Evaluation of Pentravan®, Pentravan® Plus, Phytobase®, Lipovan® and Pluronic Lecithin Organogel for the transdermal administration of antiemetic drugs to treat chemotherapy-induced nausea and vomiting at the hospital.

The objective of this study was to evaluate five commercial ready-to-use transdermal vehicles (Phytobase®, Lipovan®, Pentravan®, Pentravan® Plus and Pluronic Lecithin Organogel (PLO)), for the compounding of three antiemetic drugs (ondansetron, dexamethasone and aprepitant) and their administration in combination to treat chemotherapy-induced nausea and vomiting (CINV) at the hospital. Drugs were individually formulated in these vehicles and in mixture in Pentravan® Plus using different penetration enhancers. Quality control of the forms has demonstrated that formulation process was mastered and convenient for the hospital (time required: 20min). Diffusion experiments through synthetic membranes and pig ear epidermis performed using Franz-type diffusion cells, have shown that the release and permeation process were greater for ondansetron than for dexamethasone and aprepitant, with a release step not limiting. As permeation of aprepitant was too low, it was discarded of the study. When ondansetron and dexamethasone were compounded in combination in Pentravan® Plus, the most efficient vehicle, a permeation decrease was observed. Finally, the use of tween 20 instead of EtOH as chemical enhancer has led to 2-fold factor increase in the flux of dexamethasone, resulting in fluxes convenient for transdermal administration of ondansetron to a child, but insufficient for an adult and for dexamethasone.

Complementarity of UV-PLS and HPLC for the simultaneous evaluation of antiemetic drugs.

This work was dedicated to the development of a simple and direct multivariate UV spectrophotometric method for the simultaneous determination of three antiemetic drugs (ondansetron, dexamethasone and aprepitant) in a new organogel formulation developed for their simultaneous transdermal administration. This method that does not require separation of the drugs and sophisticated instrument will permit to control quality of this new transdermal form both during the optimization step and for a further routine control of this preparation at the pharmacy department of the hospital. Hence, a partial least squares regression model using the spectral data record from 260 to 288 nm and 5 components, has firstly been validated thanks to the evaluation of the REP% (under 7.9%) and secondly using an accuracy profile approach (acceptance limit of ±10%). Thereby, the method allows the quantitation of the drugs in the ranges (5-15 mg L(-1)), (4-8 mg L(-1)) and (20-50 mg L(-1)) for ondansetron, dexamethasone and aprepitant, respectively. An HPLC/UV reference method has also been developed. Optimal separation (2.52

Factors predictive of ten-year mortality in severe anorexia nervosa patients.

OBJECTIVE: Little is known concerning mortality and predictive factors for anorexia nervosa in-patients. This study aimed to establish mortality rates and identify predictors in a large sample of adults through a 10-year post in-patient treatment follow-up. METHOD: Vital status was established for 601 anorexia nervosa (DSM-IV) consecutive in-patients with initial evaluation at admission. Standardized mortality ratio (SMR) was calculated. Cox analyses for hypothesized predictors of mortality were performed. RESULTS: Forty deaths were recorded. SMR was 10.6 [CI 95% (7.6-14.4)]. Six factors at admission were associated with death: older age, longer eating disorder duration, history of suicide attempt, diuretic use, intensity of eating disorder symptoms, and desired body mass index at admission. CONCLUSION: Anorexia nervosa in-patients are at high risk of death. This risk can be predicted by both chronicity and seriousness of illness at hospitalization. These elements should be considered as warnings to adapt care provision and could be targeted by treatment.

Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis.

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R(s)>3.3 with analysis times<25min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated beta-cyclodextrins at pH 2.5 (R(s)>1.5 with analysis times <11min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts.

Separations of the diastereoisomers of three nucleoside 5'-phosphotriester derivatives of Ara-C (tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation (Rs>1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5'-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis.

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.

Switching to the bingeing/purging subtype of anorexia nervosa is frequently associated with suicidal attempts.

