Journal of Clinical Laboratory Analysis 6:136-142 (1992)
An Enzyme lmmunoassay for Determining Plasma Concentrations of Didemnin B T.J.G. Raybould,’t P.G. Grothaus,’ Samantha 6. Simpson,’ G.S. Bignami,’ Carolyn B. Lazo,’ and R.A. Newman* ‘Hawaii Biotechnology Group, Inc., lmmunochemistry Division, Aiea, Hawaii; ’Section of Pharmacology, Department of Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Texas Didemnin A was conjugated at the amino terminus of the N-methylleucine residue, via the linkers N-succinimidyl-3-(2-pyridyldithio)propionate and trans-l,4-maleimidomethylcyclohexane carboxylic acid, to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). The didemnin-KLH conjugates were used to hyperimmunize rabbits. The resulting high titer antisera were employedwith didemnin-BSA conjugate-coated microtiter Key words:
plate wells to develop an indirect competitive inhibition enzyme immunoassay(CIEIA) that was fully cross reactive with didemnin 6.A ClElA is described that is capable of detecting the drug in plasma from didemnin 6treated patients at concentrations down to 1-3 ng/ml. This simple, sensitive ClElA has been employedto demonstrate plasma drug clearance profiles with samples from didemnin 6-treated patients. o 1992Wiley-Liss, tnc.
didernnins, antibodies, antisera, ELISA, cancer, plasma didemnin concentrations
auspices of the NCI. These trials have been hampered by the unavailability of a suitable DB assay with which to monitor The didemnins are cyclic depsipeptides isolated from the blood DB levels (Jason Fisherman, NCI, personal commuCaribbean tunicate Trididernnumsolidurn (1-3). Five related nication, 1990). A sensitive and reliable assay would clearly compounds, given the names didemnin A, B, C , D, and E, aid in this task and enable more extensive pharmacokinetic have been isolated and assigned structures (2,4,5). Didemnin studies to be performed (1 1). A (DA), which is structurally the simplest member of this Attempts to develop such an assay have been made. Dorr class of compounds, is composed of six amino acid units and et al. tried unsuccessfully to develop a B 16 melanoma bioasone hydroxy acid unit (Fig. 1). Six of the residues form the say (12). These authors and Hartshorn et al. have used high parent macrocycle with one amino acid unit in the side chain. performance liquid chromatography (HPLC) systems with deThe other didemnins are derivatives of DA, differing by vir- tection limits of 50 m g / d (12) and 5 ng/ml, respectively (13). tue of substitution at the N-methylleucine chain terminus. Dorr et al. also reported the development of a radioimmunoasThe didemnins have been shown to exert a variety of say (RIA) with a detection limit of 20 pg/ml(12,14); however, potentially significant biological activities in vitro and in vivo, the antiserum required for this assay may no longer be available although their exact site and mechanism of action have not (Jason Fisherman, NCI, personal communication, 1990). yet been defined (6,7). These compounds are also immunoIn this study, didemnin haptens were synthesized and cousuppressive, a property which has been ascribed to their anti- pled to protein carriers for use as immunogens, and as solid proliferative activity, rather than to immunosuppression per phase coating antigen in enzyme-linked immunosorbent assay se (8). In addition, the didemnins have antiviral activity against (ELISA) systems with anti-didemnin antibodies. Anti-didemnin many DNA and RNA viruses. antibodies from hyperimmunized rabbits were used to develop Didemnin B (DB) is the most potent member of the didemnin a competitive inhibition enzyme immunoassay (CIEIA) sysclass of compounds. Initial shipboard screening of Trididernnum tem for detecting DB. This CIEIA was validated first with solidurn extracts for antiviral activity also revealed a high level pooled human plasma to which known amounts of DB had of mammalian cytotoxicity (9). DB’s antitumor activity was confirmed in the National Cancer Institute’s (NCI) in vivo murine tumor screening program (6). In addition, DB has shown activity in vitro against certain human tumors in the Received November 1, 1991;accepted January 6, 1992 Human Tumor Stem Cell Assay (1 0). These studies formed Address reprint requests to Paul G. Grothaus, Ph.D., Hawaii Biotechnolthe basis for examining the anticancer therapeutic potential ogy Group, Inc., 99-103 Aiea Heights Drive, Aiea, HI 96701. of DB in phase I and I1 clinical trials, conducted under the ‘Deceased.
