A biomarker assay validation approach tailored to the context of use and bioanalytical platform.

It is widely acknowledged by the bioanalytical and biomarker community that biomarker assay validations should be fit-for-purpose depending on the context of use. The challenge is how to consistently apply these principles in teams responsible for measuring a disparate array of biomarkers, often on multiple analytical platforms, at various stages of the drug discovery and development pipeline and across diverse biology focus areas. To drive consistency, while maintaining the necessary flexibility to allow validations to be driven by scientific rationale and taking into consideration the context of use and associated biological and (pre)analytical factors, a framework applicable across biomarker assays was developed. Herein the authors share their perspective to engage in the ongoing conversation around fit-for-purpose biomarker assay validation.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Pharmacokinetic interaction of brivaracetam on other antiepileptic drugs in adults with focal seizures: Pooled analysis of data from randomized clinical trials.

OBJECTIVE: To assess the effect of brivaracetam (BRV) on steady-state plasma concentrations of commonly prescribed antiepileptic drugs (AEDs). METHODS: Data were pooled from five randomized, double-blind, placebo-controlled efficacy studies (NCT00175929, NCT00175825, NCT00490035, NCT00464269, and NCT01261325) in which adults with refractory epilepsy, and receiving stable doses of 1-2 AEDs, initiated adjunctive treatment with BRV (or placebo) for up to 12 weeks, following a 4-8 week baseline period. Concentrations of carbamazepine, carbamazepine epoxide, clobazam, clonazepam, lacosamide, lamotrigine, levetiracetam, oxcarbazepine (MHD), phenobarbital, phenytoin, pregabalin, topiramate, valproic acid and zonisamide, were measured during baseline and during BRV or placebo evaluation periods. Log-transformed data for patients receiving BRV dosages of 50-200 mg/day (or placebo) were evaluated using repeated measures analysis of covariance. Geometric least-squares means ratios of respective AED concentrations (treatment vs baseline) and their 90% confidence intervals (CIs) were calculated. Relevant interaction of BRV on the respective AED was inferred if CIs were entirely outside of 0.80-1.25 limits. RESULTS: Within the population for analysis (n = 1402), relevant interaction was observed for carbamazepine epoxide alone which increased up to 2-fold from baseline due to inhibition of epoxide hydrolase by BRV, and the effect size was not influenced by concomitant valproic acid. Relevant interaction was not observed for other AEDs. CONCLUSION: In adults with focal seizures, adjunctive BRV treatment does not affect plasma concentrations of the evaluated AEDs but increases carbamazepine epoxide metabolite. Carbamazepine dose reduction should be considered if tolerability issues arise.

European Bioanalysis Forum recommendation on the best practices to demonstrate processed sample stability.

The European Bioanalysis Forum is providing a proposal on experiments to be conducted if processed samples are to be stored for a period of time, either after an initial injection or if processed samples are to be stored for a period of time prior to their first injection. Using survey information and extensive discussion an insight was gained as to whether the current practices employed during method validation were conducted to scientifically validate actual sample handling conditions or to simply meet a perceived regulatory expectation. The goal of this report is to recommend the adoption of scientifically justified validation experiments that exactly reproduce and processed test sample handling and storage procedures and to introduce some uniformity across the industry.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 2 - Hybrid LBA/LCMS and input from regulatory agencies).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Brivaracetam, a selective high-affinity synaptic vesicle protein 2A (SV2A) ligand with preclinical evidence of high brain permeability and fast onset of action.