OBJECTIVE: Anorexia nervosa has the highest suicide mortality ratio of psychiatric disorders, suicide being associated with many factors. We assessed the first lifetime occurrence of these factors taking into account their possible overlap. METHOD: Three hundred and four in- and out-patients with anorexia nervosa (DSM-IV) were systematically recruited in three hospitals of Paris suburbs, between December 1999 and January 2003. Patients were assessed by a face-to-face interview (DIGS). Current eating disorder dimensions were measured, and patients interviewed by a trained clinician to assess minimal BMI and, retrospectively, the age at which anorexia nervosa, major depressive disorder, anxiety disorders and switch to bingeing/purging type occurred for the first time, if applicable. RESULTS: Major depressive disorder (p<0.001) and subtype switch from the restrictive to the bingeing/purging type (p<0.001) were the two factors significantly more frequently occurring before suicidal attempts, and remained involved when a multivariate analysis is performed, whether syndromic or dimensional measures are being used. Taking into account lifetime occurrence with a survival analysis, the switch to bingeing/purging type of anorexia appears as a major predictive factor, with a large increase of the frequency of suicidal attempts (OR=15) when compared to patients with neither major depressive disorder nor bingeing/purging type. CONCLUSIONS: Bingeing/purging type of anorexia nervosa is largely associated with suicidal attempts, and may deserve specific attention. If confirmed on a prospectively designed study, these results would argue for early detection and/or more intensive and specific therapeutic intervention on this aspect of bingeing and purging behaviors.

Determination of pKa values of benzoxa-, benzothia- and benzoselena-zolinone derivatives by capillary electrophoresis. Comparison with potentiometric titration and spectrometric data.

Acidity constants of benzoxa-, benzothia- and benzoselena-zolinone derivatives were determined by capillary electrophoresis, potentiometry and spectrophotometry experiments. These three analytical techniques gave pK(a) results that were in good agreement. A convenient, accurate and precise method for the determination of pK(a) was developed to measure changes in acidity constants induced by heteroatom or 6-benzoyl substituted derivatives. pK(a) values were determined simultaneously for two compounds characterized by different electrophoretic mobility (micro(e)) and pK(a) value and in the presence of an analogous neutral marker.

Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.

A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.

Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs. Application to cellular extract analysis.

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12-0.20 and 0.40-0.67 microm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract.

Determination of ionization constants of N-imidazole derivatives, aromatase inhibitors, using capillary electrophoresis and influence of substituents on pKa shifts.

Capillary electrophoresis (CE) was used as a method to determine the acidity constants of eight aromatase inhibitors. This method was validated by comparison of results obtained with a traditional method, UV spectroscopy, and additionally with computational calculations. We confirmed here, with our series of compounds, that capillary electrophoresis is an attractive method for pKa measurements which is based on migration time or mobilities of the ionic species over a range of pH values. The precision of pKa measurements of N-imidazole derivatives is useful to observe pKa shifts induced by chemical modifications introduced on adjacent aromatic rings such as heterocycle (benzoxa- or benzothiazolinone) or substituted benzyle. The knowledge of these pKa values is a great interest to predict migration of solutes and qualitative interactions with ionized cyclodextrines as chiral selectors in further enantioseparative CE studies.

[Hyperactivity and anorexia nervosa: behavioural and biological perspective]. / Aspects comportementaux et biologiques de l'hyperactivité dans l'anorexie mentale.

