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M C Garnett

Publications and source records attributed to M C Garnett.

At least 37 records · Page 2Linked to original sources
PubMed1997

Long circulating biodegradable poly(phosphazene) nanoparticles surface modified with poly(phosphazene)-poly(ethylene oxide) copolymer.

The biodistribution of biodegradable poly(organo phosphazene) nanoparticles surface modified by adsorption of a novel poly(organo phosphazene)-poly(ethylene oxide) copolymer with a 5000 M(W) PEO chain (PF-PEO[5000]), following intravenous administration in rats and rabbits, is described. The data are compared to the biodistribution of poly(organo phosphazene) and poly(lactide-co-glycolide) nanoparticles coated with a tetrafunctional copolymer of poly(ethylene oxide)-poly(propylene oxide) ethylenediamine, commercially available as Poloxamine 908. This copolymer has a PEO chain of the same size as the poly(organo phosphazene)-PEO derivative used. The results in the rat model reveal that poly(organo phosphazene) nanoparticles with a Poloxamine 908 coating were mainly captured by the liver, although a retardation in clearance from the systemic circulation was seen. In contrast, the poly(organo phosphazene) nanoparticles coated with PF-PEO(5000) showed a prolonged blood circulating profile, with only a small amount of the nanoparticles sequestered by the liver. This indicates the importance of the nature of both the anchoring group and the particle surface on the biological performances of the system. Study of the biodistribution of the PF-PEO(5000)-coated poly(organo phosphazene) nanoparticles in the rabbit model also indicated a prolonged systemic circulation lifetime and reduced liver uptake, whereby a significant amount of the administered nanoparticles was targeted to the bone marrow.

Animals↗
PubMed1995

Design, synthesis and in vitro testing of methotrexate carrier conjugates linked via oligopeptide spacers.

A range of methotrexate (MTX)-spacer-human serum albumin (HSA) conjugates have been prepared. They have been designed to release one of the following materials: MTX, MTX-Lys (alpha-epsilon) or MTX-Lys (gamma-epsilon). These materials have previously been shown to be the low-molecular-weight products released on hydrolysis of a MTX-HSA conjugate by rat liver tritosomes. Preliminary studies were carried out using a lysine residue, linked by the epsilon-amino group to the oligopeptide chain, as a model for the polymer. This use of this model system indicated that an Ala-Leu-Ala-Leu spacer was cleaved by lysosomal enzymes with release of the appropriate derivative. Analysis of the lysosomal digestion products of the MTX-spacer-HSA conjugates showed that the model system appeared to work well. A maximum rate of release of 100% MTX-Lys (alpha-epsilon) from a MTX-spacer-HSA within 48 h was obtained, as compared to 5% in 48 h obtained for MTX-HSA. Determination of the in vitro cytotoxicity of the conjugates showed that release of 'free' MTX was 10-fold more cytotoxic than either of the MTX-Lys derivatives, but the level of release of MTX from MTX-HSA was 10-fold less than that of the MTX-Lys materials.

Amino Acid Sequence↗
PubMed1995

Studies on the mechanism of action of an MTX-HSA-MoAb conjugate.

The active principles of a monoclonal antibody (791T/36)-human serum albumin-methotrexate (MoAb-HSA-MTX) conjugate have been investigated and identified. This drug-carrier conjugate has previously been shown to be selective for the target cell line, more potent than the 'free' drug, to be internalized into the lysosomes of the cell and to work by a lysosomotropic mechanism. Digestion of MTX-HSA by lysosomal enzymes showed three peaks by HPLC assay. Using authentic standards prepared by solid-phase peptide synthesis, these peaks were identified as 'free' MTX, MTX-Lys (alpha-epsilon) and MTX-Lys (gamma-epsilon). Optimization of the digestion conditions allowed for a maximum total release of MTX-containing material of 5% after 48 h. Of this released material, only 10% was in the form of 'free' MTX. It was shown that increasing the ratio of drug to carrier improved the efficiency of release of drug, and these results were complemented by in vitro cytotoxicity assays. Such a low level of drug release associated with a conjugate which has been shown to be superior, in terms of cytotoxicity, to 'free' drug against the target cell line was an unexpected finding. The consequences of these results are discussed.

Animals↗
PubMed1994

Surface modification of poly(lactide-co-glycolide) nanospheres by biodegradable poly(lactide)-poly(ethylene glycol) copolymers.