0 1992 Wiley-Liss, Inc.
Enzyme lmmunoassay for Plasma Didemnin B
&%4? w @+* 0
(SPDP) pyr. 25 “c. 18 h
Fig. 1. Synthesis of didemnin haptens
been added, then by demonstrating typical drug clearance profiles with plasma samples from DB-treated patients.
MATERIALS AND METHODS DA and DB were a gift from Professor Kenneth Rinehart of the University of Illinois. DB (NSC 325319) for construction of standard curves with human plasma was supplied by the National Cancer Institute. HPLC analyses were performed with a Gilson HPLC system (Gilson Medical Electronics, Inc., Middleton, WI). All HPLC analyses and purifications were performed under the following conditions: C18 column (Phenomenex, Torrance, CA); 77:23:0.01 methanol/water/ triethylamine; adjusted to pH 7.5 with acetic acid; 3.0 ml/min. Nuclear magnetic resonance (NMR) spectra were acquired on a GE Omega 500.
for 15 min and a solution of DA (10 mg, 10.62 pmol) in CH2C12(1 .0 ml) was added. After being stirred for 48 hr at ambient temperature, the reaction was diluted with CH2C12 (10 ml) and washed with H20 (3 x 5 ml). The CH2C12 layer was dried (Na2S04) and concentrated to yield a gummy yellow residue. The product (TR 27 min) was separated from unreacted DA (TR 20 min) by preparative reversed phase HPLC .
Thiolation of Keyhole Limpet Hemocyanin (KLH)
A sample of KLH was purified on a Sephadex (3-25 column that had been equilibrated with 25 mM borate buffer, pH 9.0. The protein concentration of the purified KLH was determined from the extinction (OD) of the sample at 280 nm. The KLH was mixed with a 50-fold molar excess of 2-iminothiolane (IMT) in borate buffer and stirred for 1 hr at room temperature. The resulting thiolated KLH (KLH-SH) Synthesis of 3’-(2”-Pyridyldithio)Propionyl-Didemnin was purified over Sephadex (3-25 that had been equilibrated A (DA-PDP) with 50 mM sodium phosphate buffer, pH 6.6. The concenN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (3.65 tration of KLH-SH was determined from the OD of the sammg, 11.68 pmol) was added to a solution of DA (10 mg, ple at 280 nm and the molar ratio of sulfhydryl groups per 10.62 pmol) in pyridine (2.0 ml). After being stirred for 18 KLH was estimated by the 2,2’-dithiodipyridine (2-DTDP) hr at ambient temperature, the solvent was removed in vacuo. assay (16). The residue was dissolved in CH2C12 (10 ml) and washed with H 2 0 (3 X 5 ml).The CH2C12layer was dried (Na2S04)and Thiolation of Bovine Serum Albumin (BSA) concentrated to yield a gummy yellow residue. The product A sample of BSA in 25 mM borate buffer, pH 9.0, was (retention time (TR] 36 min) was separated from unreacted mixed with a 2.5-fold molar excess of IMT in borate buffer DA (TR 20 min) by preparative reversed phase HPLC. and stirred for 1 hr at ambient temperature. The resulting
Synthesis of 4’-(N”-Maleimidomethyl)Cyclohexane1-Carboxyl-Didemnin A (DA-MCC) 1-( 3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (2.44 mg, 12.74 pmol) was added to a solution of trans-l,4-maleimidomethylcyclohexane(MCC) (15) (2.77 mg, 1 1.68 pmol) in CH2C12 (1 .0 ml). This mixture was stirred
thiolated BSA (BSA-SH) was purified, and its concentration determined, using the methods employed for KLH-SH.
DA-PDP-KLH and DA-PDP-BSA Conjugation To a freshly prepared and characterized sample of KLH-SH or BSA-SH was added a 5-fold molar excess (relative to the net protein thiolation) of an ethanol solution of DA-PDP.
