Assessment of early therapeutic response to sorafenib in renal cell carcinoma xenografts by dynamic contrast-enhanced and diffusion-weighted MR imaging.

OBJECTIVE: To investigate the feasibility of dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DWI) in monitoring early therapeutic response to sorafenib in renal cell carcinoma (RCC) xenograft models. METHODS: Sorafenib (40 mg kg(-1)) was administered orally to BALB/c nude mice (n = 9) bearing subcutaneous tumours of human RCC ACHN xenografts. DCE-MRI and DWI were obtained 0, 1, 3 and 7 days after therapy, and DCE-MRI parameters (K(trans) and ve) and apparent diffusion coefficient (ADC) values were calculated. Tumour size and volume changes were correlated with changes in DCE-MRI parameters or ADC values after therapy. RESULTS: Following therapy, K(trans) showed a significant decrease over time (p = 0.005), whereas ve did not demonstrate significant changes between time points (p = 0.97). ADC values showed a progressive increase over time (p = 0.004). Compared with pre-therapy, K(trans) showed a significant decrease after 3 days of therapy (p = 0.039), and ADC values increased significantly after 7 days (p = 0.039). Tumour size and volume did not show significant changes during 7 days. Tumour size and volume changes were not associated with changes in DCE-MRI parameters or ADC values. CONCLUSION: DCE-MRI and DWI may show early physiological changes within 1 week after initiating sorafenib treatment on human RCC xenografts. ADVANCES IN KNOWLEDGE: The quantitative parameters of DCE-MRI and DWI may offer the potential for assessing early therapeutic response to sorafenib in clinical trials.

Hepatic veno-occlusive disease in children after hematopoietic stem cell transplantation: incidence, risk factors, and outcome.

Four hundred and sixty-seven hematopoietic stem cell transplantations (HSCTs) (217 autologous and 250 allogeneic HSCT) were performed in 374 children at four pediatric HSCT centers in Korea from January 2005 to December 2007. Among 467 transplants, veno-occlusive disease (VOD) developed in 72 transplants (15.4%) at a median of 10 days after HSCT. Multivariate analysis showed that BU or TBI-containing regimen (P=0.002), VOD prophylaxis without lipo-prostaglandin E1 (PGE1) (P=0.012), number of previous HSCT (P=0.014), and pretransplant serum ferritin (P=0.018) were independent risk factors for developing VOD. Mean serum ferritin levels were significantly higher in HSCT with VOD (2109.6+/-2842.5 ng/ml) than in HSCT without VOD (1315.9+/-1094.4 ng/ml) (P<0.001). The relative risk of death within 100 days of HSCT in transplants with VOD compared with transplants without VOD was 3.39 (confidence interval: 1.78-6.45). Our results suggest that lipo-PGE1 might have a protective effect against the development of VOD, and pretransplant serum ferritin could act as a risk factor for VOD. A larger prospective study is needed to confirm a possible role of lipo-PGE1 and iron chelation therapy in reducing the incidence of VOD.

The heat-shock protein 90 inhibitor, geldanamycin, induces apoptotic cell death in Epstein-Barr virus-positive NK/T-cell lymphoma by Akt down-regulation.

