[Study of the efficiency of cellular accumulation of doxorubicin supplied with a targeted delivery system based on phospholipid nanoparticles with integrin-directed peptide]. / Issledovanie éffektivnosti kletochnogo nakopleniia doksorubitsina, snabzhennogo targetnoi sistemoi dostavki na osnove fosfolipidnykh nanochastits c integrin-napravlennym peptidom.

Chemotherapeutic agents containing targeted systems are a promising pathway to increase the effectiveness of glioblastoma treatment. Specific proteins characterized by increased expression on the surface of tumor cells are considered as possible targets. Integrin αvß3 is one of such proteins on the cell surface. It effectively binds the cyclic Arg-Gly-Asp (cRGD) peptide. In this study, the cRGD peptide-modified doxorubicin (Dox) phospholipid composition was investigated. The particle size of this composition was 43.76±2.09 nm, the ζ-potential was 4.33±0.54 mV. Dox was almost completely incorporated into the nanoparticles (99.7±0.58%). The drug release increased in an acidic medium (at pH 5.0 of about 35±3.2%). The total accumulation and internalization of Dox used the composition of phospholipid nanoparticles with the targeted vector was 1.4-fold higher as compared to the free form. In the HeLa cell line (not expressing αvß3 integrin) this effect was not observed. These results suggest the prospects of using the cyclic RGD peptide in the delivery of Dox to glioblastoma cells and the feasibility of further investigation of the mechanism of action of the entire composition as a whole.

Nanoliposomes as drug delivery systems: safety concerns.

The review highlights the safety issues of drug delivery systems based on liposomes. Due to their small sizes (about 80-120 nm, sometimes even smaller), phospholipid nanoparticles interact intensively with living systems during parenteral administration. This interaction significantly affects both their transport role and safety; therefore, special attention is paid to these issues. The review summarises the data on the basic factors affecting the safety of nanoliposomes: composition, size, surface charge, stability, the release of an incorporated drug, penetration into tissues, interaction with the complement system. Attention is paid to the authors' own research of unique phospholipid nanoparticles with a diameter of 20-30 nm. The influence of technological processes of nanoliposome production on their properties is considered. The article also discusses the modern safety assessment criteria contained in the preliminary regulatory documents of the manufacturing countries for new nanoliposome-based drugs being developed or used in the clinic.

[The effect of lipid derivative of anti-tumor drug sarcolysin embedded in phospholipid nanoparticles in the experiments in vivo]. / Deistvie lipidnogo proizvodnogo protivoopukholevogo lekarstva sarkolizina, vkliuchennogo v fosfolipidnye nanochastitsy, na opukholi v éksperimentakh in vivo.

To improve the therapeutic properties of the antitumor agent Sarcolysin, we have previously developed and characterized a dosage form representing its ester conjugate with decanol embedded in ultra-small phospholipid nanoparticles less than 30 nm in size ("Sarcolysin-NP"). The effect of the resulting composition was investigated in vivo in comparison with the free substance of sarcolysin. The composition intravenous administration to mice showed an improvement in the pharmacokinetic parameters of sarcolysin associated with its initial higher (by 22%) level in the blood and prolonged circulation, which was also observed in mice with P388 tumor. In mice with three types of tumors - lymphocytic leukemia P388, lymphocytic leukemia L1210, and adenocarcinoma of the mammary gland Ca755 - administration of two doses of sarcolysin over a period of 7 days showed its predominant antitumor effect. The maximum tumor growth inhibition was noted for lymphocytic leukemia L1210 and adenocarcinoma of the mouse mammary gland Ca755 (at a dose of Sarcolysin-NP - 8,4 mg/kg), which was higher in comparison with free substance by more than 24% and 17%, respectively. Differences in the life span of the treated animals were revealed significantly at a dose of 10 mg/kg and amounted to 25% and 17,4% for lymphocytic leukemia P388 and L1210, respectively, and 11% for adenocarcinoma Ca755. In an experiment on rats, acute toxicity of Sarcolysin-NP administered intravenously showed that an average LD50 value 2-3 times exceeded a similar parameter for commercial preparations of free sarcolysin (Melphalan and Alkeran), which indicates its lower toxicity.

Composite phospholipid-gold nanoparticles with targeted fragment for tumor imaging.

