PubMed Health⌕ Search

Biomedical subjects

C J Springer

Publications and source records attributed to C J Springer.

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

Antibody-directed enzyme prodrug therapy: pharmacokinetics and plasma levels of prodrug and drug in a phase I clinical trial.

Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase absorption and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m2, achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 microM for the "therapeutic window" of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 +/- 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 +/- 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites.

Antibodies, Monoclonal↗
PubMed1997

Biodistribution of an antibody-enzyme conjugate for antibody-directed enzyme prodrug therapy in nude mice bearing a human colon adenocarcinoma xenograft.

The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5-0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1-0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg.

Adenocarcinoma↗
PubMed1997

A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy.

The potential for expressing the bacterial enzyme carboxypeptidase G2 (CPG2) tethered to the outer surface of mammalian cells was examined for use in gene-directed enzyme prodrug therapy. The affinity of CPG2 for the substrate methotrexate was unaffected by three mutations required to prevent N-linked glycosylation. Breast carcinoma MDA MB 361 cells expressing CPG2 internally showed only a very modest increase in sensitivity to the prodrug CMDA because the prodrug did not enter the cells. Cells expressing surface-tethered CPG2, however, became 16-24-fold more sensitive to CMDA and could mount a good bystander effect. Systemic administration of CMDA to mice bearing established xenografts of the transfected cells led to sustained tumor regressions or cures.

3T3 Cells↗
PubMed1997

Gene-directed enzyme prodrug therapy: a review of enzyme/prodrug combinations.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising, new, two-step treatment for cancer chemotherapy. In the first step, the gene for a foreign enzyme is administered and is directed to the tumour, where it is expressed by the use of specific promoters. In the second step, injected prodrugs are activated by the foreign enzyme. The design and synthesis of prodrugs able to undergo enzymatic activation in such systems is an essential component. This review focuses on the requirements which must be fulfilled by the components of GDEPT systems in order for this therapy to be considered a realistic possibility. A special emphasis is placed on the description of the prodrugs used in GDEPT protocols and the requirement for a bystander effect is also discussed.

Journal Article↗
PubMed1996

Gene-directed enzyme prodrug therapy with a mustard prodrug/carboxypeptidase G2 combination.

The gene for the bacterial enzyme carboxypeptidase G2 (CPG2) was expressed internally in mammalian cells. Mammalian-expressed CPG2 had kinetic properties indistinguishable from bacterially expressed CPG2. Human tumor cell lines A2780, SK-OV-3 (ovarian adenocarcinomas), LS174T, and WiDr (colon carcinomas) were engineered to express constitutively either CPG2 or bacterial beta-galactosidase. These cell lines were subjected to a gene-directed enzyme prodrug therapy regime, using the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). The lines which expressed CPG2 had enhanced sensitivity to CMDA. Comparing IC50S, WiDr-CPG2 and SK-OV-3-CPG2 were 11-16-fold more sensitive, whereas A2780-CPG2 and LS174T-CPG2 were approximately 95-fold more sensitive than the corresponding control lines. CPG2-expressing cells and control cells were mixed in differing proportions and then treated with prodrug. Total kill occurred when only approximately 12% of cells expressed CPG2 with the WiDr and SK-OV-3 lines and when only 4-5% of cells expressed CPG2 with the LS174T and A2780 lines, indicating a substantial bystander effect. These results establish this CPG2 enzyme/CMDA prodrug system as an effective combination for the gene-directed enzyme prodrug therapy approach.

Animals↗
PubMed1996

ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts.

ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.

Animals↗
PubMed1996

New mustard prodrugs for antibody-directed enzyme prodrug therapy: alternatives to the amide link.

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.

Aniline Mustard↗
PubMed1996

Prodrugs of thymidylate synthase inhibitors: potential for antibody directed enzyme prodrug therapy (ADEPT).

