Safety, pharmacokinetics and efficacy of factor VIIa formulated with PEGylated liposomes in haemophilia A patients with inhibitors to factor VIII--an open label, exploratory, cross-over, phase I/II study.

Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding episodes in haemophilia patients with inhibitors to factor VIII (FVIII) and factor IX. The pharmacological effect of FVIIa is short-lived and therefore with the recommended dose of 90 µg kg(-1), a bleeding episode is treated with multiple injections. A long-acting form of FVIIa that can ensure adequate haemostasis with a single infusion, without increasing the thrombotic risk, would therefore be beneficial. PEGylated liposomes (PEGLip) have been shown to bind FVIIa and to improve haemostatic efficacy in preclinical experiments. In the present phase I/II clinical trial, we assessed the safety and efficacy of PEGLip-formulated FVIIa in severe haemophilia A patients (FVIII≤1%) with inhibitors to FVIII. Each patient received one prophylactic infusion of standard FVIIa and one prophylactic infusion of PEGLip-formulated FVIIa. The order of the infusions was randomized and the two infusions were separated by a ten-day washout period. Efficacy assessed by thromboelastography revealed that PEGLip-FVIIa induced significantly shorter clotting times and produced higher clot firmnesses than standard FVIIa. Thrombin generation assays showed that PEGLip-FVIIa induced faster thrombin generation and higher peak levels of thrombin than standard FVIIa. These effects lasted up to 5 h postinfusion. Measurements of D-dimer, prothrombin fragment 1+2 and fibrinogen showed no significant differences between the PEGLip-FVIIa and standard FVIIa treatments. PEGLip-FVIIa therefore showed improved haemostatic efficacy without increased risk of thrombosis and may be further developed for the treatment for bleeding episodes in haemophilia patients with inhibitors.

Enhancement of haemostatic efficacy of plasma-derived FVIII by formulation with PEGylated liposomes.

We have shown previously that PEGylated liposomes (PEGLip) bind recombinant FVIII (rFVIII) with high affinity and specificity. This binding resulted in a significant extension of the biological activity of rFVIII as demonstrated in animal models and in clinical trials. In the present study we found that PEGLip bind plasma-derived factor VIII (pdFVIII). PEGLip binding did not affect potency or stability in vitro and did not alter levels of FVIII activity in vivo immediately after injection. However, formulation of pdFVIII with PEGLip led to several important improvements. Twenty-four and 30 hours after injection, FVIII activity levels were significantly higher in haemophilic mice injected with PEGLip-pdFVIII than in mice injected with standard pdFVIII. Half life, area under the curve and mean residence time were increased while clearance was decreased. In vivo efficacy was evaluated in a tail vein transection assay performed in haemophilic mice. Prophylactic treatment with PEGLip-pdFVIII was much more effective in prolonging survival in this assay than similar treatment with standard pdFVIII. These results suggest that formulation of pdFVIII with PEGLip has the potential to improve patient care by prolonging the biological efficacy of pdFVIII and reducing the frequency of FVIII infusions.

Enhancement of factor VIIa haemostatic efficacy by formulation with PEGylated liposomes.

Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia patient with inhibitors and acquired haemophilia. However, on account of its relatively short half-life (HL), achieving therapeutic efficacy with FVIIa requires repeated injections. The development of a long-acting FVIIa product would therefore be beneficial. The formulation of factor VIII with PEGylated liposomes (PEGLip) was previously shown to extend the bleeding-free period in haemophilia patients. We report here an enhancement of haemostatic efficacy by similarly formulating FVIIa with PEGLip. Surface plasmon resonance analysis indicated that FVIIa binds non-covalently but with high affinity and specificity to PEGLip. A one-stage clotting assay demonstrated that formulation of FVIIa with PEGLip does not affect FVIIa activity and stability. A pharmacokinetic study in rats demonstrated that PEGLip formulation of FVIIa extends circulation time and results in higher FVIIa levels several hours after injection. Thromboelastography experiments indicated that PEGLip-FVIIa induces faster clot formation and higher clot stability than standard formulated FVIIa. These results suggest that formulation of FVIIa with PEGLip may lead to a safe and effective long-acting FVIIa that improves the care of haemophilic patients with inhibitors and acquired haemophilia.

A modified chromogenic assay for the measurement of very low levels of factor VIII activity (FVIII:C).