Gold nanoparticles and their conjugates have significant potential in the field of diagnosis of various diseases due to their SPR, which enhances light scattering and absorption. Conjugates of gold nanoparticles with various ligands can be used for imaging biomolecules or detecting malignant neoplasms at an early stage. This study focuses on the construction of composite (or hybrid) phospholipid-gold nanoparticles using soy phosphatidylcholine and a targeted ligand (folic acid derivative) to attach specific targeting properties. According to the method of dynamic light scattering, the diameter of the obtained nanoparticles was less than 100 nm, the results of the MTT test indicated their moderate cytotoxicity. In vitro and in vivo experiments showed a significant increase in the accumulation of phospholipid-gold nanoparticles with a targeted fragment compared to those without a targeted fragment both in HeLa cells and in a tumor (in BDF mice with an injected LLC tumor). The resulting nanoparticles are suitable for specific delivery into tumor cells and visualization of various malignant neoplasms, including at early stages, due to the increased expression of the folate receptor characteristic of cells of a wide range of tumors.

[Plasma high density lipoproteins phospholipds as an indirect indicator of their cholesterol efflux capacity - new suspected atherosclerosis risk factor]. / Uroven' fosfolipidov lipoproteinov vysokoi plotnosti plazmy kak kosvennyi pokazatel' ikh kholesterin-aktseptiruiushchei sposobnosti.

High density lipoproteins (HDL) are a unique natural structure, protecting the body from the development of atherosclerotic vascular lesions and cardiovascular diseases due to this ability to remove cholesterol from cells. Plasma HDL level estimated by their cholesterol content, is a common lipid parameter, and its decrease is considered as an established atherosclerosis risk factor. However, a number of studies have shown the absence of positive clinical effects after drug-induced increase in HDL cholesterol. There is increasing evidence that not only HDL concentration, but also HDL properties, considered in this review are important. Many studies showed the decrease of HDL cholesterol efflux capacity in patients with coronary heart diseases and its association with disease severity. Some authors consider a decrease of this HDL capacity as a new additional risk factor of atherosclerosis. The review summarizes existing information on various protein and lipid components of HDL with a primary emphasis on the HDL. Special attention is paid to correlation between the HDL cholesterol efflux capacity and HDL phospholipids and the ratio "phospholipids/free cholesterol". The accumulated information indicates importance of evaluation in the HDL fraction not only in terms of their cholesterol, but also phospholipids. In addition to the traditionally used lipid criteria, this would provide more comprehensive information about the activity of the reverse cholesterol transport process in the body and could contribute to the targeted correction of the detected disorders.

[Targeted drug delivery system for doxorubicin based on a specific peptide and phospholipid nanoparticles]. / Sistema adresnoi dostavki dlia doksorubitsina na osnove spetsificheskogo peptida i fosfolipidnykh nanochastits.

Doxorubicin is one of the widely known and frequently used chemotherapy drugs for the treatment of various types of cancer, the use of which is difficult due to its high cardiotoxicity. Targeted drug delivery systems are being developed to reduce side effects. One of the promising components as vector molecules (ligands) are NGR-containing peptides that are affinity for the CD13 receptor, which is expressed on the surface of many tumor cells and tumor blood vessels. Previously, a method was developed for preparing a composition of doxorubicin embedded in phospholipid nanoparticles with a targeted fragment in the form of an ultrafine emulsion. The resulting composition was characterized by a small particle size (less than 40 nm) and a high degree of incorporation of doxorubicin (about 93%) into transport nanoparticles. When assessing the penetrating ability and the degree of binding to the surface of fibrosarcoma cells (HT-1080), it was shown that when the composition with the targeted fragment was added to the cells, the level of doxorubicin was almost 2 times higher than that of the liposomal form of doxorubicin, i.e. the drug in the system with the targeted peptide penetrated the cell better. At the same time, on the control line of breast adenocarcinoma cells (MCF-7), which do not express the CD13 receptor on the surface, there was not significant difference in the level of doxorubicin in the cells. The data obtained allow us to draw preliminary conclusions about the prospects of targeted delivery of doxorubicin to tumor cells when using a peptide conjugate containing an NGR motif and the further need for its comprehensive study.

Preclinical Study of Pharmacological Properties of Doxorubicin-NPh.

Preclinical study of therapeutic properties of an innovative drug Doxorubicin-NPh (doxorubicin in the form of ultrafine suspension of phospholipid liposomes) in comparison with free doxorubicin (Doxorubicin-Teva) and protected doxorubicin (Caelyx) was performed on transplanted murine tumor models. All these drugs were efficient in Ca755 breast carcinoma model (tumor growth inhibition ≈100%, increase in lifespan 90.6-114.3%). In P388 lymphocytic leukemia and LLC lung carcinoma, advantages of the protected doxorubicin by the benefit/risk ratio (width of therapeutic interval) were demonstrated: Caelyx>Doxorubicin-NPh>Doxorubicin-Teva. Doxorubicin-NPh and Caelyx exhibited similar therapeutic activity in the LLC model, especially when administered 3 times with 3-day intervals; for Doxorubicin-Teva, the optimal interval between the injections was 7 days.