NK/T-cell lymphoma (NKTL) is strongly associated with latent Epstein-Barr virus (EBV) infection. Recently, latent membrane protein 1 (LMP1), an EBV oncoprotein, was reported to activate the phosphatidylinositol-3 kinase (PI3K)/Akt pathway for cell survival. Because geldanamycin (GA) and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG), exhibit anti-tumour activity by degrading HSP90 client proteins, including Akt, we investigated the effect of GA and 17-AAG on the survival of NKTL cell lines. EBV-positive NKTL cell lines, Hank-1 and NK-YS, and an EBV-negative NK leukaemia cell line, NK-L, were treated with PI3K and Akt inhibitors, GA, and 17-AAG, and were subjected to apoptosis and cell viability assays, and immunoblot analysis. EBV-positive B-lymphoblastoid cell lines IM9 and LMP1-transfected IM9 (IM9-LMP1) were also included. Hank-1 and NK-YS cell viability was compromised and apoptosis was induced by LY294002 (PI3K inhibitor) or Akt inhibitor II. GA or 17-AAG administration resulted in the apoptosis of NKTL cells, accompanied by Akt and pAkt down-regulation, caspase 3 activation, and mitochondrial membrane potential disruption. The intrinsic level of pAkt was higher in EBV-positive NKTL cells than in EBV-negative NK-L, and GA or 17-AAG decreased the viability of NKTL cells more efficiently than NK-L. Moreover, IM9-LMP1 was more sensitive to Akt inhibitor II or HSP90 inhibitors than IM9. Importantly, GA showed little effect on the viability of normal peripheral NK cells as non-neoplastic counterparts for comparison. In conclusion, this study suggests that the PI3K/Akt pathway is frequently activated in EBV-positive NKTL and that therapeutic modalities based on targeting the PI3K/Akt pathway with HSP90 inhibitors could be useful for achieving NKTL control.

Effect of dietary supplementation with conjugated linoleic acid, with oleic, linoleic, or linolenic acid, on egg quality characteristics and fat accumulation in the egg yolk.

The effects of conjugated linoleic acid (CLA) with other fatty acids on the fatty acid composition of egg yolk and on egg quality characteristics were studied in 5 groups: 1) CLA 0% (control), 2) CLA 2%, 3) CLA 2%+oleic acid (OA) 2% (CLA+OA), 4) CLA 2%+linoleic acid (LA) 2% (CLA+LA), and 5) CLA 2%+alpha-linolenic acid (LNA) 2% (CLA+LNA). Some parameters of egg quality such as shell thickness, shell strength, yolk color, yolk index, egg diameter, and Haugh units were aggravated when CLA was fed alone, but the quality was improved when CLA was combined with some other fatty acids. The egg production rate, which was decreased by feeding CLA alone, was improved by co-supplementation with LA or OA. An increase in CLA content was observed in all the dietary groups fed CLA for 2 wk. Feeding hens with CLA+LNA led to a linear increase in CLA content in the egg yolk after the fourth week of the feeding trial. Egg yolks from hens given CLA had considerably higher amounts of saturated fatty acids and lower amounts of monounsaturated fatty acids than egg yolks from the control group. The pattern of change in CLA concentration during the feeding trial was similar to the level of C18:0, which was inversely correlated with the level of C18:1. The unsaturated fatty acid co-supplementation strategy applied in this study offers insight into the mechanism of CLA accumulation in the egg yolk without apparent adverse effects on egg quality and egg production.

Inhibition of TPA-induced cyclooxygenase-2 expression and skin inflammation in mice by wogonin, a plant flavone from Scutellaria radix.

Wogonin (5,7-dihydroxy-8-methoxyflavone), isolated from Scutellaria radix, was previously reported to inhibit the expression and activity of the enzyme cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated cells of a mouse macrophage cell line, RAW 264.7. Here, in order to find in vivo effects, inhibition by wogonin of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 expression and anti-inflammatory activity in vivo were investigated. When applied topically to the dorsal skin of mice, wogonin at doses of 50-200 microg/site/treatment (total of five treatments in 3 days) inhibited cyclooxygenase-2 expression and prostaglandin E2 production induced by multiple treatments with TPA. At 200 microg/site/treatment, wogonin caused a 55.3% reduction of prostaglandin E2 production on the dorsal skin compared with an increased production in the TPA-treated control group. The same compound significantly inhibited mouse ear edema induced by TPA in both preventive (58.1% inhibition) as well as curative treatment (31.3% inhibition) schedules at 200 microg/ear/treatment. Inhibition of neutrophil infiltration was also observed. Therefore, wogonin may be beneficial for cyclooxygenase-2-related skin disorders.