Prodrugs of quinazoline antifolate thymidylate synthase (TS) inhibitors have been designed and synthesized for use in antibody-directed enzyme prodrug therapy (ADEPT). The syntheses of the alpha-linked dipeptides of two potent thymidylate synthase inhibitors, ZD1694 [N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid] and ICI198583 ¿N-[4-[N-[(2-methyl-3,4-dihydro-4-oxo-6-quinazolinyl) methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid¿ are described. The alpha-carboxyl of the glutamic acid has been linked through an amide bond to an L-alanine or an L-glutamic acid. The alpha-linked L-dipeptide prodrugs were designed to be activated to their corresponding thymidylate synthase inhibitors at a tumour site by prior administration of a monoclonal antibody conjugated to the enzyme carboxypeptidase A (CPA). The viability of a colorectal cell line was monitored with the potential prodrugs in the presence or absence of CPA or with the parent drugs alone. All the dipeptides had greatly decreased cytotoxicity, with a deactivation of approximately 100-fold for the ZD1694 prodrugs and approximately 20-200-fold for the ICI198583 prodrugs. Activation of the alpha-linked L-alanine dipeptides with CPA led to a cytotoxicity enhancement of approximately 10-100 fold.

Antineoplastic Agents↗
PubMed1995

Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT).

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.

Antibodies, Monoclonal↗
PubMed1995

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954.

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.

3T3 Cells↗
PubMed1995

Anti-tumour effects of an antibody-carboxypeptidase G2 conjugate in combination with phenol mustard prodrugs.

ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl-L- glutamic acid (PGP) and (S)-2-[N-[4-[N,N-bis(2-chloroethyl)amino]- phenoxycarbonyl]amino]-4-(5-tetrazoyl)butyric acid (PTP), which are cleaved by the bacterial enzyme CPG2 to release the 4-[N,N-bis(2-chloroethyl)amino] phenol drug. In vitro, both prodrugs are approximately 100- to 200-fold less potent than the parent drug (1 h IC50 = 1.4 microM) in LoVo colorectal tumour cells. These prodrugs have been evaluated for utility in ADEPT when used in combination with a conjugate of CPG2 and the F(ab')2 fragment of the anti-CEA monoclonal antibody, A5B7. The conjugate was shown to localise specifically to established LoVo tumour xenografts growing in nude mice and optimal tumour-normal tissue ratios were achieved after 72 h. Administration of either prodrug, at doses which cause 6-8% body weight loss, 72 h after administration of the A5B7-CPG2 conjugate to the LoVo tumour-bearing mice resulted in tumour regressions and growth delays of 14-28 days. The PTP prodrug in combination with a high dose of conjugate (10 mg kg-1) gave the best anti-tumour activity despite being a 10-fold worse substrate for CPG2 than PGP. Prodrug alone, active drug alone or prodrug in combination with a non-specific conjugate had minimal anti-tumour activity in this tumour model.

Aniline Mustard↗
PubMed1995

Antibody-directed enzyme prodrug therapy (ADEPT) with mustard prodrugs.

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step targeting procedure designed to improve the selectivity of anti-tumour agents. The approach is based on the activation of specially designed prodrugs by enzyme-antibody conjugates targeted to tumour-associated antigens. This review concerns ADEPT using nitrogen mustard prodrugs and carboxypeptidase G2 (CPG2) as the activating enzyme. The specific structural features required of the nitrogen mustard prodrugs, their design, syntheses, physicochemical properties, biological characteristics and activation to the corresponding drugs are reviewed. The ADEPT clinical trial with a nitrogen mustard prodrug is also discussed.

Animals↗
PubMed1994

Regression of established breast carcinoma xenografts with antibody-directed enzyme prodrug therapy against c-erbB2 p185.