[Chlorine e6 in phospholipid nanoparticles with specific targeting and penetrating peptides as prospective composition for photodynamic therapy of malignant neoplasms]. / Khlorin e6 v fosfolipidnykh nanochastitsakh so spetsificheskimi adresnymi i pronikaiushchimi peptidami kak perspektivnaia kompozitsiia dlia fotodinamicheskoi terapii zlokachestvennykh novoobrazovanii.

Cytotoxic and photoinduced activity of chlorine e6 (Ce6) in phospholipid nanoparticles with specific tumor targeting and cell-penetrating peptides was studied in vitro using human fibrosarcoma cells HT-1080. It was shown, that the binding of cell-penetrating peptide R7 - alone or combined with the peptide containing specific targeting motif NGR (Asn-Gly-Arg) - resulted in 3-fold decrease of Ce6 photoinduced activity as compared with that in nanoparticles without peptides (IC50 values were 0.7 µg/ml and 2.1 µg/ml, respectively). The NGR influence was unexpectedly low - less than 20% (IC50 1.7 µg/ml). This suggests the more importance of Ce6 cell penetration in this case, than of NGR-mediated targeting. The effect of inclusion of both peptides on the total cytotoxicity of Ce6 was minimal (10-16 times less than on the specific photoinduced activity). The obtained results - together with earlier shown effects on improvement of the pharmacokinetics of Ce6 in vivo after its embedding into phospholipid nanoparticles - indicate the prospects of using the obtained phospholipid nanoparticles system for photodynamic therapy.

[Prednisolone in phospholipid nanoparticles: prolonged circulation and increased antiinflammatory effect]. / Prednizolon v fosfolipidnykh nanochastitsakh: prolongirovannaia tsirkuliatsiia i povyshennoe protivovospalitel'noe deistvie.

Along with modern new drugs, many therapeutic schemes also include known effective drugs, particularly, glucocorticoids. One of the most distributed of them is prednisolone that has pronounced anti-inflammatory properties. Its disadvantage is short-term circulation, resulting in a number of side effects. For this reason the development of its more effective and safe formulations is carried out. We have obtained the formulation of prednisolone included in nanoparticles from soy phosphatidylcholine with an average diameter of 20 nm. With oral administration to rats and analysis by HPLC an increase in prednisolone maximal concentration in of plasma and the duration of circulation as compared with free drug administration were shown. The experiment with mice with conconavalin A induced inflammation was also carried out: conconavalin A was injected subplantary in an hour after oral administration of both prednisolone formulations in several doses. The index of the inflammatory reaction (determined by the edema degree) was suppressed more effectively in the case of prednisolone in nanoparticles. Maximal suppression (62.2% as compared with 49.6% for free prednisolone) was observed even at a minimal dose (2.5 mg/kg), at which the free drug did not act at all. The results indicate an increase in the efficiency of prednisolone included in phospholipid nanoparticles, that makes it possible to diminish its administered doses and thereby reduce the risk of side effects.

Effect of Cell-Penetrating Arginine Peptide on Interaction of Photosensitizer Chlorin e6 Incorporated into Phospholipid Nanoparticles with Tumor Cells.

We studied the possibility of increasing the efficiency of photodynamic therapy by improving delivery of photosensitizers chlorin e6 into tumor cells. Previous studies showed that incorporation of chlorin e6 onto phospholipid nanoparticles with a diameter <20 nm reduces its cytotoxicity due to accelerated elimination from organs [8]. A heptapeptide R7 synthesized and added to this combination promoted internalization of chlorin e6 into HepG2 cells in comparison with initial nanoparticles without peptide R7. The observed effect of peptide R7 can be explained by activation of endocytosis and/or macropinocytosis (bearing in mind the interaction of arginine with carboxyl groups of e6. The development of this transporting system is a promising trend in photodynamic therapy of cancer diseases.

[Increase of antituberculosis efficiency of rifampicin embedded into phospholipid nanoparticles with sodium oleate]. / Povyshenie protivotuberkuleznogo deistviia rifampitsina pri vstraivanii v fosfolipidnye nanochastitsy c oleatom natriia.