The modification of surface properties of biodegradable poly(lactide-co- glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethylene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da. respectively) were studied. The results reveal the formation of a PLA:PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Preparation of sterically stabilized human serum albumin nanospheres using a novel Dextranox-MPEG crosslinking agent.

Human serum albumin (HSA) nanospheres with a size less than 200 nm in diameter were prepared using a modified coacervation method and crosslinking with methyl polyethylene glycol modified oxidized Dextram (Dextranox-MPEG) which created a sterically stabilizing polyethylene oxide surface layer surrounding the nanospheres. The crosslinking efficiency and the surface characteristics of glutaraldehyde and Dextranox-MPEG crosslinked HSA nanospheres were determined and compared. The zeta potential of the Dextranox-MPEG crosslinked particles was significantly lower than that of glutaraldehyde stabilized particles. The existence of a hydrated steric barrier surrounding the nanospheres was confirmed by an electrolyte and pH induced flocculation test. The Dextranox-MPEG crosslinked nanospheres showed a significantly reduced plasma protein adsorption on the particle surface compared with glutaraldehyde crosslinked nanospheres.

Chemistry, Pharmaceutical↗
PubMed1994

The acidic groups of the neocarzinostatin protein play an important role in its biological activity.

Despite the extensive literature on the mechanism of action of the anticancer agent neocarzinostatin (NCS), the role of the protein moiety is still not clear. The model involving endocytosis of intact holo-NCS has been dismissed in favor of a theory proposing entry of free dissociated chromophore. However, the fact that the NCS protein has a certain affinity for cell membranes cannot be disregarded. In the present work the protein moiety has been modified by transformation of the carboxyl groups into isopropylamide groups. This modification resulted in a broad shift of the protein pI towards higher values, accompanied by a marked reduction of toxicity in vitro and reduced binding of protein to cells. Coincubation of modified NCS with inactive native apo-NCS led to restoration of the biological activity of native holo-NCS. It has been shown chromatographically that the modified NCS is capable of transferring active chromophore to native apo-NCS. The inactive form (apoprotein) of the charge-modified NCS, however, is capable of inhibiting the toxicity of the active form, as has been described for the corresponding native pair. Binding experiments with tritium-labeled proteins revealed that the modified protein has diminished affinity for membranes, in comparison with native NCS.

Agar↗
PubMed1993

Synthesis of 2'-deoxyuridine and 5-fluoro-2'-deoxyuridine derivatives and evaluation in antibody targeting studies.

Derivatives of 2'-deoxyuridine and of the anticancer agent 5-fluoro-2'-deoxyuridine (FdUR) were linked indirectly via a human serum albumin carrier (HSA) to the murine antiosteosarcoma monoclonal antibody 791T/36. Starting from the 2'-deoxyuridines 1a and 1b, the new nucleosides containing 5'-succinamic acid 7 and 5'-maleamic acid 8 spacers were synthesized from the key intermediate 5'-aminonucleoside 4, and the ribofuronamidobenzoic acid 13 from ribofuranuronic acid 10. These nucleosides were linked via their spacer functionality to HSA. High molar substitution ratios (MSR: moles of drug/mole of HSA) of 25-40 for these derivative-HSA conjugates were achieved. All derivatives were less cytotoxic than the parent drug against both antigen positive osteogenic sarcoma 791T and antigen negative bladder carcinoma T24 cell lines; no IC50 was achieved with any derivative against 791T cells. The fluorodeoxyuridine-HSA conjugates were then further linked via a stable thioether bond to the mouse monoclonal antibody 791T/36. The optimum fluorinated 5'-succinamic acid immunoconjugate exhibited an IC50 of 1 microM against 791T and T24 cells, slightly better than that of fluorodeoxyuridine. The unconjugated derivative 7 was much less cytotoxic than immunoconjugate, with an IC50 of 62 microM on T24 cells, and failed to reach 50% inhibition of 791T cell growth at 290 microM concentration. Derivative 7-HSA conjugate was 10-fold less cytotoxic than the immunoconjugate against both cell lines. Immunoconjugates synthesized with the other 5-fluoro derivatives were unable to effect 50% inhibition of growth of cell lines. Nonfluorinated derivatives and their HSA conjugates and immunoconjugates exhibited no cytotoxicity.

Antibodies, Monoclonal↗
PubMed1991

Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action.

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

Ammonium Chloride↗
PubMed1991

Increased serum stability and prolonged biological half-life of neocarzinostatin covalently bound to monoclonal antibodies.

