Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.

Pegylated brain-derived neurotrophic factor shows improved distribution into the spinal cord and stimulates locomotor activity and morphological changes after injury.

The neurotrophin brain-derived neurotrophic factor (BDNF) shows promise for the treatment of central nervous system (CNS) trauma and disease. Effective delivery methods are required, however, for BDNF to be useful as a therapeutic agent. To this end, we examined the penetration of intrathecally infused N-terminal pegylated BDNF (peg-BDNF) compared to similar infusion of native BDNF after spinal cord injury (SCI). Pegylation dramatically improved delivery of BDNF to the spinal cord and induced the expression of Fos in spinal cord neurons. To test whether enhanced delivery would improve the modest effects on behavioral recovery and axonal outgrowth observed with native BDNF infusion, we assessed the efficacy of 2-week 25 microg/day peg-BDNF treatment, beginning 12-24 h (early) or 15 days (delayed) after midthoracic spinal contusion. Similar to native BDNF, early treatment with peg-BDNF accelerated the recovery of stepping in the open-field and acutely stimulated locomotor central pattern generator activity, as seen by the activation of hindlimb airstepping during either period of administration. The infusion of peg-BDNF, regardless of the timing of delivery, was related to enhanced sprouting of putative cholinergic fibers, like that observed after high dose native BDNF treatment. Despite improved delivery, however, neither axonal responses nor the extent of locomotor recovery were enhanced compared to native BDNF treatment. This suggests that alternative strategies, such as neurotrophin treatment in conjunction with cell transplantation techniques, or treatment nearer the cell bodies of target neurons might be employed in an attempt to effect significant repair after SCI.

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans.

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

Characterization and stability of N-terminally PEGylated rhG-CSF.

PURPOSE: The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue. METHODS: The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules. RESULTS: Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation. CONCLUSIONS: We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.

Systemic hematologic effects of PEG-rHuMGDF-induced megakaryocyte hyperplasia in mice.

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices. In addition to the expected thrombocytosis after 7 to 10 days of daily injection of high doses of PEG-rHuMGDF, a transient decrease in peripheral red blood cell numbers and hemoglobin is noted accompanied in the bone marrow by megakaryocytic hyperplasia, myeloid hyperplasia, erythroid and lymphoid hypoplasia, and deposition of a fine network of reticulin fibers. Splenomegaly, an increase in splenic megakaryocytes, and extramedullary hematopoiesis accompany the hematologic changes in the peripheral blood and marrow to complete a spectrum of pathologic features similar to those reported in patients with myelofibrosis and megakaryocyte hyperplasia. However, all the PEG-rHuMGDF-initiated hematopathology including the increase in marrow reticulin is completely and rapidly reversible upon the cessation of administration of PEG-rHuMGDF. Thus, transient hyperplastic proliferation of megakaryocytes does not cause irreversible tissue injury. Furthermore, PEG-rHuMGDF completely ameliorates carboplatin-induced thrombocytopenia at a low-dose that does not cause the hematopathology associated with myelofibrosis.

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

Prolonged circulation of recombinant human granulocyte-colony stimulating factor by covalent linkage to albumin through a heterobifunctional polyethylene glycol.

PURPOSE: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF. METHODS: Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 micrograms/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA. RESULTS: The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 +/- 0.121 ml/min/kg for rhG-CSF to 0.172 +/- 0.013 ml/min/kg for RSA-PEG-rhG-CSF and 0.141 +/- 0.005 ml/min/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37 degrees C decreased five fold. CONCLUSIONS: The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.

The pulmonary absorption of aerosolized and intratracheally instilled rhG-CSF and monoPEGylated rhG-CSF.

PURPOSE: The objective of this study was to highlight differences in the pulmonary absorption of a monoPEGylated rhG-CSF and rhG-CSF after intratracheal instillation and aerosol delivery. METHODS: Male Sprague Dawley rats (250 g) were anesthetized and intratracheally instilled (IT) with protein solution or were endotracheally intubated and administered aerosol for 20 min via a Harvard small animal ventilator. A DeVilbiss "Aerosonic" nebulizer containing 5 ml of protein solution at approximately 3 mg/ml was used to generate aerosol. The volume of protein solution deposited in the lung lobes was estimated to be approximately 13 microliters after delivery of Tc-99m HSA solutions. The PEGylated proteins consisted of a 6 kDa (P6) or 12 kDa PEG (P12) linked to the N-terminus of rhG-CSF. rhG-CSF also was administered IT in buffers at pH 4 and pH 7 and in dosing volumes ranging from 100 to 400 microliters. Blood samples were removed at intervals after dosing and the total white blood cell counts (WBC) were determined. Plasma was assayed for proteins by an enzyme immuno assay. RESULTS: The plasma protein concentration v. time profiles were strikingly different for aerosol v. IT delivery. The Cmax values for rhG-CSF and P12 after aerosol delivery were greater than found after IT (Aerosol: 598 +/- 135 (ng/ml) rhG-CSF; 182 +/- 14 P12 v. IT: 105 +/- 12 rhG-CSF; 65.9 +/- 5 P12). Similarly, Tmax was reached much earlier after aerosol administration (Aerosol: 21.7 +/- 4.8 (min) rhG-CSF; 168 +/- 31 P12 v. IT: 100 +/- 17 rhG-CSF; 310 +/- 121 P12). Estimated bioavailabilities (F(lung)%) were significantly greater via aerosol delivery than those obtained after IT (Aerosol: 66 +/- 14 rhG-CSF; 12.3 +/- 1.9 P12 v. IT: 11.9 +/- 1.5 rhG-CSF; 1.6 +/- 0.1 P12). An increase in circulating WBC counts was induced by all proteins delivered to the lungs. The rate and extent of absorption of rhG-CSF was not influenced by the pH employed nor the instilled volume. CONCLUSIONS: Estimates of bioavailability are dependent upon the technique employed to administer drug to the lungs. Aerosol administration provides a better estimate of the systemic absorption of macromolecules.

Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro.

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.

Synthesis and properties of modified oligonucleotides.

Phosphorothioate oligonucleotide analogs conjugated to cholesteryl by a neutral, 6 atom linker are more effective inhibitors of HIV-1 in cell culture than the corresponding analogs conjugated via a phosphorothioate group. The antiviral activity correlates with the hydrophobic character of the oligonucleotide. Some new synthetic methodology is also discussed.

[High molecular weight L-asparaginase derivatives based on a water-soluble carboxymethyl cellulose: biological properties]. / Vysokomolekuliarnye proizvodnye L-asparaginazy na osnove vodorastvorimoi karboksimetiltselliulozy: biologicheskie svoistva.

L-asparaginase, covalently bound with water-soluble CM-cellulose, exhibited the elevated antileukemic activity in mice with inoculated lymphoid leukemia L5178y as compared with the native enzyme. The antileukemic activity of the immobilized enzyme was shown to depend on the content of the polymer bound with the enzyme; the polymer amount may be altered during the enzyme modification. The prolonged effect of immobilized L-asparaginase was observed in rabbit circulation.