A precise and sensitive chromogenic assay for the measurement of very low levels of factor VIII (FVIII) in plasma has been developed. The assay is based on modifications of a commercially available chromogenic assay. The modifications include reduction of sample final dilution factor and prolongation of the development period. The modified assay allows accurate and precise measurement of FVIII in the range of 0.001-0.02 IU mL(-1). The detection limit is 0.0005 IU mL(-1) and the quantitation limit is 0.0015 IU mL(-1). This assay can be used in research and to study the clinical efficacy of low circulating levels of FVIII in haemophilia A patients.

Size exclusion chromatography on soft and semi-rigid packing materials in the dynamic axial compression mode.

The effect of dynamic axial compression within a range of up to 5 bar upon the structure of the bed packed with soft and semi-rigid packing materials (Sephadex G-25, Bio-Gel P2 and Toyopearl HW-40) and the associated chromatographic parameters were studied for size exclusion chromatography. Continuous packing compression is accomplished by use of a special column with controlled external pressure applied to the packing. Compression has been shown to favor an overall increase in the resolution with pressure optima observed in some cases.

[Synthesis and antibacterial activity of analogues of the N-terminal fragment of the sarcotoxin IA antimicrobial peptide]. / Sintez i antibakterial'naia aktivnost' analogov N-kontsevogo fragmenta antimikrobnogo peptida sarkotoksina IA.

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1-18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.

Pressure monitoring of continuous-flow solid-phase peptide synthesis.

We describe the noninvasive real-time pressure monitoring of Boc- and Fmoc-based peptide synthesis. Pressure was measured using a resistance strain gauge attached to the inlet of a continuous-flow reactor of variable volume. In the assembly of the 'difficult' polyalanine sequence, it was shown that pressure monitoring can reveal structural variations of the peptide-resin, e.g. the onset, development and termination of aggregation. This method provided washing minimization that favored substantial saving of solvents. The obtained results demonstrated the advantage of pressure monitoring over swellographic monitoring.

In vivo liver-directed gene transfer in rats and pigs with large anionic multilamellar liposomes: routes of administration and effects of surgical manipulations on transfection efficiency.

Conventional large (500-800 nm) multilamellar liposomes encapsulating DNA have been used in vivo as gene vectors into rat and pig liver. By using the intraportal vein route, high dose DNA (10 mg/kg) provided low efficiency and transient luciferase gene expression in the liver. This gene expression was, however, increased by liver resection (> 50%), ischemia (20 min) or orthotopic transplantation. As evidenced by histochemical analysis of beta-galactosidase expression, the gene transfection mainly ensued in Kupffer cells, but spleen and lung were contaminated. In comparison, injection into the bile duct of even 25-fold lower dose of liposome-encapsulated DNA (0.4 mg/kg) produced higher (100-fold) and long-lasting (during 6 days, at least) luciferase expression in rat liver. The gene expression was restricted to the liver and enhanced by liver resection. By this route, transgene-expressing cells were mainly hepatocytes. A treatment with colchicine prior to the administration of the vector allowed the persistence of relative high gene expression for at least 7 days. In pigs, qualitatively similar, but quantitatively less efficient gene expression was obtained by either the portal vein or the bile duct route. These results indicate that intrabile duct route might render large non-viral vectors applicable to gene transfer into the hepatocytes. The efficiency of liposome-mediated gene transfer into the liver can be increased by liver resection, ischemia or transplantation performed before DNA injection.

Spectrophotometric monitoring in continuous-flow Boc-based solid-phase peptide synthesis.

It has been demonstrated that SPPS with Boc amino group protection can be monitored spectrophotometrically if it is performed in a continuous-flow reactor of variable volume. It is shown that this approach provides useful and adequate evidence on the beginning/end-point of most steps of the SPPS cycle. At the deprotection step the spectrophotometric monitoring enables real-time recording of the initial and final moments of the process. When synthesizing a 'difficult' polyalanine sequence, we were able to monitor variation in the deprotection dynamics associated with the aggregation phenomena. The time actually necessary for the Boc protecting group removal appeared to be significantly smaller than that usually preset in the available Boc-SPPS protocols.

Lysosome-disrupting peptide increases the efficiency of in-vivo gene transfer by liposome-encapsulated DNA.