Raybould et al.
The concentration of KLH-SH or BSA-SH had been adjusted such that the final concentration of ethanol in the reaction mixture did not exceed 5% of the total volume of the reaction mixture. After being stirred for 18 hr at ambient temperature for DA-PDP-KLH or 4°C for DA-PDP-BSA, the conjugates were centrifuged for 10 minutes to remove particulate material. The supernatants were then transferred into a Centricon'" 30 unit (Amicon Division, W.R. Grace and Co., Beverly, MA) and centrifuged for 20 min at 3,0006.. The DA-PDP-KLH conjugate retentate was purified on a Sephadex G-25 column that had been equilibrated with 50 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl (PBS). The DA-PDP-BSA retentate was diluted with 2 ml of phosphate buffer and again centrifuged in the Centricon'" 30. This was repeated 4 times and the final DA-PDP-BSA retentate diluted to 2 ml with phosphate buffer. The conjugation ratios of the DA-PDP-KLH and DA-PDPBSA conjugates were estimated via two methods. The equivalents of pyridine-2-thione released during the conjugations were determined from the OD at 343 nm of the initial filtrates from the Centricon'" 30s. The concentrations of DA-PDPKLH and DA-PDP-BSA were estimated from the OD of each sample at 280 nm and the molar ratios of unreacted sulfhydry1 groups per conjugate molecule measured by the 2-DTDP assay ( 1 6). The decrease in measurable sulfhydryls per KLH or BSA molecule was taken to be an indirect estimate of DA conjugation to each protein.
DA-MCC-KLH and DA-MCC-BSA Conjugation To a freshly prepared and characterized sample of KLH-SH or BSA-SH was added a 5-fold molar excess (relative to the net KLH thiolation) of an ethanol solution of DA-MCC. The concentration of KLH-SH or BSA-SH had been adjusted such that the final concentration of ethanol in the reaction mixture did not exceed 5% of the total volume of the reaction mixture. After being stirred for at 4°C for 18 hr, each conjugate was dialyzed against 4 changes of 500 ml phosphate buffer at 4°C. The concentrations of DA-MCC-KLH or DA-MCCBSA and the degree of DA conjugation to each protein were estimated using the methods described in the preceding section. Immunization of Rabbits With DA-Conjugates Four New Zealand White female rabbits were immunized with the DA hapten-KLH conjugates. Priming injections (500 p g of DA-PDP-KLH or 385 p g of DA-MCC-KLH per rabbit), emulsified in an equal volume of complete Freund's adjuvant, were administered intramuscularly, subcutaneously, and intradermally at multiple sites. Booster injections (150 p g of DA-PDP-KLH or 200 p g of DA-MCC-KLH per rabbit), emulsified in an equal volume of incomplete Freund's adjuvant, were administered on days 14, 28,42, and 56 after priming. Test bleeds were taken on days 14, 21, 35, and 49 after priming, and large bleeds on day 63. Rabbits 1 and 4 were immunized with DA-PDP-KLH and rabbits 2 and 3 were immunized with DA-MCC-KLH.
Primary Indirect ELSA Screen for Antibodies to DA The primary indirect ELISA was performed by the microplate modification ( 17) of the method of Engvall and Perlmann ( 1 8). Immulon 2 microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 50 pl/well of DA-PDP-BSA or DA-MCC-BSA at a predetermined concentration in PBS, for 1 hr at room temperature. The optimum coating concentration for each batch of antigen was determined by titration. Plates were then washed three times with 0.01 M Tris, pH 7.0, 0.15 M NaCl containing 0.02% NaN3 and 0.05% (v/v) Tween-20 (TBS-T). After blocking with 200 pl/well of 1% BSA in PBS at room temperature for 1 hr, or at 4°C for I8 hr, microtiter plates were washed three times with TBS-T. Fifty microliters of the rabbit serum being tested for anti-DA antibody, titrated in 1% BSA in PBS, was added to each well, and plates were incubated at room temperature for 1 hr. Microtiter plates were washed three times with TBS-T, then 50 pl/well of alkaline phosphatase (AP)-labeled goat anti-rabbit IgG (gamma + L chain-specific) (Caltag Laboratories, Inc., San Francisco, CA) conjugate, at an optimal dilution in 1 % BSA in PBS (previously established by titration), was added. After incubation for 1 hr at room temperature, plates were washed four times with TBS-T, then 200 pl/well of 1 mg/ml p-nitrophenylphosphate (pNPP; Sigma 104, 5 mg tablets; Sigma Chemical Co., St. Louis, MO), diluted in AP substrate buffer (25 mM Trizma base, pH 9.5, 0.15 M NaCl, 5 mM MgCI2, 0.02% [w/v] NaN3, pH 9.5), was added. Plates were incubated for 1 hr at room temperature, then the absorbance of each well was read on a Titertek Multiskan MC (ICN Flow Laboratories, Inc., Costa Mesa, CA) using a sample wavelength of 414 nm and a reference wavelength of 690 nm.