The pharmacokinetics of neocarzinostatin (NCS) have been compared to NCS conjugates with monoclonal antibodies using Balb/c and tumor bearing nude mice. Data on blood and whole body clearance revealed that the high MW conjugate persists in the body far longer and at a higher level than the free drug. Excretion of the free drug occurs with an extremely rapid renal clearance and localization of the remaining drug in the kidney, whereas the NCS immunoconjugate remained in circulation far longer allowing time for tumor localization to occur without renal accumulation of drug. In addition, NCS conjugated to monoclonal antibody was found to retain its activity in human serum better than free drug, in agreement with data obtained for other NCS-derivatives. Half-time of inactivation was greatly extended when measured under relevant conditions in a DNA strand-break assay. The results indicate that two of the most important requirements for the successful targeting of NCS in vivo, decreased clearance rate and increased serum stability are achieved by conjugation to antibody. Both results increase the probability of NCS accumulating in tissue while still in its active form. Coupling of NCS to monoclonal antibody decreases clearance and inactivation rate and increases localization of the active drug in tumor tissue.

Animals↗
PubMed1989

Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments.

Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.

Animals↗
PubMed1989

Abrogation of antibody responses in rats to murine monoclonal antibody 791T/36 by treatment with daunomycin-cis-aconityl-791T/36 conjugates.

The majority of monoclonal antibodies in clinical use are of murine origin. It is now well-established that patients generate an antibody response to the mouse immunoglobulin which restricts repeated administration. Pre-sensitization of patients to mouse antibody is screened by hypersensitivity to i.d. administered antibody. This study shows that low doses of mouse antibody administered either i.d. or s.c. are highly immunogenic and suggests that a serological assay would be a safer method of screening for anti-mouse antibodies. Rats treated with monoclonal antibody linked via an acid labile cis-aconityl bond to daunomycin failed to produce a primary response to this conjugate. They were also rendered immunologically unresponsive to subsequent challenges with the unconjugated monoclonal antibody. The induced state of immunological unresponsiveness to free antibody persisted in the rats for 18 weeks and although antibody-cis-aconityl-daunomycin pre-treated animals eventually responded to the fourth challenge with free antibody, at week 25, the response was still significantly less than in the free antibody-pre-treated and challenged animals. These studies show that the use of antibody-cis-aconityl-daunomycin conjugates may provide an approach for the control of human responses to mouse immunoglobulin.

Animals↗
PubMed1988

Biodistribution and tumour localization of a methotrexate-monoclonal-antibody 791T/36 conjugate in nude mice with human tumour xenografts.

The blood kinetics and tumour localization of a conjugate of methotrexate (MTX) and MAb 791T/36 were examined in nude mice with human tumour xenografts. The antibody moiety of the conjugate was detected by labelling with 125I and the drug moiety was assayed using a radioimmunoassay for methotrexate. After radioiodination, the drug moiety was co-precipitable with the radiolabel when TCA or rabbit anti-mouse IgG antiserum was used. Following i.v. injection, serum kinetics of both the antibody and the drug moieties of the conjugate were essentially similar, and the integrity of serum-borne conjugate was confirmed by the co-precipitation of radiolabel and drug. The radiolabelled antibody moiety of the conjugate localized in tumour xenografts, with 5-7% of the injected dose being present per gram of tissue within 6 hr of injection, and the levels were maintained for up to 4 days. Analysis of tumour levels of the MTX moiety showed a progressive uptake over the 4-day observation period with up to 4% of the injected dose being present per gram of tumour when the experiment was terminated. Parallel studies with free MTX showed rapid clearance from the blood and a maximum of 0.35% of the dose/g of tumour 30 min after injection. Control immunoglobulin conjugated to MTX did not show tumour localization of either the antibody or the drug moieties. These studies confirm that in vivo MTX remains bound to antibody in this type of drug antibody conjugate and demonstrate site-specific targeting of this therapeutic agent.

Animals↗
PubMed1988

Potentiation of anti-carcinoembryonic antigen immunotoxin cytotoxicity by monoclonal antibodies reacting with co-expressed carcinoembryonic antigen epitopes.