The formulation of the antituberculosis drug rifampicin embedded into 20-30 nm nanoparticles from soy phosphatidylcholine and sodium oleate, is characterized by greater bioavailability as compared with free drug substance. In this study higher antituberculosis activity of this formulation was shown. Rifampicin in nanoparticles demonstrated more effective inhibition of M. tuberculosis H37Rv growth: minimal inhibiting concentration (MIC) was twice smaller than for free rifampicin. Administration of this preparation to mice with tuberculosis induced by M. tuberculosis Erdman revealed that after 6 weeks of oral administration the CUF value in lung was 22 times smaller for rifampicin in nanoparticles than for free drug (1.7 un. vs. 37.4 un.). The LD50 value in mice was two fold higher for rifampicin in nanoformulation.

[Pharmacological agents and transport nanosystems based on plant phospholipids].

A new generation of plant phosphatidylcholine (PC)-based pharmacological agents has been developed under academician A.I. Archakov leadership at the Institute of Biomedical Chemistry (IBMC). For their production a unique technology allowing to obtain dry lyophilized phospholipid nanoparticles of 30 nm was elaborated. The successful practical application of PC nanoparticles as a drug agent may be illustrated by Phosphogliv (oral and injection formulations). Being developed at IBMC for the treatment of liver diseases, including viral hepatitis, Phosphogliv (currently marketed by the "Pharmstandard" company) is approved for clinical application in 2000, and is widely used in medical practice. Based on the developed and scaled in IBMC technology of prerparation of ultra small size phospholipid nanoparticles without the use of detergents/surfactants and stabilizers another drug preparation, Phospholipovit, exhibiting pronounced hypolipidemic properties has been obtained. Recently completed preclinical studies have shown that PC nanoparticles of 20-30 nm activate reverse cholesterol transport (RCT) and in this context it is more active than well known foreign preparation Essentiale. Phospholipovit is now at the stage of clinical trials (phase 1 completed). PC was also used as a basis for the development of a transport nanosystem with a particles size of 20-25 nm in diameter and incorporation of various drug substances from various therapeutic groups. Using several drugs substances as an example, increased bioavailability and specific activity were demonstrated for the formulations equipped with such transport nanosystem. Formulations equipped with the transport nanosystems have been developed for such pharmacological agents as doxorubicin, rifampin, budesonide, chlorin E6, prednisone, and others.

[Nanoparticles as drug delivery system for antituberculous drugs].

The increase of tuberculosis incidence in last decade stimulated elaboration of both new antituberculous drugs and also searches ofoptimiting delivery systems for existing drugs. It is determined by their side effects and low bioavailability of effective first line drug rifampicin. Various nanosystems for transport of antituberculous drugs are considered on the basis of various polymers, liposomes, lipid nanoparticles, nanoemulsios, nanosuspensions, dendrimers, cyclodextrines. Influence of drug incorporation into nanoparticles, most often for rifampicin, on pharmacokinetics and efficiency in tuberculosis models is discussed. The most of works are devoted to polymer nanoparticles for oral administration where increased circulation time and efficiency were shown. The best results were observed after drug inclusion into solid lipid nanoparticles. The liposomes formulations were investigated mostly for inhalation and injection administrations. Positive results were also observed. Authors underline the viability of incorporation of antituberculous drugs into phospholipid nanoparticles that may increase intestinal absorption and bioavailability. It is confirmed by authors' own data that showed increase of rifampicin efficiency after their incorporation into such nanoparticles.

[Drug delivery system on the base of phospholipid nanoparticles for rifampicin].

Low bioavailability of rifampicin, one of the main antituberculous drug, stimulates searches of its new optimized formulations. The present study has showen possibility of rifampicin embedding into nanoparticles from plant phosphatidylcholine (diameter of 20-30 nm). Addition of sodium oleate to the phospholipid system caused a 2-fold increase of the percent of rifampicin incorporation. After oral administration to rats, the maximal drug observed in plasma one hour after was 0.5 and 4.2 mkg/ml for free rifampicin for rifampicin in phospholipids-oleate nanoparticles, respectively. These levels were maintained for more than two hours of the experiment. High rifampicin bioavailability in the oleate containing phospholipid nanosystem suggests prospectivity of its pharmaceutical elaboration.

Influence of doxorubicin inclusion into phospholipid nanoformulation on its antitumor activity in mice: increased efficiency for resistant tumor model.