Immunostimulatory effects of anionic alkali mineral complex solution Barodon in porcine lymphocytes.

The anionic alkali mineral complex solution, Barodon (Barodon-S.F. Corp., Korea), was evaluated for its effectiveness as a nonspecific immunostimulator in pigs. The effects of Barodon were determined by analysis of feed efficiency, growth rate, and phenotype of leukocyte subpopulations using monoclonal antibodies specific to porcine leukocyte differentiation antigens and flow cytometry (FC). The study was focused to investigate the change in proportion of the CD4+CD8+ double positive T lymphocyte subpopulation (dpp) which exists uniquely in pigs. In addition, the mitogen-stimulated lymphoproliferative response, tissue distribution in lymphoid organs and the adjuvant effect of Barodon on hog cholera vaccine efficiency were determined. The study has revealed the average daily gain rates and feed conversion rates were significantly (p<0.05) improved in either group of pigs fed with 0.05% Barodon-spray feed (Tx-1) or pigs fed with 3% Barodon-fermented feed (Tx-2) in comparison with group of pigs fed with feed containing no Barodon (control). The proportion of cells expressing CD4+ antigen in Barodon-treated group increased from 3 weeks posttreatment and was significantly higher (p<0.05) than that of control at 8 weeks posttreatment. Particularly, the significantly higher proportion was maintained from 8 weeks through 13 weeks posttreatment in Tx-1 group (p<0.05). The proportion of cells expressing CD8+ antigen was significantly higher at 3 weeks posttreatment in Tx-2 (p<0.01). Proportion of MHC class II-expressing cells was significantly higher in Tx-1 and Tx-2 group at 11 weeks and 8 weeks posttreatment (p<0.05), respectively. In addition, the proportion of Non T/Non B (N) cells was also significantly higher in Tx-2 at 3 weeks posttreatment (p<0.01) and maintained to 13 weeks posttreatment (p<0.1). Between Barodon-treated groups, the proportion of MHC class II-expressing cells was observed to be larger in Tx-2 than Tx-1 from 3 weeks to 8 weeks posttreatment (p<0.05). However, there were no significant difference in the proportions of CD2+ cells, B cells, monocytes and granulocytes between Barodon-treated and control group during the experiment. Dual-color FC analysis, study has revealed an increased proportion of dpp present in lymphocytes obtained from peripheral blood (PB) and mesenteric lymph node (MLN) of Barodon-treated group at 8 and 11 weeks posttreatment. The proportion of dpp in PB was 27.5% and 32.1% in Tx-1 and Tx-2, respectively, but only 2.2% in control group at 8 weeks posttreatment. In MLN, the proportion was 45.1% and 52.1% in Tx-1 and Tx-2, respectively, otherwise 16.5% in control group at 8 weeks posttreatment. The mitogen-stimulated activity was significantly higher in Tx-1 than in the control group at 11 weeks posttreatment when cells were stimulated with Con A and PHA, respectively (p<0.01). Also, Con A-, PHA and PWM-stimulated activity was significantly higher in Tx-2 than in the control group at the same time (p<0.05). The tissue distribution of CD4+, CD8+ and CD4+CD8+ dpp in MLN and spleen was significantly larger in Tx-1 and Tx-2 than in the control group (p<0.01). Also, a larger proportion of dpp was observed in Tx-2 than Tx-1 in spleen between Barodon-treated groups (p<0.01). In conclusion, the study has demonstrated that Barodon had an immunostimulatory effect on pigs through proliferation and activation of porcine immune cells, specially CD4+CD8+ dpp lymphocytes.

Isolation of peptide ligands that inhibit glutamate racemase activity from a random phage display library.

Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing antibiotic substances. The new agents include compounds that interfere with bacterial membrane function. The peptidoglycan component of the bacterial cell wall is synthesized by glutamate racemase, and this enzyme is responsible for the biosynthesis of d-glutamate, which is an essential component of cell wall peptidoglycan. In this study, we screened a phage display library expressing random dodecapeptides on the surface of bacteriophage against an Escherichia coli glutamate racemase, and isolated specific peptide sequences that bind to the enzyme. Twenty-seven positive phage clones were analyzed, and seven different peptide sequences were obtained. Among them, the peptide sequence His-Pro-Trp-His-Lys-Lys-His-Pro-Asp-Arg-Lys-Thr was found most frequently, suggesting that this peptide might have the highest affinity to glutamate racemase. The positive phage clones and HPWHKKHPDRKT synthetic peptide were able to inhibit glutamate racemase activity in vitro, implying that our peptide inhibitors may be utilized for the molecular design of new potential antibacterial agents targeting cell wall synthesis.

Relation between lymphocyte subpopulations of peripheral blood and immune responses of modified live hog cholera virus vaccine in pigs treated with an ionized alkali mineral complex.

Thirty-nine healthy pigs (28-32 days old) were purchased from a commercial swine farm and housed at swine pens of the College. The animals were vaccinated intramuscularly (1 ml) with an attenuated live hog cholera virus (HCV, LOM strain) and then boostered at 5 weeks after the first vaccination. The animals were divided into 4 experimental groups: 0.05% (w/w) PowerFeel-supplemented diet (T-1, n = 10); 3% (w/w) SuperFeed-supplemented diet (T-2, n = 10); diluted PowerFeel solution (1 : 500, v/v) as drinking water (T-3, n=9); control (n=10). PowerFeel is an original form of ionized alkali mineral complex (IAMC) and SuperFeed is a commercial product of IAMC. The subpopulation of lymphocyte in blood was assayed by a flow cytometry and HCV-specific antibody was determined by an indirect immunofluorescence assay. In IMAC-treated groups, the proportions of subpopulation expressing MHC-class II, CD2+, CD4+, CD8+, and surface IgM+ B lymphocytes were significantly decreased at 5-weeks after the first vaccination. Significant decreases were also observed in the proportions of MHC-class II, CD2+ and CD8+ lymphocyte at 3-weeks after the booster injection. The humoral immune responses in T-1 and T-2 groups were greater than those in T-3 or control group. These results suggest that IAMC-supplemented diets may have an HCV-specific immunostimulatory effect in pigs.

Mechanism-based design of parasite-targeted artemisinin derivatives: synthesis and antimalarial activity of benzylamino and alkylamino ether analogues of artemisinin.

Several artemisinin derivatives linked to benzylamino and alkylamino groups were synthesized in order to enhance accumulation within the malaria parasite. The in vitro antimalarial activity was assessed against the chloroquine sensitive HB3 strain and the chloroquine resistant K1 strain of Plasmodium falciparum. In general the incorporation of amino functionality enhances the activity relative to artemisinin. The most potent analogue in the series was compound 6 which was severalfold more active than artemisinin against both strains of P. falciparum used in the study.

Efficacy of fumagillin against Thelohanellus kitauei infection of Israel carp, Cyprinus carpio nudus.

The potential of fumagillin dicyclohexylamine salt to treat and prevent intestinal giant-cystic disease in Israel carp, Cyprinus carpio nudus, was monitored in field experimental studies. In experiment 1 (therapeutic), most fish were already naturally infected with more advanced stage of Thelohanellus kitauei. Fumagillin was administered to fish (mean body weight of 830 g) for a period of one month at a dose of 10.62 mg in the first group and 5.3 mg in the second group per fish per day. In experiment 2 (prophylactic), most fish also were already naturally infected with an early developmental stage of the protozoa and fish (average body weight of 484 g) were administered fumagillin for 45 days at a dose of 3.95 mg per fish per day. In both experiments, the cumulative mortalities of fish and the extrusion rates of the polar filaments of the spores were significantly decreased in a dose-independent fashion. In experiment 2 no dead fish were observed. No adverse side effects of the drug were observed among fish from any dosage group. In experiment 2, an oval or dot-like concave lesion of most cysts developed at the 7th day and the vegetative form was never observed at the 17th day postmedication and the cysts were grossly reduced in size as compared with the control group, beginning at the 24th day until the end of the study. In contrast, it was scarcely effective to the cysts in experiment 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-protein conjugates--XVII. The effect of storage on the antigenicity and immunogenicity of benzylpenicillin in the rat.