The initial step in ricin A-chain (RTA)-immunotoxin-mediated cell cytotoxicity involves binding to the target cell Ag through the antibody moiety. One of the factors influencing this is the affinity of the antibody component for the target cell Ag. Multiple epitopes on carcinoembryonic Ag have been mapped providing a range of mAb of known specificity. These have been used to show that the cytotoxicity of an immunotoxin containing RTA conjugated to an anti-carcinoembryonic Ag mAb (228-RTA) is potentiated by mAb recognizing different epitopes. The potentiating antibodies also increased the level of target cell binding of antibody 228. Cross-linking of cell bound antibody was not involved because monovalent fragments of a potentiating antibody were effective. The potentiating antibodies modified the binding affinity of 228 antibody increasing the t1/2 of antibody at the tumor cell surface. This increased the dwell time of cell bound antibody and using conjugates of 228 linked to albumin-tetramethylrhodamine it was shown to enhance conjugate endocytosis. These investigations indicate that enhanced antibody affinity leads to increased endocytosis of bound immunoconjugate and potentiates cytotoxicity.

Adjuvants, Immunologic↗
PubMed1988

Biodistribution of a monoclonal antibody-methotrexate conjugate (791T/36-MTX) in patients with colorectal cancer.

The monoclonal antibody (MAb) 791T/36 which has previously been shown to localise in colorectal cancer has been conjugated to methotrexate (MTX) for potential use as a chemotherapeutic agent in malignant disease. To examine its biodistribution and tumour localisation, 16 patients with primary colorectal cancer were injected intravenously with 131I-labelled 791T/36-MTX conjugate and imaged using a gamma camera after 48-72 hr. Serial blood samples were taken to determine the levels of circulating conjugate and samples of tumour and normal colon were assayed for uptake of radioactivity. Whole body biodistribution, visualised by gamma scintigraphy, and blood clearance of both the antibody and drug moieties of 791T/36-MTX were similar to that previously found with unconjugated antibody. Assays of tissue radioactivity showed positive tumour uptake of drug-antibody conjugate (T:NT greater than 1.9:1) in 13/15 primary tumour specimens (median T:NT = 2.9:1). These studies indicate that 791T/36-MTX conjugates may have potential in the treatment of metastatic colorectal carcinoma.

Aged↗
PubMed1987

Sensitivity of newly established colorectal cell lines to cytotoxic drugs and monoclonal antibody drug conjugates.

A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies recognising tumour antigens may overcome these difficulties by providing access of active agents to the tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer, 791T/36, has been investigated as a potential targeting antibody. Eight cell lines were established from surgically resected material and were shown to bind 791T/36 antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although 5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines. However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested. Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a drug monoclonal antibody carrier.

Antibodies, Monoclonal↗
PubMed1987

Conjugates of monoclonal antibody 791T/36 with methotrexate in cancer therapy.

The design and evaluation of drug-antibody conjugates for tumor therapy is considered with respect to the use of the anti-human osteogenic sarcoma monoclonal antibody designated 791T/36. Conjugates have been constructed by linking antibody to methotrexate; the agent has been linked to antibody, either directly or via a human serum albumin bridging agent. In each case, conjugates have been assessed for retention of antibody reactivity with osteogenic sarcoma target cells by comparing their capacity with that of unmodified antibody in competing with fluorescein isothiocyanate-labeled 791T/36 binding, as measured by flow cytometry. Retention of drug cytotoxicity was evaluated by in vitro cytotoxicity and colony inhibition assays. Conjugates with acceptable retention of drug and antibody reactivities were then examined for in vivo suppression of human tumor xenografts.

Animals↗
PubMed1987

Imaging of primary and metastatic colorectal carcinoma with monoclonal antibody 791T/36 and the therapeutic potential of antibody-drug conjugates.

Monoclonal antibody 791T/36, prepared against a tumour-associated 72,000 dalton glycoprotein, reacted with cells from primary and metastatic colorectal carcinomas. I-131 or In-111-labelled antibody localized in xenografts of colorectal carcinomas established from in vitro clonogenic populations. Clinically, with I-131-labelled antibody, 8/11 colonic tumours imaged positively. Imaging was negative in four patients with benign colon disease. 5/11 rectal tumours were positively imaged, but excreted I-131 in the bladder obscured tumours in several studies. In-111-labelled antibody gave superior images and positively imaged primary and metastatic sites in 13/14 patients. Prospectively in the detection of recurrent disease, I-131 or In-111-antibody detected 29/33 separate sites in 24 patients. Seven negative patients remain disease free. There were 3 false positives; overall sensitivity was 88%, with 70% specificity. Specific localization of radiolabel was confirmed immunochemically and by counting radioactivity in resected specimens. Antibody conjugates with methotrexate, vindesine and daunomycin retained drug activity and antibody function, including xenograft localization and conjugates were therapeutically effective against xenografts. 791T/36 antibody has potential for immunodetection of primary and recurrent colorectal carcinoma and for targeting of therapeutic agents.

Animals↗