ClElA for DA/DB Immulon 2 microtiter plates were coated, blocked, and washed using the method described in the preceding paragraph, except that 100 pl/well of DA-PDP-BSA or DA-MCCBSA was employed. After washing, 50 pllwell of DA or DB, diluted in bovine calf serum (HyClone Laboratories, Inc., Logan, UT) and 50 pl/well of rabbit anti-DA antibody, at an optimal dilution in 1 % BSA in PBS (previously established by titration), were added. Plates were incubated at room temperature for 1 hr and the assay completed using the method described in the preceding paragraph, except that 100 pllwell of AP-labeled goat anti-rabbit IgG conjugate was added.
Data Analysis All samples tested by ELISA and CIEIA were normally run in triplicate and the mean result of each set of replicates was calculated. BSA-coated wells treated with AP-conjugate and substrate were included on each plate to measure background color development, and the mean OD414of these wells
Enzyme lmmunoassayfor Plasma Didemnin B
was subtracted from the mean OD414 of each set of control and test replicates before data analysis. For CIEIAs, standard curves were constructed for each experiment using a set of DA or DB standards. B/Bo values for each standard curve were calculated by dividing the mean OD414 of a given set of replicates containing DA or DB inhibitor by the mean OD414 of all the wells containing no inhibitor. Unknown didemnin concentrations in samples under test were calculated from the OD414 of the sample dilution(s) that fell within the linear portion of the standard curve. The 50% inhibitory concentration (ICso) and IC20values from standard curves were used to establish the sensitivity and cross-reactivity levels of the CIEIA system.
RESULTS Synthesis of Didemnin Haptens Two didemnin haptens, DA-PDP and DA-MCC, were prepared via acylation of the N-methyl-D-leucyl terminus of DA.
Synthesis of DA-PDP DA-PDP was synthesized by treatment of DA with SPDP in pyridine (Fig. I). The structure of DA-PDP was conclusively proven by homonuclear H-COSY (2-dimensional protonproton correlated spectroscopy). Comparison of the COSY data for DA (19) and DA-PDP confirmed the presence of a monosubstituted pyridine ring and a X-CH2CH2-Ygrouping as well as the disappearance of an amide proton. In addition, the COSY spectra confirmed that the signal for the a-H of the N-MeLeu residue shifted from 6 4.1 to 6 5.15, thus confirming that acylation occurred on the nitrogen of the N-MeLeu residue.
Synthesis of DA-MCC DA-MCC was synthesized via an EDC mediated coupling of DA with MCC (Fig. 1). Comparison of the COSY data for DA (19) and DA-MCC confirmed the presence of the cyclohexane grouping as well as the disappearance of an amide proton. In addition, the COSY-NMR spectra revealed that the signal for the a - H of the N-MeLeu residue shifted from 6 4.1 to 6 5.1, thus confirming that acylation occurred on the nitrogen of the N-Me-Leu residue.
