Immunogenicity of thrombopoietin mimetic peptide GW395058 in BALB/c mice and New Zealand white rabbits: evaluation of the potential for thrombopoietin neutralizing antibody production in man.

Administration of exogenous proteins and peptides as therapeutics carries with it the potential for immune system recognition and the development of neutralizing antibodies to endogenous regulatory proteins. PEGylation of proteins typically reduces their immunogenicity in vivo. GW395058 is a PEGylated peptide thrombopoietin receptor (TPOr) agonist being evaluated for the treatment of chemotherapy-induced thrombocytopenia. Although GW395058 shares no homology with TPO, it does compete with TPO for binding to a common receptor, and a similarity in local structure could result in shared epitopes. Thus GW395058 could elicit TPO-neutralizing antibodies. In this study, we evaluated the immunogenicity of GW395058 in mice, the potential of rabbit antibodies elicited by immunizations with the non-PEGylated parent peptide AF15705 to cross-react with recombinant human (rHu) TPO, and the potential of mouse anti-rHuTPO antibodies elicited by repeated dosing with rHuTPO to cross-react with AF15705. GW395058-dosed mice failed to produce antibodies to AF15705 or rHuTPO. Mouse anti-rHuTPO did not cross-react with AF15705 and rabbit anti-AF15705 antibodies failed to cross-react with rHuTPO. GW395058 caused no immune-mediated lesions in mice, but rHuTPO suppressed megakaryocytopoiesis and caused B-lymphocyte hyperplasia in lymphoid tissues consistent with antigenic stimulation. These data suggest that the potential for an immune response to GW395058 in man would be low.

Folate-mediated drug delivery: effect of alternative conjugation chemistry.

When utilized as a macromolecular drug targeting ligand, folic acid (Pte-Glu) has traditionally been coupled to peptides, proteins and lipids via one of its two carboxylate groups fortuitously located within a distal glutamyl moiety. It has been assumed in the literature that the gamma-glutamyl carboxylate of Pte-Glu is the preferred conjugation site for macromolecules enduring endocytosis via the folate-binding protein receptor. However, it is also possible that the steric placement of the attached macromolecule around the vitamin's pteridine moiety may be the more influential parameter controlling this delivery mechanism. Using solid-phase chemistries, we have synthesized dipeptide derivatives of pteroic acid for the purpose of identifying the preferred site onto which a macromolecule can be chemically attached without compromising its endocytosis potential. Thus, using fluorescent and radiolabeled conjugates, we have determined that macromolecules attached to Pte-Glu by either an alpha- or gamma-glutamyl linkage could associate with receptor-bearing cells at virtually identical levels. We further discovered that removal of the remaining un-conjugated glutamyl carboxylate had no inhibitory effect on cell uptake; and, the cytotoxicity of related momordin toxin conjugates were comparable among the various pteroate derivatives tested. From these observations we suggest that the preparation of endocytosis-competent pteroate-macromolecule conjugates is strongly influenced by the steric environment around the ligand's para-aminobenzoic acid moiety, and that no selective isomeric (i.e. alphaGlu versus gammaGlu) conjugation requirement necessarily exists.

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

Enteral bioavailability of human granulocyte colony stimulating factor conjugated with poly(ethylene glycol).

PURPOSE: The focus of this paper is to demonstrate that pegylation of a therapeutic protein, recombinant human granulocyte colony stimulating factor (PEG-G-CSF), results in an increase in stability and in retention of in vivo bioactivity when administered by the intraduodenal route and may, therefore, be a suitable form of the protein for inclusion in an oral delivery formulation. METHODS: The ability of PEG-G-CSF to elicit a therapeutic response from the enteral route was investigated by two methods of intraduodenal dosing in an in vivo model to determine the optimal dosing method: by slow, constant infusion, or by a single bolus administration. RESULTS: Circulating levels of the proteins confirmed that PEG-G-CSF was delivered into the systemic circulation from the enteral route and that biological activity was retained. Bioavailability from the enteral route by the constant infusion method was calculated from the intravenous administration of the proteins to be between 1.8 and 3.5% while un-modified G-CSF failed to elicit a quantifiable response by this method. Bolus administration of PEG-G-CSF also resulted in biological activity although responses were short lived and significantly lower than with the pegylated formulation. CONCLUSIONS: The possible mechanisms of enteral delivery of PEG-G-CSF are discussed. Our results indicate that oral delivery of pegylated G-CSF may be possible and in fact, preferable to using the un-modified form of the therapeutic.

Rat stem-cell factor induces splenocytes capable of regenerating the thymus.

Cytokine regulation of prethymic T-lymphoid progenitor-cell proliferation and/or differentiation has not been well-defined, although much is known of cytokine regulation of hemopoietic stem- and progenitor-cell development. Here we use a recently identified hemopoietic growth factor, stem-cell factor (SCF) (a form of the c-kit ligand), and a transplant model of thymocyte regeneration to assess the effect of SCF on the in vivo generation of prethymic, thymocyte progenitor-cell activity. We show that recombinant rat SCF (rrSCF164) administered to weanling rats selectively induces an increase in thymocyte progenitor activity in the spleens of treated rats as compared to rats treated with vehicle, polyethylene glycol (PEG)-conjugated rat albumin, or recombinant human granulocyte colony-stimulating factor (rhG-CSF). These data demonstrate that administration of SCF in vivo affects extrathymic-origin thymocyte regenerating cells and may influence, directly or indirectly, early prethymic stages of T-cell lymphopoiesis in addition to its known effect on early stages of myelopoiesis and erythropoiesis.