OBJECTIVE: Rapid distribution to the brain is a prerequisite for antiepileptic drugs used for treatment of acute seizures. The preclinical studies described here investigated the high-affinity synaptic vesicle glycoprotein 2A (SV2A) antiepileptic drug brivara-cetam (BRV) for its rate of brain penetration and its onset of action. BRV was compared with levetiracetam (LEV). METHODS: In vitro permeation studies were performed using Caco-2 cells. Plasma and brain levels were measured over time after single oral dosing to audiogenic mice and were correlated with anticonvulsant activity. Tissue distribution was investigated after single dosing to rat (BRV and LEV) and dog (LEV only). Positron emission tomography (PET) displacement studies were performed in rhesus monkeys using the SV2A PET tracer [11C]UCB-J. The time course of PET tracer displacement was measured following single intravenous (IV) dosing with LEV or BRV. Rodent distribution data and physiologically based pharmacokinetic (PBPK) modeling were used to compute blood-brain barrier permeability (permeability surface area product, PS) values and then predict brain kinetics in man. RESULTS: In rodents, BRV consistently showed a faster entry into the brain than LEV; this correlated with a faster onset of action against seizures in audiogenic susceptible mice. The higher permeability of BRV was also demonstrated in human cells in vitro. PBPK modeling predicted that, following IV dosing to human subjects, BRV might distribute to the brain within a few minutes compared with approximately 1 h for LEV (PS of 0.315 and 0.015 ml/min/g for BRV and LEV, respectively). These data were supported by a nonhuman primate PET study showing faster SV2A occupancy by BRV compared with LEV. SIGNIFICANCE: These preclinical data demonstrate that BRV has rapid brain entry and fast brain SV2A occupancy, consistent with the fast onset of action in the audiogenic seizure mice assay. The potential benefit of BRV for treatment of acute seizures remains to be confirmed in clinical studies.

Cross-Species Differences in the Preclinical Pharmacokinetics of CT7758, an α4ß1/α4ß7 Integrin Antagonist.

CT7758, a carboxylate containing α4ß1/α4/ß7 integrin antagonist, was characterized for its pharmacokinetic profile in various in vitro and in vivo assays in support of clinical development. The oral bioavailability of CT7758 was 4% in mice, 2% in rats, 7-55% in dogs, and 0.2% in cynomolgus monkeys. The low bioavailability in rodents and monkey results from low intestinal absorption as evidenced by a low fraction absorbed in the rat portal vein model (3%), low-to-medium permeability in Caco-2 cells (≤1.3 × 10(-6) cm/s) with evidences of polarized efflux, and high polar surface area (104 Å). In rodents and cynomolgus monkeys, the total plasma clearance was moderate to high (≥50% hepatic blood flow QH) and associated with a short elimination half-life (≤1 hour). This contrast with the dog data which showed a much lower clearance (6% QH) and a longer t1/2 (2.4 hours). The volume of distribution (Vz) also varied significantly across species with value of 5.5, 2.8, 0.24, and 0.93 l/kg in mouse, rat, dog, and cynomolgus monkey, respectively. In vitro assays demonstrated that active hepatic uptake accounted for most of the in vivo clearance and was the source of the large species variability. In vitro uptake assays predicted a total plasma clearance in humans in the low range (33% QH), a finding subsequently confirmed in the clinic. Assays in OAPT1B1-transfected cells demonstrated active uptake transport through this transporter. The prospect of limited absorption in human prompted the synthesis an ethyl ester prodrug, CDP323, which demonstrated higher in vitro permeability, increased oral bioavailability, as well as efficient in vivo release of its active moiety CT7758.

Anti-ictogenic and antiepileptogenic properties of brivaracetam in mature and immature rats.

OBJECTIVE: Brivaracetam (BRV) is a new antiepileptic drug candidate rationally designed for high affinity and selectivity for the synaptic vesicle protein 2A. This study explored anti-ictogenic and antiepileptogenic effects of BRV in rats at different stages of development. METHODS: Using a rapid kindling model in P14, P21, P28, and P60 rats, we studied two doses of BRV: 10 and 100 mg/kg injected intraperitoneally 30 min before afterdischarge assessment. We also assessed blood and brain concentrations of BRV 30 min after the injection. RESULTS: BRV 100 mg/kg significantly increased the afterdischarge threshold (ADT) at all ages, whereas BRV at 10 mg/kg increased ADT in P60, P28, and P21 rats. BRV also shortens the afterdischarge duration (ADD), achieving statistical significance with 10 and 100 mg/kg at P60 and with 100 mg/kg at P21. At P60, BRV increases the number of stimulations required to achieve a stage 4-5 seizure in a dose-dependent manner. At P28 and P21, BRV increased the number of stimulations required to develop a stage 4-5 seizure in a dose-dependent manner with almost complete elimination of stage 4-5 seizures. In contrast, at P14, BRV had no effect on the number of stage 4-5 seizures. An age-related decrease in blood and brain concentrations of BRV was observed 30 min after injection of BRV 10 mg/kg, whereas with 100 mg/kg there were no significant age-correlated differences in brain and serum BRV concentrations. SIGNIFICANCE: BRV exerted dose-dependent anti-ictogenic effects from P60 to P14 independent of brain maturation. BRV also exhibited antiepileptogenic effects at P60, whereas this effect need to be further evaluated at P28 and P21. We did not observe any effect on epileptogenesis at P14 at either dose.