Antibody Production and ClElA Development Sera from bleeds from each rabbit were initially tested by an indirect ELISA system using microtiter plate wells coated with either DA-PDP-BSA, DA-MCC-BSA, BSA, or KLH. The endpoint titer against each coating antigen was noted, together with the serum dilution that resulted in an OD414 of about 0.5, 1 hr after substrate addition on DA-PDP-BSA and DA-MCC-BSA coated wells. A working dilution of half this dilution was employed for each serum in the CIEIA system. Bleeds taken from all animals on days 21 and 28 had extremely high endpoint titers against KLH in the indirect ELISA system, indicating that KLH conjugated DA was not significantly immunosuppressive. These sera had insignificant reactivity against BSA. The responses of all four rabbits exhibited virtually equal cross-reactivity with DA-PDP-BSA and DA-MCC-BSA. Rabbits 2 and 3 , immunized with DA-MCC-KLH, exhibited much higher titer antibody responses than rabbits 1 and 4, immunized with DA-PDP-KLH. The response of rabbit 4 was significantly lower than that of rabbit 1 and did not reach a level where its serum was of any value for immunoassay development. Binding of serum antibody from the day 21 and 35 bleeds of rabbit 1 to solid phase coating antigen was specifically inhibited by free DA in the CIEIA, with an ICso of 12-31 ng/ml. However, the level of inhibition of this response by free DB dropped in later bleeds from this rabbit. Sera from rabbits 2 and 3 had extremely high CIEIA working dilutions and continued to be specifically inhibited by free DA in this system, with in the 15-30 ng/ml range. Serum antibody from the day 63 large bleeds taken from rabbits 2 and 3 was clearly usable in a sensitive CIEIA for DA. Further optimization of the CIEIA system was achieved using final bleed serum from rabbit 3 at a dilution of 1 in 32,000, AP-goat anti-rabbit IgG conjugate at a dilution of 1 in 1,000 and microtiter plate wells coated with DA-PDP-BSA. The use of microtiter plate wells coated with DA-PDP-BSA in the CIEIA resulted in greater sensitivity than use of those coated with DA-MCC-BSA. The cross-reactivity of this optimized CIEIA system with DB was investigated. The system was fully cross-reactive with DA and DB with an IC50 of 3 ng/ml (0.16 pmol) and an IC20 of 0.8 nglml (0.04 pmol) (Fig. 2).
Conjugationof Didemnin Haptensto Protein Carriers
Variation Between Assays and Within a Single Assay
The DA-PDP and DA-MCC haptens were each conjugated to KLH and BSA to prepare didemnin immunogens and ELISA coating antigens respectively. The KLH and BSA were prepared for conjugation via thiolation of their lysine residues with 2-IMT. Subsequent treatment with DA-PDP or DA-MCC resulted in formation of stable disulfide or thioether linkages, respectively, between the haptens and carrier proteins.
Variation between assays run on five separate days was examined by constructing daily CIEIA standard curves of DB diluted in bovine calf serum. The standard curve range extended from 0.1 ngiml to 300 ng/ml. Coefficients of variation for the mean values from the standard curve points in its linear portion typically were less than 20%. Although concentrations as low as 0.8 ng/ml (at IC20)were detectable, the coefficient of variation (CV) produced by analyses on this portion of the stan-
----D-Human Piasma 1
. . . . . . I
. . ......
. . . . . . I
[DBI w m l )
Fig. 2. Comparison between standard curves for detection of didemnin B in bovine calf serum and human plasma by the CIEIA. DB was titrated in bovine calf serum and in two different human plasma samples through the concentration range shown in the graph, and samples tested, with controls containing no DB, in duplicate by the CIEIA. B/Bo values were calculated by dividing the mean of each set of replicates containing DB inhibitor by the mean of all the wells containing no inhibitor, and plotted against DB concentration. Error bars are shown for the bovine calf serum titration curve only, and represent t I standard deviation.
dard curve (53%)was considered to be unacceptable. Variation within a single assay was examined by testing 15 replicates containing 10 ng/ml and 15 replicates containing 5 ngiml of DB by the CIEIA system. Mean DB concentrations were calculated to be 9.5 ng/ml and 6.0 ng/ml, with standard errors of 0.41 ngiml (CV = 11%) and 0.28 ng/ml (CV = 1 l%), respectively. Variation between assays using the CIEIA was examined by constructing a standard curve from DB standards diluted in bovine calf serum on five separate days. The mean and ICzOfrom the five standard curves were 3.02 nglml and 0.77 ng/ml, with standard errors of 0.23 nglml (CV = 17%) and 0.18 ng/ml (CV = 53%), respectively.
