The humoral immune response to macrocyclic chelating agent DOTA depends on the carrier molecule.

UNLABELLED: The chelating agent 1,4,7,10-tetraazacyclododecane-N,N', N",N"'-tetraacetic acid (DOTA) is used to label monoclonal antibodies (mAbs) and peptides with (90)Y. DOTA allows the generation of clinically useful stable metallic radioconjugates for the treatment of a variety of tumors, but its immunogenicity has remained controversial. In this study, we evaluated the immune response to DOTA in a preclinical mouse model and in patients entered in a clinical trial. METHODS: Sera were obtained from BALB/c mice injected intraperitoneally or subcutaneously with different doses and formulations of syngeneic and xenogeneic mAbs or peptide (murine mAb Mov19 [mM19]; its chimeric version; murine V/human C ChiMov19 [cM19]; or Tyr(3)-octreotide)-DOTA conjugates. Sera from patients with neuroendocrine tumors, enrolled in a protocol for somatostatin receptor-mediated radionuclide therapy with (90)Y-DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC), were also collected before and after each treatment. Levels and specificity of antibody response to relevant (Mov19, ChiMov19, or Tyr(3)-octreotide) and nonrelevant (human serum albumin) DOTA targets were tested by enzyme-linked immunosorbent assay and competition assays. An anti-DOTA mAb (IgG1) derived from a ChiMov19-DOTA immunized mouse was used, in a competitive radioimmunoassay, to determine the efficiency of DOTA presentation on the different carriers. RESULTS: Depending on the immunogenicity and dosage of the mAb, a specific anti-DOTA response was revealed in the preclinical system. However, DOTA-peptide conjugate induced no immune-detectable response against either chelator or carrier. DOTA was poorly presented on small peptides, as determined using the anti-DOTA mAb. CONCLUSION: A humoral response against DOTA is possible, but only as a consequence of the response elicited against the carrier. Octreotide was not immunogenic. Thus, (90)Y-DOTATOC can be considered a safe and useful tool for receptor-mediated radionuclide therapy of somatostatin receptor-overexpressing tumors.

111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-lys(8)-vasotocin: a new powerful radioligand for oxytocin receptor-expressing tumors.

We developed a radioactive ligand for tumors expressing oxytocin receptors (OTRs) by linking the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) to Lys(8)-vasotocin (LVT), an analogue of oxytocin with high affinity for OTRs. The new reagent (DOTA-LVT) retained high affinity for human OTRs, as proved by in vitro affinity binding to cells endogenously expressing OTRs, such as MCF7 breast carcinoma and MOG-U-V-W glioblastoma cells lines, as well as to transiently transfected COS7 cells. In in vivo experiments, DOTA-LVT carrying (111)In showed specific binding activity to OTR-positive TS/A mouse mammary tumors. The present study opens new perspectives for imaging and, possibly, therapy of OTR-positive human tumors such as breast and endometrial carcinomas, neuroblastomas, and glioblastomas.

Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.

Orally administered ranitidine plasma concentrations before and after biliopancreatic diversion in morbidly obese patients.

BACKGROUND: Patients undergoing biliopancreatic diversion (BPD) may develop gastric ulcers, particularly within the first postoperative year. The prophylactic use of antisecretory compounds at the usual therapeutic doses, mainly conventional H2-receptor antagonists such as ranitidine, may reduce the incidence of this complication, which occurs in approximately 5% of patients after BPD. METHODS: The authors measured the plasma concentrations of ranitidine (300 mg orally) in obese patients, before and 8 months after BPD, and in control subjects of normal weight. The study included 11 obese patients undergoing BPD (age 45+/-14 years; preoperative and postoperative weights 124+/-21 and 92+/-11 kg) and 10 normal-weight subjects (age 37+/-13 years, weight 67+/-9 kg). RESULTS: Postoperative ranitidine plasma concentrations showed only minor differences from preoperative levels, with slightly higher maximum concentrations occurring sooner. The mean area under the curve was on the average 30% higher than preoperatively. All parameters, however, were similar to those in control subjects. CONCLUSIONS: BPD per se does not greatly affect the pharmacokinetic behavior of ranitidine, and therefore a conventional dosage regimen appears adequate for the prophylaxis and therapy of gastric ulcers associated with this operation.