The disposition and immunogenicity of freshly prepared and stored solutions of benzylpenicillin (BP) and benzylpenicillenic acid (BPE), a degradation product of BP, were studied. No IgG anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA) following daily i.p. or i.m. administration to male Wistar rats of BP (2.7 mmol/kg) freshly dissolved in 0.5% glucose, for 4 consecutive days at 4-week intervals. In contrast, IgG anti-BPO antibodies were detected following both chronic i.p. and i.m. administration of BP (2.7 mmol/kg) stored for 24 hr at room temperature in 0.5% glucose. An IgG anti-BPO response was obtained only after the high dose, following daily i.m. administration of BPE (27 mumol/kg, 2.7 mumol/kg, 0.24 mumol/kg). The specificity of the IgG antibody for the BPO-determinant was confirmed by ELISA inhibition with BPO-amino-caproate. Circulating BPO plasma-protein antigens were detected by a modified ELISA following i.p. and i.m. administration of both stored and fresh BP. Significantly lower BPO-antigen levels were detected in serum following BPE administration. Irreversible binding of BP to 75% rat plasma proteins was of the same magnitude when freshly dissolved in phosphate buffer or in 0.5% glucose (2.63 +/- 0.32% and 2.55 +/- 0.25% bound, respectively after 3 hr incubation at 37 degrees). Irreversible binding was significantly greater (P less than 0.05) when the BP was stored prior to incubation with the protein (3.81 +/- 0.27%). The major degradation product of stored BP was benzylpenicilloic acid; a small amount of BPE (0.2% of incubated BP) was detected in stored but not fresh BP. Thus, the increased immunogenicity of BP stored for 24 hr at room temperature may be due to the formation of reactive degradation products such as BPE in vitro, which can then form immunogenic drug-protein conjugates in vivo. These experiments also show that although BP and BPE form drug-protein conjugates in vivo, circulating levels of antigen do not relate to the immunogenicity of either of the compounds.

An investigation of the pharmacological response to vitamin K1 in the rabbit.

1. The relationship between pharmacological response and disposition of a dose of vitamin K1 (10 mgkg-1, i.v.) in normal rabbits and in rabbits treated with the coumarin anticoagulant brodifacoum, has been studied. 2. High performance liquid chromatography (h.p.l.c.) with electrochemical detection (EC) was used to determine concentrations of vitamin K1 in plasma, whole liver homogenate, and liver microsomes. 3. After intravenous administration of vitamin K1, plasma concentrations of the vitamin declined in a tri-exponential fashion. There were no differences between the two groups over the first 24 h of the experiment. However, between 24 h and the end of the study, plasma concentrations of vitamin K1 in the presence of brodifacoum were significantly (P less than or equal to 0.05) below those of vehicle-treated rabbits. 4. Seventy-two hours after administration of vitamin K1, plasma concentrations of the vitamin were not different from normal. 5. Three hours after administration of vitamin K1, the concentrations of the vitamin in whole liver were 46.6 +/- 4.3 micrograms g-1 in the presence of brodifacoum, and 32.8 +/- 6.4 micrograms g-1 in the absence of brodifacoum; and were significantly (P less than or equal to 0.05) greater than normal (127.7 +/- 44.3 ng g-1). Likewise, microsomal concentrations of vitamin K1 (4.00 +/- 2.38 micrograms mg-1 protein, and 2.65 +/- 1.01 micrograms mg-1 protein, in the presence and absence of brodifacoum, respectively) were significantly (P less than or equal to 0.01) greater than normal (16.0 +/- 3.5 ng mg-1 protein). 6 In conclusion, there appears to be no direct effect of coumarins on clearance of vitamin K1 from either plasma or liver; the need for large doses of vitamin K1 during coumarin poisoning is due to a greatly increased requirement for the vitamin.