of Medical Oncology of the University of Texas M.D. Anderson Cancer Center. These patients had received 6.3 mg/m2 of DB administered in 50 cc of sterile normal saline by intravenous infusion over a period of 30 min. Blood samples were taken prior to infusion, at the end of infusion, and at 5, 10, 30, 60, 120, 240, and 360 min post-infusion. For assay by the CIEIA method, duplicate plasma samples were tested undiluted, and diluted 10- and 50-fold in pre-infusion plasma. For construction of standard curves, DB standards at concentrations ranging between 0.3 and 300 nglml were prepared in triplicate in pre-infusion plasma from each patient and also tested. B/Bo values were calculated by dividing the mean of each set of replicates containing DB inhibitor by the mean of wells containing no inhibitor, and plotted against DB concentration. Test sample dilutions that gave B/Bo values between 0.3 and 0.7 were used to calculate plasma DB levels by interpolation from the standard curve. The calculated DB concentration in each plasma sample was then plotted against time, as shown in Figure 3. The non-linear log DB concentration versus time profile observed appears typical of drugs which fit a two-compartment pharmacokinetic model, which is characterized by a rapid distribution phase and a more prolonged elimination phase (20). More extensive analysis of DB pharmacokinetics using the CIEIA system will be reported elsewhere.
Validation of ClElA System by Recovery of Added DB From Pooled Human Plasma l
The efficacy of the CIEIA system for measuring DB in human plasma was investigated. Standard concentrations of DB were added to aliquots of two plasma samples drawn from different volunteers, and each aliquot was tested by the CIEIA. Figure 2 shows that the CIEIA standard curves for detecting DB in human plasma were not significantly different from the standard curves for detecting DB in bovine calf serum.
Determinationof the Efficacy of the ClElA for Measuring DB in Plasma Samples From DB-Treated Patients Plasma samples from two patients (patients 1 and 2) who had been treated with DB were supplied by the Department
, . , . 30 60
, . 90
1 2 0 150
180 2 1 0 240
270 300 330 360
Fig. 3. Change in didemnin B concentration with time in plasma from two patients after infusion with didemnin B, as measured by the CIEIA. Plasma samples, taken prior to infusion, at the end of infusion, and at 5, 10, 30, 60, 120, 240, and 360 min post-infusion were tested, in duplicate, undiluted and diluted 10- and 50-fold in pre-fusion plasma, by the CIEIA method. For construction of standard curves, DB standards at concentrations from 0.3 to 300 nglml were prepared in triplicate in pre-infusion plasma from each patient and also tested; BIB, values were calculated by dividing the mean of each set of replicates containing DB inhibitor by the mean of wells containing no inhibitor, and plotted against DB concentration. Test sample dilutions that gave BIB, values between 0 . 3 and 0.7 were used to calculate plasma DB by interpolation from the standard curve. The calculated DB concentration in each plasma sample was then plotted against time, as shown in the above graph.