Anorexia nervosa is an eating disorder defined by a symptomatic triad, anorexia, emaciation and amenorrhoea. This disease mainly affects young women. Besides these three symptoms, hyperactivity is often associated with anorexia nervosa. Hyperactivity can be considered as a strategy to lose weight, but studies on animal models have shown that it could be explained by more complicated mechanisms. Hyperactivity is defined by an excess of physical activity, which can induce social, professional and family consequences. Hyperactivity can take different forms, most striking is the restless one. Patients with anorexia nervosa are not all hyperactive. Brewerton et al. have compared patients with anorexia nervosa and hyperactivity to patients without hyperactivity. Hyperactive patients are more dissatisfied by their body image, they use less means of purging (laxatives, vomiting), and they start starving earlier than patients without hyperactivity. Many factors can promote the emergence and maintenance of hyperactivity, especially social and cultural requirements, sports environment, family influences. Various models can explain the links between excessive exercise and anorexia nervosa. Epling and Pierce have exposed a behavioural model which shows how hyperactivity can lead to starvation, creating a self-maintained cycle. Eisler and Le Grande have described four models to explain the links between hyperactivity and anorexia nervosa. First, excessive exercise can be considered as a symptom of anorexia nervosa. It can also promote the development of eating disorders. Anorexia nervosa and hyperactivity can be a manifestation of an other psychiatric disorder. At least, hyperactivity can be a variant of anorexia nervosa, which has the same effects, as weight loss. Hyperactivity can also be considered as a kind of obsessive compulsive disorder. Hyperactivity and obsessive compulsive disorders actually share some clinical and neurochemical characteristics. An other model consists in comparing excessive exercise in anorexia nervosa to an addictive behaviour. Self-starvation exacerbated by hyperactivity can be considered as an addiction to endogenous opioid. Few studies are carried out in order to estimate the prevalence of high level exercise in the eating disorders. Davis et al. have achieved a prevalence study. The results indicate that a large majority of patients with anorexia nervosa (80,8%) were exercising excessively during an acute phase of the disorder. Research on animals, specially on rats, brings us an interesting model explaining interactions between anorexia nervosa and hyperactivity. With animal models, we have noticed that, when rats with access to a running wheel, are restricted in their food intake, they become excessively active, and paradoxically reduce food consumption. Many searchers have tried to explain this phenomenon. Morse et al. have pointed from animal models that the level of hyperactivity was linked to the severity of food restriction. This result can be explained by a failure of a part of the brain involved in rest and activity regulation. Animal research brings us explanations about the effects of starvation on the endocrine system and the neurotransmitters. Broocks et al. have shown that corticosterone concentration in plasma was synergistically increased by semi starvation and exercise, and the reduction of triiodothyronine by semi starvation was significantly greater in the running wheel group. An other study of Broocks et al. has revealed an increased hypothalamic serotonin metabolism with the combined effect of hyperactivity and food restriction. Tryptophan, an amid acid involved in serotonin synthesis, can also play a role in the maintenance of anorexia nervosa. In starvation conditions, opioid releasing caused by physical exercise would decrease food intake. Exner's study and Adan's one have shown that leptin would be involved in semi starvation induced hyperactivity mechanisms. In spite of animal models can not be entirely generalized to human, they are useful to try to explain biological supports of hyperactivity. Hyperactivity is not only a strategy to lose weight, but also a specific symptom which completes the clinical triad. Animal studies have led to promising results; we might use medicine, such as serotonin reuptake inhibitors or opioid antagonists in the treatment of hyperactivity in anorexia nervosa.

[Schizophrenia and eating disorders]. / Schizophrénie et troubles du comportement alimentaire (TCA).

The comorbidity of schizophrenia and eating disorders is understudied. In the early nineteenth century, Eugen Bleuler has reported cases of schizophrenia with eating disorders that were related to delusional ideas. Potomania, merycism and pica have often been described in schizophrenic patients. Schizophrenic patients with eating disorders usually do not meet all criteria for typical eating disorders and are therefore classified as "eating disorders not otherwise specified" (EDNOS). It may even be difficult to recognize schizophrenia in patients with eating disorders associated to delusional ideas and distorted cognitions related to food or body perception. In any case, the diagnosis of schizophrenia should preferably be made and is only valid after renutrition is achieved. The prevalence of schizophrenia in samples of patients with eating disorders is generally below 10% but reaches 35% in males, the most frequent form being hebephrenia. Cognitive behavioural therapies for eating disorders need to be adapted in cases of comorbid schizophrenia. The new antipsychotic medications seem helpful in patients with eating disorders with or without schizophrenia. They reduce anxiety towards eating and bring in better adherence to treatments.

Positively charged templates for labeling internalizing antibodies: comparison of N-succinimidyl 5-iodo-3-pyridinecarboxylate and the D-amino acid peptide KRYRR.

Receptor-mediated internalization of monoclonal antibodies (mAbs), such as those specific for the epidermal growth factor receptor variant III (EGFRvIII), can lead to rapid loss of radioactivity from the target cell. In the current study, the anti-EGFRvIII mAb L8A4 was radioiodinated using two methods -N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) and via a D-amino acid peptide LysArgTyrArgArg (D-KRYRR). Paired-label internalization assays performed on EGFRvIII-expressing U87DeltaEGFR cells in vitro demonstrated that labeling L8A4 using D-KRYRR resulted in significantly higher retention of radioiodine in the intracellular compartment. In athymic mice with D256 human glioma xenografts, tumor uptake was similar for both labeling methods through 24 hr. However, an up to fourfold higher tumor retention was observed for mAb labeled with the D-amino acid peptide at later time points. Radiation absorbed dose calculations based on these biodistribution data indicated that L8A4 labeled using D-KRYRR exhibited better tumor-to-normal-organ radiation dose ratios, suggesting that this labeling method may be of particular value for labeling internalizing mAbs.