Drug-protein conjugates--XV. A study of the disposition of D-penicillamine in the rat and its relationship to immunogenicity.

The disposition of [14C]D-penicillamine (PA) was investigated in vitro and in vivo with male Wistar rats. Irreversible binding of [14C]PA to isolated rat plasma proteins in vitro reached a maximum of 20.6% of total radioactivity at 6 hr. Irreversibly bound [14C]PA could be dissociated with dithiothreitol, demonstrating that conjugation was via disulphide linkage. Three hours after i.v. administration of [14C]PA (27 mumol/kg) to rats 100% of plasma radioactivity was irreversibly bound, representing approximately 3.5% of the dose. Further studies on the disposition of PA-plasma protein conjugates showed that dissociation occurred readily in vivo: the plasma half-life of the conjugate was approximately 3 hr. Free [14C]PA was the major urinary metabolite after administration of both free and conjugated drug. These studies show that the disposition of PA is similar to that reported for the structurally related sulphydryl drug captopril (CP). Free PA (340 mumol/kg and 3.4 mmol/kg) administered i.p. and i.m. daily for 4 days at one monthly intervals, and also PA-KLH conjugate (100 micrograms/rat) administered by single i.p. injection at monthly intervals with and without Freund's complete adjuvant, failed to induce PA-specific IgG or IgM antibody responses detectable by ELISA. In contrast, CP (270 mumol/kg), administered by the same protocol as free PA, induced a CP-specific IgG antibody response after the third series of monthly injections. These data suggest that the difference in immunogenicity between PA and CP arises from a difference in the intrinsic immunogenicity of the haptens, rather than from their disposition.

Drug-protein conjugates--XII. A study of the disposition, irreversible binding and immunogenicity of penicillin in the rat.

The disposition, irreversible binding and immunogenicity of benzylpenicillin (BP) were studied in male Wistar rats. [3H]BP, administered i.v. to anaesthetized rats at two doses (27 mumol/kg, 2.7 mmol/kg), showed dose-dependent kinetics: plasma and tissue concentrations of total BP were disproportionately increased at the higher dose. BP was rapidly cleared from the plasma at both doses (less than 0.05% of administered dose/ml plasma after 3 hr). In spite of the disproportionately elevated levels of total BP after the higher dose, covalent binding to plasma proteins was quantitatively similar as a percentage of the dose at both doses. Three hours after i.v. injection of 27 mumol/kg and 2.7 mmol/kg of the drug, 5.6% +/- 1.7% and 3.3% +/- 1.1% respectively of circulating BP was covalently bound, representing less than 0.004% of the administered dose bound per ml of plasma in each case. Covalent binding of BP to rat plasma proteins in vitro was of a similar magnitude to that observed in vivo: 1.6% +/- 0.4% of BP was bound to 25% rat plasma after 3 hr incubation at 37 degrees. In a separate series of experiments the immunogenicity of BP was studied by chronic administration of the drug to rats. Following daily i.v. or i.m. administration of BP (27 mumol/kg, 270 mumol/kg, 2.7 mmol/kg) for 4 consecutive days at 4-week intervals (three series of injections) neither IgG nor IgM anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of the high dose of BP was discontinued after the first series of injections due to local necrosis. In contrast to free BP, BPO-keyhole limpet haemocyanin (BPO-KLH, 42 nmol BP bound/mg KLH) administered by single i.v. injection at 4-week intervals at two doses (20 and 200 micrograms conjugate/kg, corresponding to 0.84 and 8.4 nmol BPO/kg) readily induced IgG and IgM anti-BPO antibody responses (median IgG titres were 872 and 5470 one week after the third injection of the low and high dose of conjugate respectively; corresponding IgM titres were 4513 and 22,866). The specificity of the IgG and IgM antibodies for the BPO determinant was confirmed by ELISA inhibition with BPO-aminocaproate. These experiments show that BP binds irreversibly, but to a limited extent, to plasma proteins in vivo, and that such a degree of conjugation appears to be insufficient to elicit a detectable anti-BPO antibody response.