The main limitation of liposome-mediated gene transfer is its low efficiency, a result of degradation of the transferred DNA in the lysosome compartment. In an effort to overcome this problem, lysosome-disrupting peptide was co-encapsulated with a luciferase expression vector in liposomes. The encapsulation level of the peptide was high (> 80%) and did not affect DNA encapsulation efficiency. Liposomes encapsulating DNA were injected into mice and the efficiency of gene transfer and expression were measured. Polymerase chain reaction (PCR) analysis of DNA purified from mouse livers and spleens indicated that co-encapsulation of lysosome-disrupting peptide with DNA significantly increased the amount of transferred DNA found 5 days post-injection in the organ cells. Luciferase activity at 5 days post-injection in spleens of mice that were injected with liposomes containing luciferase expression vector and lysosome-disrupting peptide was significantly higher than that in mice injected with liposomes containing only luciferase expression vector or liposomes containing luciferase expression vector and control peptide. These results indicate that the efficiency of in-vivo liposome-mediated gene transfer can be significantly increased by co-encapsulation of lysosome-disrupting peptide with the transferred DNA.

Liposome-encapsulated DNA-mediated gene transfer and synthesis of human factor IX in mice.

Hemophilia B is an X-chromosome-linked recessive disorder that is caused by a deficiency of biologically active clotting factor IX (FIX). In this work, liposomes (Lip) were used for non-viral, in vivo gene transfer of the human FIX gene into mouse organs. Plasmid DNA, containing the human FIX cDNA under the control of the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was encapsulated in 1-2-microns multilamellar Lip composed of egg phosphatidylcholine (EPC). The percentage of Lip-associated DNA was 47%, and 72% of the Lip DNA was protected from DNase I digestion. The Lip-encapsulated (Len) DNA was injected intravenously into Balb/c mice, and at various times post-injection, various tissues were examined for the presence of the exogenous DNA. Plasmid DNA was detected by Southern blot analysis mainly in the liver and spleen, but small amounts were also detected in the lungs, heart and kidneys. The plasmid DNA was retained in mouse liver cells for at least 7 days post-injection, and remained in an episomal state. The levels of human FIX protein in the mouse plasma were 190-650 pg per ml for 2 to 7 days post-injection. Treatment of mice with chloroquine (Cq) and colchicine (Cc) prior to Lip injection significantly increased the amount of plasmid DNA found in the liver cells, as well as the level of human FIX in the plasma. These results demonstrate the potential use of Len DNA for gene transfer into liver and spleen, and for gene therapy of inherited and acquired disorders.

[The automated "SynChrom" system for solid-phase synthesis of peptides and liquid column chromatography. I. Principles of design and structural constituents]. / Avtomatizirovannyi kompleks dlia tverdofaznogo sinteza peptidov i zhidkostnoi kolonochnoi khromatografii "SinKhron". I. Printsipy postroeniia i strukturnye sostavliaiushchie.

An automatic modular SynChrom system was designed for solid phase synthesis and chromatographic purification of peptides. Structural constituents and organization and operation of the system in solid phase peptide synthesis and liquid column chromatography modes are described. Swellographic monitoring of the course of synthesis was used. Hydraulic diagrams, operation algorithm and software description are presented.

[The automated "SynChrom" system for solid-phase synthesis of peptides and liquid column chromatography. II. Use in the solid-phase synthesis of peptides and liquid column chromatography]. / Avtomatizirovannyi kompleks dlia tverdofaznogo sinteza peptidov i zhidkostnoi kolonochnoi khromatografii "SinKhrom". II. Ispol'zovanie v tverdofaznom sinteze peptidov i zhidkostnoi kolonochnoi khromatografii.

Automatic solid phase peptide synthesis using the SynChrom system is described. Problems of swellographic monitoring are discussed. Combined monitoring (swellographic, spectrophotometric, and manometric) of all steps of the synthetic cycle are suggested. Potential applications of the system to liquid column chromatography were demonstrated.

[Optimization of a protocol for automatic solid phase synthesis of peptides using a variable volume flow reactor]. / Optimizatsiia protokola tverdofaznogo avtomaticheskogo sinteza peptidovs ispol'zovaniem protochnogo reaktora peremennogo ob''ema]

The protocol for solid phase peptide synthesis using automatic synthesizers MilliGen/Biosearch 9500 and 9600 with built-in variable volume flow-reactors has been elaborated. The data of swellographic monitoring were used for optimization of process parameters. The peptides involving from 10 to 21 amino acid residues were synthesized using developed protocol. Significant economy of reagents and solvents has been demonstrated for synthesis of good quality peptides.

Retroviral-mediated in vivo gene transfer into muscle cells and synthesis of human factor IX in mice.