Direct separation of the stereoisomers of methoxytetrahydronaphthalene derivatives, new agonist and antagonist ligands for melatonin receptors, by liquid chromatography on cellulose chiral stationary phases.

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct separation of the stereoisomers of disubstituted tetralin derivatives with two chiral centers, new agonist and antagonist ligands for melatonin receptors. The separations were made using normal-phase methodology with a mobile phase consisting of n-hexane-alcohol (methanol, ethanol, 1-propanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes on the discrimination between the stereoisomers were examined. Baseline separation (Rs>1.5) was easily obtained in many cases.

Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody.

The mutant epidermal growth factor receptor variant III (EGFRvIII) has been found on gliomas and other tumors but not on normal tissues, including those that express the wild-type receptor. Monoclonal antibodies (mAbs) specific for EGFRvIII are rapidly internalized and degraded after binding to EGFRvIII-expressing cells. If anti-EGFRvIII mAbs are to be useful for radioimmunotherapy, then methods for trapping radionuclides in target cells after mAb processing are required. Because lysosomes are known to retain positively charged molecules, we have evaluated a new reagent for this purpose that uses a polycationinc peptide composed of D-amino acids (D-Lys-D-Arg-D-Tyr-D-Arg-D-Arg; D-KRYRR). D-KRYRR was first labeled using lodogen and then coupled to the murine anti-EGFRvIII mAb L8A4 via maleimido bond formation in 60% yield. In vitro assays with the U87deltaEGFR cell line indicated that internalized and total cell-associated activity for the 125I-labeled D-KRYRR-L8A4 conjugate were up to 4 and 5 times higher, respectively, than for L8A4 labeled with 131I using Iodogen. Paired-label comparisons in athymic mice with s.c. U87deltaEGFR xenografts demonstrated up to 5-fold higher tumor uptake for mAb labeled using D-KRYRR. Higher levels of radioiodine activity also were observed in kidney when L8A4 was labeled using D-KRYRR. Another paired-label study directly compared L8A4 labeled using radioiodinated D-KRYRR and L-KRYRR, and confirmed the role of D-amino acids in enhancing tumor uptake. These results suggest that D-KRYRR is a promising reagent for the radioiodination of internalizing mAbs, such as the anti-EGFRvIII mAb L8A4.

Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications.

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate.

Monoclonal antibodies (MAbs) such as the anti-epidermal growth factor variant III (EGFRvIII) MAb L8A4 are rapidly internalized, which can lead to rapid loss of radioactivity from the tumor cell. The aim of this study was to evaluate the potential utility of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate ([211At]SAPC) for labeling murine L8A4 with 211At. SAPC was synthesized by astatodestannylation of N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate and then coupled to L8A4 in approximately 50% yield. The affinity and immunoreactive fraction for 211At-labeled L8A4 were comparable to those obtained when the MAb was labeled with 131I via N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). Paired-label comparisons of the 211At- and 131I-labeled MAbs demonstrated similar internalization and catabolism by EGFRvIII-positive cells in vitro, and with the exception of the stomach, similar tissue distribution in athymic mice with EGFRvIII-expressing U87MGdeltaEGFR xenografts. These results suggest that SAPC may be a useful reagent for labeling L8A4, and possibly other internalizing proteins, with 211At.

Radioiodinated antibody targeting of the HER-2/neu oncoprotein: effects of labeling method on cellular processing and tissue distribution.

Monoclonal antibody (MAb) internalization can have a major effect on tumor retention of radiolabel. Two anti-HER-2/neu MAbs (TA1 and 520C9) were radioiodinated using the iodogen, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), and tyramine-cellobiose (TCB) methods. Paired-label studies compared internalization and cellular processing of the labeled MAbs by SKOv3 9002-18 ovarian cancer cells in vitro. Intracellular radioiodine activity for 520C9 was up to 2.6 and 3.0 times higher for SIPC and TCB labeling, respectively, compared with iodogen. Likewise, intracellular activity for TA1 was up to 2.3 and 2.9 times higher with the SIPC and TCB methods compared with iodogen labeling. Unfortunately, similar advantages in tumor accumulation were not achieved in athymic mice bearing SKOv3 9008-18 ovarian cancer xenografts.