A specific enzyme-linked immunosorbent assay for definition of the IgG antibody response to disulphide-conjugated D-penicillamine in the rabbit.

An enzyme-linked immunosorbent assay (ELISA) has been developed for unambiguous detection of antibodies against the sulphydryl drug D-penicillamine (PA) and its disulphide-conjugated metabolites. Disulphide-linked PA human serum albumin (PA-HSA) conjugates for use as coating antigens were prepared by a range of procedures employing oxidation with either potassium ferricyanide (0.1 M) or cupric sulphate (5 ppm). A satisfactory degree of conjugation was achieved by both oxidative procedures. Hapten density and antigenicity were increased when urea-denatured rather than non-denatured HSA was used. In 2 out of 3 rabbits, a specific IgG anti-PA response was detected following monthly injection of PA keyhole limpet haemocyanin (PA-KLH) in Freund's complete adjuvant. In the third rabbit, any anti-PA activity was obscured by a high level of binding to HSA. The anti-PA response was slow to develop in the 2 responder rabbits (requiring four injections) and was of low intensity (antibody titres less than 6,000). In contrast, the IgG antibody response to the structurally related drug captopril (CP), administered under identical conditions, was rapid in onset and of greater intensity (titres greater than 6,000 after one injection of CP-KLH). The hapten specificity of the IgG anti-PA-HSA antisera was defined by ELISA inhibition assays. Binding of IgG to PA-HSA was inhibited by PA, PA disulphide, PA cysteine and disulphide-linked PA-HSA conjugates, but not by PA acetone (thiazolidine ring-linked PA), CP, or unconjugated HSA. The inhibitory preparations were inactive in unrelated ELISAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-protein conjugates--IX. Immunogenicity of captopril-protein conjugates.

The immunogenicity of captopril (CP), conjugated to heterologous proteins, was investigated in male New Zealand White rabbits by monthly injections of CP-protein conjugates in Freund's Complete Adjuvant. Anti-CP antibody activity was readily detected by immunodiffusion in sera of rabbits immunized with the amide-linked CP-HSA (23:1) conjugate. Hapten inhibition studies revealed that the antigenic determinant contained CP and a lysine residue from the protein carrier. When rabbits were immunized with disulphide-linked CP-S-S-HSA (9:1) and CP-S-S-KLH (160:1) conjugates, anti-CP antibody activity was detected by a sensitive ELISA method, but not by immunodiffusion and radioligand binding assays. The specificity of the serum IgG anti-CP activity after immunization with disulphide-linked CP-S-S-protein conjugates was confirmed since anti-CP activity was inhibited by preincubation of the antisera with CP conjugated to an unrelated protein carrier (CP-S-S-OVA), but not by the corresponding unconjugated protein, nor by penicillamine-S-S-protein conjugates. These results show that disulphide-linked CP-protein conjugates are sufficiently stable to induce humoral (B lymphocyte) anti-hapten responses under the experimental conditions employed. In a separate study, delayed-type skin hypersensitivity reactions to topically applied CP were demonstrated in the guinea pig. The specific and sensitive immunochemical technique (ELISA) described here could be useful in future studies for determining whether or not patients taking CP produce antibodies to the drug.