Muscle cells, when transduced with a factor IX (FIX) expression vector, are capable of producing FIX protein and secreting it into the blood. These cells can thus serve as appropriate targets for gene transfer in hemophilia B patients. In this work, we studied the potential of retrovirus-mediated in vivo gene transfer of the FIX gene into muscle cells. Producer cells, which produce recombinant retroviruses containing the human FIX cDNA under the transcriptional control of the chicken beta-actin promoter, were treated with the cytostatic drug mitomycin C (MC), and injected into the rectus femoris muscle of 6-week-old CB-17/1cr severe combined immune-deficient (SCID) mice. As a control, SCID mice were injected with NIH 3T3 cells, which were stably transformed by the retroviral vector described above and therefore secreted FIX, but did not produce retroviruses. The injected MC-treated cells did not cause any tumours to develop in the mice, and the amount of human FIX in the plasma of mice injected with the producer cells was significantly higher, at any of the time points measured beyond 10 days postinjection, than the amount of human FIX in the plasma of mice injected with FIX-secreting 3T3 cells. These results indicate that muscle cells transduced by retroviruses produced human FIX and secreted it into the blood. In vivo gene transfer to muscle cells by injection of retrovirus-producing cells, or by direct injection of recombinant retroviruses, could prove to be important for the gene therapy of hemophilia B.

A swellographic approach to monitoring continuous-flow solid-phase peptide synthesis.

A new type of practical low pressure continuous-flow reaction system has been developed, allowing continuous "dead volume free" handling of polystyrene-based peptide-resins (PR) under conventional Boc/Bzl solid-phase peptide synthesis (SPPS). The reaction system offers a unique opportunity for direct recording of bed volume changes accompanying chemical and physical manipulations with PRs. A new monitoring principle, called "swellography," based on a straightforward interpretation of PR bed volume dynamics, is described. It seems to provide a basis for a novel, automated, non-invasive feedback control system. This novel technique does not complicate SPPS practice; and, moreover, it provides useful information about the swelling behavior of PRs in the real time of SPPS. Although the approach is not directly applicable for quantitation of deprotection and coupling reactions, it does give a good opportunity for real-time optimization of solvent and re-agent usage during the washing and neutralization steps of SPPS. A number of 8- to 16-membered peptides were synthesized manually with complete swellographic monitoring. The practical merits of the new approach are discussed in some detail.

Induction of polyomavirus DNA replication by carcinogens in polyomavirus-transformed rat cells: evidence that the viral enhancer is not the primary target in the induction pathway.

In the polyomavirus (Py)-transformed rat cell line designated LPT, replication of the integrated Py DNA can be induced by exposure of the cells to carcinogens. In view of the observation that enhancer elements are essential components of the Py origin of replication, it appeared plausible that the induction is triggered by synthesis or modification of an enhancer-binding protein which is required for activation of the viral origin. To test this hypothesis, we have used a plasmid containing a modified Py origin (test plasmid), in which the Py enhancer has been replaced with five repeats of the yeast GAL4 upstream activating sequence, and a plasmid encoding the GAL4 transcriptional activator protein. Previous studies in which these two plasmids were cotransfected into mouse cells that are permissive for Py showed that the GAL4 protein can transactivate the modified Py origin and cause replication of the test plasmid. When similar cotransfection assays were performed in LPT cells, no replication of the test plasmid was observed unless the cells were exposed to the carcinogen mitomycin C subsequent to the transfection, in which case replication of the test plasmid was induced. Control experiments showed that even though the GAL4 protein was required for the induction, its concentration was not affected by the exposure to mitomycin C. These results indicate that the primary target in the induction pathway is not an enhancer-binding protein; instead, the induction appears to be triggered by changes in other components of the replication initiation complex which may be associated with the origin core.

The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells.

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.

Induction of polyomavirus DNA replication by cyclic AMP and a tumor promoter.

The effects of reagents that stimulate the intracellular level of cyclic AMP (cAMP), and of a tumor promoter, on polyomavirus (Py) DNA replication were examined in a Py-transformed rat cell line, designated the LPT line. cAMP-stimulating reagents (forskolin and 3-isobutyl-1-methyl xanthine) were found to induce replication of Py DNA in normally growing LPT cells and to enhance replication of the viral DNA in cells exposed to suboptimal doses of UV light and mitomycin C (which have been previously shown to induce Py DNA replication in LPT cells). The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate did not cause a significant induction in Py DNA replication when it was applied to the cells alone, but enhanced replication of the viral DNA when it was applied together with the cAMP-stimulating reagents. Cycloheximide, an inhibitor of protein synthesis, was found to inhibit the induction of Py DNA replication by the cAMP-stimulating reagents. Based on these data, and on previous studies of the Py enhancer, it is proposed that cAMP may induce synthesis of an enhancer-binding protein that causes Py DNA replication in LPT cells by binding and activating an enhancer element which is a component of the Py origin.