Enzyme lmmunoassay for Plasma Didemnin B
DISCUSSION In this study, rabbit antibodies, produced against characterized DA-haptens conjugated to KLH, were used to develop a simple, sensitive CIEIA system that employs DA-haptens conjugated to BSA, for quantitating DA and DB in plasma samples. The CIEIA detected DB with an ICs0 of 3 ng/ml and IC20 of 0.8 ng/ml, and had a total assay time of 5-6 hr, including coating and blocking microtiter plate wells and all technical manipulations. DA was chosen as the starting material for the synthesis of didemnin haptens because it was more readily available in the quantities necessary for synthetic chemistry than DB. In addition, immunogens conjugated via the amino terminus of DA were expected to elicit antibodies against the common structural feature of DA and DB, the macrocyclic ring. Such antibodies would be expected to be cross-reactive with any early DB metabolites possessing an intact ring. The DA-MCC-KLH conjugate proved to be considerably more successful as an immunogen than DA-PDP-KLH. Conversely, the use of DA-PDP-BSA coating antigen provided a CIEIA system with greater sensitivity than the use of DA-MCCBSA. The optimal CIEIA system therefore employed rabbit antibody against DA-MCC-KLH and microtiter plate wells coated with DA-PDP-BSA. This combination would be expected to provide the most specific system, as it avoids reaction between linker and anti-linker antibodies. It might be predicted that the SPDP-linkage would be less stable than the SMCC linkage, particularly in vivo. The DA-MCC-KLH conjugate might therefore be expected to retain its integrity better than the DA-PDP-KLH conjugate during slow release from the adjuvant depot in injected rabbits. This hypothesis is supported by the observation that the antibody responses of the DA-PDP-KLH-injected rabbits against DA were much lower than the responses of those injected with DA-MCC-KLH. Rabbit 4 did not respond satisfactorily at any time, and both the CIEIA working dilution and the capacity for inhibition by DA of serum from rabbit 1 actually decreased as the immunization schedule progressed. This phenomenon could have been caused by instability of the DA-PDPKLH conjugate in vivo, resulting in free DA causing immunosuppression in the rabbits injected with this conjugate. The immunosuppressive properties of DB have been described (21,22), and DA may exhibit similar properties. The experiments performed to determine the level of variation with the CIEIA between assays and within a single assay indicated that both the inter- and intra-assay variation were acceptable. The ICsos of the standard curves constructed on five different days had a CV of less than 20%, while the IC20 had a CV of 53%. The high % CV of the ICz0s reflects the lower level of accuracy with results obtained from the nonlinear extremes of the standard curve, rather than the cen-
tral, linear portion. If CIEIA results on a patient sample fell on the non-linear extremes of the standard curve, the sample would be rerun at a less dilute concentration to bring results within the central, linear portion. The mean DB concentrations of 9.5 ng/ml and 6.0 ng/ml, determined from a standard curve for 15 aliquots containing 10 ng/ml and 5 ng/ml, respectively, are within the level of accuracy and precision expected from an enzyme immunoassay system. The level of intra-assay variation, reflected by the CVs of 11% for both the 10 ng/ml and 5 ng/ml samples, is also acceptable for this type of system. The CIEIA system was developed using DA and DB diluted in bovine calf serum rather than buffer, as the ultimate aim of this project was to develop an assay that could be performed using human plasma. Calf serum had been selected because of its similarity to human plasma, because it is readily available, and because it is free from many of the hazards associated with human material of unknown origin. The sensitivity of the CIEIA was not significantly different for detecting DB in bovine calf serum (Fig. 2). The sensitivity of the CIEIA with plasma samples from DB-treated patients was satisfactory and seemed to compare well with the RIA of Dorr et al. (12). Despite administration of different drug levels to the patients in these two studies, the distribution phase pharmacokinetic profile is quite similar. The slopes of the plasma decay curves shown in Figure 3 virtually parallel the curve published in their paper, for time points between 0 and 30 min post-infusion. The DB pharmacokinetic profile determined by CIEIA approximates a two-compartment model with a rapid distribution phase and a prolonged elimination phase (20). Dorr et al. also describe a rapid distribution of drug out of the plasma, with nearly 50% of DB removed from plasma within 60 min. In addition, DB is apparently rapidly metabolized, producing uncertain levels of plasma DB metabolites. Cautious interpretation of the results presented in Figure 3 is therefore necessary, since the cross-reactivity of the CIEIA with DB metabolites has not yet been characterized. The presence of cross-reacting DB metabolites in these plasma samples would influence the apparent level of DB measured, and therefore the shape of the plasma DB concentration vs. time curve.
ACKNOWLEDGMENTS The authors thank Professor Kenneth Rinehart of the University of Illinois for his generous gift of didemnin A and didemnin B. The authors also acknowledge the valuable contributions of Dr. Jason Fisherman of the National Cancer Institute, Bethesda, MD, and Dr. Martin Raber, Department of Medical Oncology at MD Anderson Cancer Center, for making this collaboration possible. This publication was supported by grant IR43 CA47034-
Raybould et al.
OlAl from the National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
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