Acid-catalysed hydrolysis and benzodiazepine-like properties of 5-(dialkylamino)- and 5-(alkylthio)-substituted 8-chloro-6-phenyl-6H-[1,2,4]triazolol[4,3-a][1,5]benzodiazepines in mice.

The in-vitro and in-vivo hydrolysis of two benzodiazepine compounds has been studied to evaluate their in-vivo activity in mice. Compounds RL 218 and RL 236, selected as representative examples of N,N-dialkyl-8-chloro-6-phenyl-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiaz epin-5-amines (1) and of their 5-(alkylthio) substituted analogues (2), were rapidly hydrolysed to the corresponding 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5(6H )-one 3 (RL 214) in aqueous acidic solution at pH 1.5. This reaction also occurred extensively in mice when compounds RL 218 and RL 236 were given orally but not intraperitoneally. Both compounds were active against pentylenetetrazole-induced lethal convulsions in mice only when administered orally. After administration of pharmacologically effective oral doses (ED50, the dose protecting 50% of mice), at the time of assessment of the anti-pentylenetetrazole activity, mean brain concentrations of RL 218 and RL 236 were below the limits of sensitivity of the analytical procedure whereas brain concentrations of their metabolite RL 214 were comparable with that present after an oral equiactive dose of this compound itself. RL 214 but not RL 218 or RL 236 had in-vitro affinity for brain benzodiazepine receptors. These results indicate that the anticonvulsant activity of RL 218 and RL 236 in mice depends essentially on their in-vivo transformation into the common active metabolite RL 214 which most probably arises as a result of acid catalysed hydrolysis in the gastric juice.

Brain-to-blood partition and in vivo inhibition of 5-hydroxytryptamine reuptake and quipazine-mediated behaviour of nefazodone and its main active metabolites in rodents.

The brain/plasma partition of nefazodone, hydroxynefazodone (OHNFZ) and m-chlorophenyl-piperazine (mCPP) and their antagonism of p-chloroamphetamine (PCA)-induced 5-hydroxytryptamine (5-HT) depletion and quipazine-induced head twitches were compared in rodents. Nefazodone (30 mg kg(-1), i.p.) rapidly entered the brain but concentrations were exceeded by mCPP, the metabolic ratio being 47 and 10 in the mouse and rat respectively. OHNFZ was detectable in plasma but never in brain. Brain concentrations of OHNFZ in the mouse (30 mg kg(-1), i.p.) were less than 10% of those in plasma, confirming a poor blood-brain barrier penetration. Concentrations of its metabolite mCPP were similar to those after 5 mg kg(-1)(i.p.) mCPP. In the mouse, nefazodone (30 mg kg(-1)) antagonized the 5-HT depleting effect of PCA 2 h after dosing, when it had disappeared from brain but when mCPP concentrations were similar to those after 5 mg kg(-1) (i.p.) mCPP. However, mCPP antagonized PCA less than nefazodone. In the rat, nefazodone pretreatment (30 mg kg(-1), 15 min) prevented (97% of inhibition) quipazine-induced head twitches. The effect was weaker (65% of inhibition) but significant when only mCPP was detected in brain. Analysis of brain concentrations of the two compounds after their ED50 against quipazine indicated that both contributed to the effect, although nefazodone was more active than mCPP in terms of concentrations required to obtain a comparable reduction of twitches. These findings show that mCPP concentrates in the brain following injection of nefazodone and may play a role in preventing quipazine-induced behaviour and PCA-induced 5-HT depletion. In contrast OHNFZ poorly enters the brain and its in vivo activity is mostly due to its biotransformation to mCPP.