The enzyme carboxypeptidase G2 (CPG2) was conjugated to the rat IgG2a monoclonal antibody (mAb) ICR12, which recognizes the external domain of the human c-erbB2 protooncogene product. The conjugate retained antigen-binding and enzyme activity. Radiolabeled conjugate localized efficiently and stably to MDA MB 361 breast carcinoma xenografts, which overexpress the c-erbB2 gene product p185. Radiotracer determinations and plasma enzyme activity studies in nu/nu mice gave conjugate blood clearance rate half-lives of approximately 4 days. In separate antibody-directed enzyme prodrug therapy regimes, one dose of the 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid prodrug was administered to nu/nu mice bearing established MDA MB 361 tumors (mean volume, 170-260 mm3). In mice which had received ICR12-CPG2 12-14 days previously, sustained dose-dependent tumor stasis or regressions were effected, which in some cases persisted throughout observation periods of up to 90 days. In control mice which had received the isotype-matched irrelevant mAb ICR16-CPG2 conjugate, tumors grew progressively, as did those in mice treated with prodrug alone, or treated simultaneously with ICR12-CPG2 and prodrug at the maximum tolerated dose. Control chemotherapy with conventional drugs proved toxic and induced only minimal growth delays.

Animals↗
PubMed1994

Novel prodrugs of alkylating agents derived from 2-fluoro- and 3-fluorobenzoic acids for antibody-directed enzyme prodrug therapy.

The synthesis of six novel fluorinated potential prodrugs for antibody-directed enzyme prodrug therapy is described. The [2- and 3-fluoro-4-[bis(2-chloroethyl)amino]benzoyl]-L-glutamic acid (9 and 21), their bis(mesyloxy)ethyl derivatives (7 and 19), and their chloroethyl (mesyloxy)-ethyl derivatives (8 and 20) are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to the glutamic acid residue. These compounds were designed to be activated to their corresponding benzoic acid alkylating agents at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2). The synthesis of the analogous novel parent drugs 2- and 3-fluoro-4-[bis(2-chloroethyl)amino]benzoic acid (12 and 24), their bis(mesyloxy)ethyl derivatives (10 and 22), and their chloroethyl (mesyloxy)ethyl derivatives (11 and 23) is also described. The viability of a colorectal cell line was monitored with the six potential prodrugs in the presence of CPG2 and with the parent drugs alone. Compounds 19-21 demonstrated substantial prodrug activity, with activation by CPG2 leading to cytotoxicities comparable to those of 22-24, respectively. The Km and kcat values for 7-9 and 19-21 were determined for CPG2. All potential prodrugs except 7 proved to be excellent substrates. A comparison of the relative chemical reactivity of the compounds as determined by their half-life measurements showed that the 2-fluoro substituent deactivated while the 3-fluoro substituent activated the alkylating moieties.

Alkylating Agents↗
PubMed1994

Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy.

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two- and three-phase system in which an antibody to a tumor-associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic-antigen antibody fragment A5B7-F(ab')2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate-specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor-to-blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT.

Animals↗
PubMed1994

Analysis of antibody-enzyme conjugate clearance by investigation of prodrug and active drug in an ADEPT clinical study.

Antibody-directed enzyme prodrug therapy (ADEPT) separates the cytotoxic function from the targeting function (5). An antibody-carboxypeptidase G2 (CPG2) enzyme is delivered prior to the nontoxic prodrug, CMDA, which is converted to a cytotoxic drug by the action of the localized conjugate at the tumor site. An indirect in vitro assay was developed to detect the presence of functional CPG2 in the plasma of patients in an ADEPT clinical trial. Compounds in the plasma of patients were characterized using liquid chromatography-mass spectrometry. Plasma at three different time points (prior to treatment, post-antibody-enzyme conjugate, and post-galactosylated anti-enzyme antibody clearing agent) was added to the CMDA prodrug and analyzed. Conversion of the CMDA prodrug to its active drug indicates that CPG2-conjugate remains in the plasma. This technique will provide essential data for the timing of prodrug administration in ADEPT.

Antibodies, Monoclonal↗
PubMed1994

Antibody-directed enzyme prodrug therapy (ADEPT). A three-phase study in ovarian tumor xenografts.

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with 125I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab')2 fragment showed localization in these xenografts. The AB57-F(ab')2 fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with 125iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab')2-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.

Animals↗