Influence of BCG infection on growth of 3-methylcholanthrene-induced rat sarcomas.
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Animals↗
Biomedical subjects
M V Pimm
Publications and source records attributed to M V Pimm.
Carrier design: biodistribution of branched polypeptides with a poly(L-lysine) backbone.
The biodistribution has been examined in mice of a range of synthetic branched polypeptides which are based on a polylysine backbone but which differ in ionic charge, side-chain structure, and molecular size. Polycationic polypeptides, regardless of their size or primary structure at the branches, were cleared rapidly from the circulation, the liver being the major site of clearance. Polypeptides with glutamic acid in the side chain, which would be amphoteric under physiological conditions, showed a significantly prolonged blood survival, and this was seen with polypeptides in the range of molecular weights of 46,000 up to 213,000. Such polypeptides provide a useful system with which to investigate the effect of structural parameters on the pharmacokinetic properties of carrier molecules and would allow the selection of candidate carriers for a variety of uses.
Amino Acid Sequence↗
Influence of carrier on biodistribution and in vitro cytotoxicity of methotrexate-branched polypeptide conjugates.
Methotrexate (MTX) has been conjugated to various structurally related, synthetic, branched polypeptides containing a poly(L-Lys) backbone by the aid of water-soluble carbodiimide. The average degree of MTX incorporated was found to be dependent on the size of the polymer and on the identity of the terminal amino acid residue of the side chains. Consequently the average molar substitution ratio was in the range of 4.9-72.0 MTX per carrier molecule. CD spectra of conjugates showed significant differences in solution conformation correlating with the identity of the side-chain-terminating amino acid. Polycationic conjugates XAK-MTX (X = Leu or D-Leu) assumed essentially ordered (helical) secondary structure, while the CD spectrum of the amphoteric conjugate (X = Glu) corresponded to only a partially ordered conformation in PBS. The covalent attachment of MTX to branched polypeptides results in a reduction of drug in vitro cytotoxicity influenced by the carrier structure. Conjugation to amphoteric polymers, depending on the configuration and position of glutamic acid (XAK-MTX vs AXK-MTX type conjugates) resulted in a decrease of anti-791T cell activity. However polycationic conjugates bearing L-Leu at the side chain terminal position (LAK-MTX) produced a compound with cytotoxicity only about 60 times less effective than free MTX. The biodistribution in mice has been characterized by blood clearance, whole-body retention, and tissue distribution 24 h after iv administration. Blood clearance of MTX-branched polypeptides could be significantly prolonged by incorporation of glutamic acid into the side chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.
Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.
Animals↗
Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.
For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.
Adjuvants, Immunologic↗
Host responses in adjuvant contact suppression of experimental rat tumours.
BCG (Glaxo) and C. parvum (Wellcome CN 6134) have been examined for suppression of growth of a range of syngeneically transplanted rat tumours, both carcinogen-induced and of spontaneous origin. Treatment by locally applied adjuvant controlled growth of both immunogenic and non-immunogenic tumours, and the response to highly immunogenic tumours was not abolished by host immunosuppression with whole body irradiation known to abrogate induction of tumour-specific immunity. In addition, rat tumour xenografts in congenitally athymic mice were suppressed by admixture with BCG or C. parvum. In contrast to these findings in immunoincompetent animals, host phagocytic cell depletion, by systemic administration of silica, a known macrophage poison, abrogated adjuvant contact suppression of tumours in syngeneic rats and athymic mice. These findings suggest that tumour suppression by regionally applied adjuvants may be more dependent upon local activation of host macrophages than upon generalized stimulation of lymphocyte mediated responses. This is further supported by the market correlation, within a range of tumours, between susceptibility to regionally applied BCG and normal levels of host macrophage infiltration, and in addition the facilitation of tumour suppression achieved by macrophage enrichment of tumour cell: BCG inocula.
Animals↗
Comparison of BCG and C. parvum preparations for adjuvant contact therapy of a rat sarcoma.
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Animals↗
Influence of syngeneic (anti-idiotypic) antibody responses on biodistribution and tumour localisation of murine monoclonal antibodies and fragments.
Balb/c mice were immunised with three different syngeneic monoclonal antibodies (Mabs). Following injection of radiolabelled Mabs into mice with anti-antibody responses they were cleared from the circulation, predominantly to the liver, due to the formation of immune complexes. Anti-antibody titres as low as 1/100 were virtually as effective as 1/3,000 in perturbing biodistribution of Mabs. There were no effects against isotype matched control Mabs. There was a much lesser effect against Fab or F (ab')2 of one Mab, although these did form immune complexes in the circulation of immune mice. With Fab of another Mab its blood survival was actually prolonged, due to immune complex formation. With this Fab, although blood survival was prolonged, localisation into human tumour xenografts expressing its target antigen in Nude mice was reduced by pre-treatment with immune serum. These studies show that syngeneic (anti-idiotypic) responses have a detrimental effect on the pharmacokinetics of Mabs, but with more diverse effects on their fragments. With fragments the indication is that even when biodistribution is not grossly perturbed, tumour recognition may still be restricted.
Animals↗