term immobilization stress (group 2). The animals of both series and control ones were exposed to severe hypoxic hypoxia in acute experiment: they were anesthetized and inhaled a gas mixture which contained 6% O2. In the first two hours of the experiment the death rate was 65% in control, 10% in adaptation to stress, and 29% in adaptation to hypoxia. Principal parameters of respiration, circulation, and basic-acid equilibrium were monitored during the experiment. They allowed us to establish that both the variants of adaptation increased the tissue oxygen consumption; furthermore, adaptation to hypoxia preserved a higher level of oxygen consumption than adaptation to stress. Despite this, in adaptation to stress the death rate was threefold higher than in adaptation to hypoxia. This result was explained by the accumulation of heat shock proteins in cells during adaptation to stress and the resultant development of a phenomenon of adaptive stabilization of structures (PhASS). In adaptation to hypoxia the PhASS did not develop.

[Super-resistance to hypoxic hypoxia in adaptation to stress exposures: its possible mechanisms]. / Superrezistentnost' k gipoksicheskoi gipoksii pri adaptatsii k stressornym vozdeistviiam: ee vozmozhnye mekhanizmy.

Adaptation of animals to repeated immobilization stress is shown to stimulate development of super-resistivity to severe hypoxic hypoxia. This phenomenon manifests itself by the fact that breathing hypoxic gas mixture containing 60% O2 causes 65% mortality among control rats and only 10% mortality among adapted animals within 2 hrs of experiment. Inquiry into mechanisms responsible for this multiple rise in resistivity showed that adaptation to stress increases rat tolerance of hypoxia due to economization of breathing and circulation, considerable strengthening of the tissue abilities to take up O2 from blood despite expressed hypoxemia, and due to limited acidosis. Adaptation also significantly suppresses hypoxia-induced processes of damaging cellular membranes, namely, activation of lipolysis and lipid peroxidation. The role of stress-limiting systems and stabilization of structures in the cross defense effect of adaptation to stress is discussed.

[Development of super-resistance to hypoxic hypoxia during adaptation to short-term stress]. / Razvitie superrezistentnosti k gipoksicheskoi gipoksii pod vliianiem adaptatsii k kratkovremennym stressornym vozdeistviiam.

Adaptation to intermittent short-term immobilization stress exposure increases the resistance of rats to hypoxic hypoxia. This was evidenced by the fact that in acute experiments in respiration with gas-mixture containing 6% of oxygen, the mortality of control rats was 65% compared with 10% of that of adapted rats. It has been estimated that this protective cross-effect of adaptation to stress is due to more stable and efficient mobilization of external respiration in adapted animals, and to a greater extent, to the fact that adapted rats tissues have gained a capacity of extracting more oxygen from hypoxemic blood compared to control animals. The role of previously stated phenomenon of adaptive stabilization of structures in superresistance to hypoxic hypoxia is discussed.

Severe hypoxia activates lipid peroxidation in the rat brain.

The level of lipid peroxides, concentration of lactic acid in the blood and brain tissue homogenates were measured in rats under hypoxic hypoxia and hemorrhage. Significant correlation was found between the O2 supply-consumption ratio, the concentration of lactate and the level of lipid peroxides. It is suggested that the peroxidation process is activated by hypoxia.

[The physiological mechanisms of the antihypoxic action of the liposomes]. / Nekotorye fiziologicheskie mekhanizmy antigipoksicheskogo deistviia liposom.

The effect of phospholipids introduced into the vascular bed in the form of liposomes, was studied in pats in conditions of breathing with hypoxic gas mixture containing 7% of oxygen in nitrogen. The liposomes were shown to improve the oxygen diffusion from the blood into tissues and from air to the blood. In the result of this, the degree of the tissue hypoxia is considerably reduced, the process of peroxide oxidation of lipids is inhibited, and the efficacy of external respiration and gas exchange is increased.

[The mechanisms of the disorder of external respiration in hypoxia]. / Mekhanizmy narusheniia vneshnego dykhaniia pri gipoksii.

Anesthetized rats breathed during 1 hr with gas mixtures containing 16 to 6 per cent of oxygen. At 6 per cent of oxygen, the hypoxia of pulmonary tissue and its processes of peroxide oxidation of lipids entails a sharp increase in elastic resistance against breathing. This is combined with insignificant disorders in tidal volume which is not compensated by an increase in the breathing rate. General mechanisms of external respiration disorders in various types of hypoxia and in stress, were revealed.

[Biological effect of liposomes in hypoxic conditions of various etiologies]. / Biologicheskii éffekt liposom pri gipoksicheskikh sostoianiiakh razlichnoi étiologii.

The antihypoxic and antioxidative effects of phosphatidyl choline liposomes have been studied in hypoxic hypoxia, pneumonia, and acute blood loss. It was demonstrated that the body tolerance of persisting hypoxia increased on liposome administration due to elimination of lactate-acidosis, inhibition of lipid peroxidation and higher rate of oxygen diffusion through the biological barriers. The antihypoxic properties of the vesicles are determined by their remedial effect on the key mechanisms responsible for the development of hypoxic organ damage.

[Disorders of external respiration and oxygen transport and utilization in stress]. / Narusheniia vneshnego dykhaniia, transporta i utilizatsii kisloroda pri stresse.

A specific hypoxic condition characterized by a combination of signs of hypoxic, circulatory, and primary tissue hypoxia develops in rats after 6-hour immobilization stress. Post-stress hypoxia is accompanied with accumulation of lactate, decompensated metabolic acidosis, and activation of lipid peroxidation processes in the blood and tissues. Besides, a complex of changes of external respiration, its regulation and biomechanics, inhibition of pulmonary gas exchange is encountered after the stress, which is linked with the development of specific stress damage to the lungs which can be characterized as the stress lung syndrome.

[Automatic installation for measuring the volume-time parameters of external respiration and gas exchange in small laboratory animals]. / Avtomatizirovannaia ustanovka dlia izmereniia ob''emno-vremennykh parametrov vneshnego dykhaniia i gazoobmena u melkikh laboratornykh zhivotnykh.

Automatic installation based on the personal computer is suggested which permits fast digital measurements of basic indices of respiration and pulmonary gas exchange in small experimental animals.

[External respiration, gas exchange and blood acid-base status in dogs with hyperthermia]. / Vneshnee dykhanie, gazoobmen i kislotno-osnovnoe sostoianie krovi pri gipertermii u sobak.

Experiments on dogs have shown that hyperthermia intensifies respiration, increases oxygen consumption, induces pronounced discrepancy of the alveolar ventilation to carbon dioxide elimination, severe hypocapnia and decompensated respiratory alkalosis.

[Use of liposomes for correction of respiratory hypoxia in experimental pneumonia]. / Primenenie liposom dlia korrektsii respiratornoi gipoksii pri éksperimental'noi pnevmonii.

White laboratory rats, weighted 100-160 g, with acute pneumonia, inhale (from 4-th to 8-th days of developing the disease) liposomes. It was shown, that introduction of liposomes promoted normalization of external breath, acid-base state of blood, increased diffusion ability of lung for O2 and decreased arterial hypoxia. It was noted, that liposomes are an effective antihypoxant, inhibited the process of lipid peroxidation and, evidently, the destruction of cell membranes.