AIM: The new formulation of doxorubicin on the base of phospholipid nanoparticles (particle size <30 nm) is elaborated in the Institute of Biomedical Chemistry (Russian Academy of Medical Sciences) on the base of plant phospholipids. The aim of study is to investigate an antitumor effect of this nanoformulation in mice with two cancer models with various sensitivity to chemotherapy ­ lymphoid malignancy P-388 and Lewis lung carcinoma (LLC). METHODS: Nanophospholipid (NPh) formulation of doxorubicin was prepared by homogenization of soybean phosphatidylcholine and doxorubicin hydrochloride. The effect of this formulation was studied in experiments with single or threefold drug administration. Percents of tumor growth inhibition in mice under influence of free or NPh doxorubicin forms were compared. RESULTS: Single administration of both free and NPh doxorubicin in mice with P-388 resulted in the same quick severe inhibition of tumor growth (60­90% depending from dose), with further gradual decrease of inhibition degree. However for more resistant tumor, LLC, the obvious advantage of NPh doxorubicin form was shown. The little effect of free doxorubicin began to reveal only after 11 days, but NPh formulation induced significant inhibition of tumor growth (40%) from the first experimental point (6 days after administration). The advantages of NPh doxorubicin was manifested particularly in low drug doses, 2 and 4 mg/kg. In other experiment design in mice with LLC, with threefold weekly drug administration, NPh doxorubicin appeared to be 2.5 times more active than free drug. The reason of the same actions of free and NPh doxorubicin form in P-388 is suggested the high drug sensitivity of this model, that gives quick high drug response for any doxorubicin form. CONCLUSION: Doxorubicin in phospholipids nanoformulation revealed higher antitumor efficiency as compared with free doxorubicin in mice with LLC carcinoma. The mechanism of such changes is supposed to be caused by increase of doxorubicin availability for cancer cells.

[The increase of bioavailability and anti-inflammatory effect of indomethacin loaded into phospholipid nanoparticles].

The ultrafine formulation on the base of plant phosphatidylcholine and antiinflammatory remedy indomethacin with nanoparticles less than 50 nm was obtained. Drug bioavailability after its peroral administration to rats was more than 2 fold higher as compared with free indomethacin. Increased antiinflammatory activity of indomethacin in phospholipids nanoparticles as compared with its free form was shown in two models of inflammation - adjuvant arthritis in rats and conconavalin A induced edema in mice. The increased bioavailability of indomethacin after administration of its phospholipid formulation allows to decrease a dose for achievement of therapeutic effect, that reduces risks of occurrence of collateral displays.

[New domestic phospholipid preparation "Fosfogliv" as an effective treatment for patients with acute viral hepatitis]. / Novyi otochestvennyi fosfolipidnyi preparat "Fosfogliv" kak effektivnoe sredstvo pri lechenii bol'nykh s ostrymi virusnymi gepatitami.

Patients with acute viral hepatitis B, A and mixed hepatitis B + C were treated in two independent clinics with phosphogliv--a new hepatoprotective drug based on polyunsaturated phosphatidylcholine and glycyrrhizic acid salt. Phosphogliv removed some symptoms of intoxication (nausea, weakness, jaundice, etc.) quicker than basic therapy. Among biochemical hepatitis markers, serum bilirubin level was most responsive to phosphogliv. Standard therapy decreases bilirubin by 30% on the average for 5 days, phosphogliv reduces bilirubin for one more week to half those values observed in control patients. At that point low aminotransferase activities were seen in phosphogliv treated patients. No side effects were seen. The new hepatoprotector phosphogliv which repairs biomembranes represents drugs of new generation compared to phospholipid drug essential.

[Hepatoprotective action of phosphogliv in surgery of patients with chronic calculous cholecystitis]. / Gepatoprotektornoe deistvie fosfogliva u bol'nykh, operirovannykh po povodu khronicheskogo kal'kuleznogo kholetsistita.

32 patients with chronic calculous cholecystitis were treated by a new preparation. Phosphogliv (on the base of polyunsaturated phospholipids)--5 days before planned surgery and 5 days after. The increase of alanine and asparagine aminotransferases activities and of bilirubin concentration (2-2.5 fold) was observed in all the patients, cirespective of the drug treatment. However, the essential difference between treated and untreated patients was revealed in the course of post-operative period. In the control group asparagine aminotransferase activity and bilirubin level remained high, and alanine aminotransferases activity even more increased. In contrast, both two aminotransferases activities and bilirubin level fell substantially in Phosphogliv group, that may show more active liver regeneration after surgery. Besides, in this group operation did not result even in short-time changes of plasma protein fractions ratios--opposite to control. It may testify on some protective action of Phosphogliv on liver protein-synthesis system. The observed action of new phospholipid preparation Phosphogliv, together with the absence of side effects, allows to recommend it as an adjuvant in the surgery of patients with chronic calculous cholecystitis. Besides, the above results broaden usage field of polyunsaturated phosphatidylcholine as cell membranes repair agent: it appears to be effective not only for liver diseases treatment, but also for soonest overcome of post-surgery liver changes.