N-alkylprotoporphyrin formation and hepatic porphyria in dogs after administration of a new antiepileptic drug candidate: mechanism and species specificity.

A new antiepileptic synaptic vesicle 2a (SV2a) ligand drug candidate was tested in 4-week oral toxicity studies in rat and dog. Brown pigment inclusions were found in the liver of high-dose dogs. The morphology of the deposits and the accompanying liver changes (increased plasma liver enzymes, increased total hepatic porphyrin level, decreased liver ferrochelatase activity, combined induction, and inactivation of cytochrome P-450 CYP2B11) suggested disruption of the heme biosynthetic cascade. None of these changes was seen in rat although this species was exposed to higher parent drug levels. Toxicokinetic analysis and in vitro metabolism assays in hepatocytes showed that dog is more prone to oxidize the drug candidate than rat. Mass spectrometry analysis of liver samples from treated dogs revealed an N-alkylprotoporphyrin adduct. The elucidation of its chemical structure suggested that the drug transforms into a reactive metabolite which is structurally related to a known reference porphyrogenic agent allylisopropylacetamide. That particular metabolite, primarily produced in dog but neither in rat nor in human, has the potential to alkylate the prosthetic heme of CYP. Overall, the data suggested that the drug candidate should not be porphyrogenic in human. This case study further exemplifies the species variability in the susceptibility to drug-induced porphyria.

In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium.

BACKGROUND: At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. RESULTS: The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. CONCLUSION: The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.

(123)I-Interleukin-2 uptake in squamous cell carcinoma of the head and neck carcinoma.

INTRODUCTION: Information obtained on the IL-2 receptor status of tumour infiltrating lymphocytes in patients suffering from squamous cell carcinoma of the head and neck (SSCHN) before and after IL-2 treatment may lead to a better understanding of the immunological changes and related kinetics induced at the tumour level and ultimately to a strategy that allows selection of those patients that will benefit from IL-2 therapy. This study set out to assess the relationship between (123)I-IL2 single-photon emission computed tomography (SPECT) findings and the presence of IL-2 receptors (CD25 staining) on tumour-infiltrating lymphocytes as well as on SCCHN tumour cells in patients suffering from SCCHN. MATERIALS AND METHODS: Seventeen consecutive patients (12 men; mean age, 57 years) highly suspected to suffer from SSCHN were prospectively included in the study. All patients underwent planar and whole body (123)I-IL2 scintigraphy and underwent surgery or had a biopsy taken within 1 week from imaging. Surgical resected primary lesions as well as biopsy material from primary tumours were histologically analysed with respect to the presence and intensity of CD25 expression on tumour infiltrating lymphocytes and tumour cells (HSCORE). Tumor-to-background (T/N) ratios of the primary tumour derived from planar and tomographic (123)I-IL2 scintigraphy were related to the results derived from histology. RESULTS: All patients suffered from SSCHN. T/N ratios derived from SPECT images were significantly correlated with CD25 lymphocyte HSCOREs (r = 0.66; p = 0.03), but not with CD25 tumour cell HSCOREs. CONCLUSIONS: (123)I-IL-2 SPECT imaging allows for non-invasive imaging of the relative amount of IL-2 receptors present on tumour infiltrating lymphocytes in SCCHN.

Optimization by experimental design of precursor synthesis and radiolabeling of 2-iodo-L-phenylalanine, a novel amino acid for tumor imaging.

Various radiolabeled amino acids show promising results in tumor detection, as applied in the management of cancer patients. We synthesized the precursor 2-iodo-L-phenylalanine for easier kit labeling of [123/125I]- 2-iodo-L-phenylalanine, using the Cu1+ -assisted nucleophilic halogen exchange. Precursor synthesis was optimized by experimental design: Eight parameters were initially screened by a quarter fractional design. The resulting most important parameters (i.e., temperature, CuSO4, NaI) were further optimized using a full three-factor, three-level factorial design. The final conclusion for the optimal values for temperature, reaction time, and concentration of 2-bromo-L-phenylalanine, NaI, CuSO4, SnSO4, C6H6O7, and C7H6O4 were 180 degrees C, 24 hours, 61 mM, 485 mM, 10 mM, 90 mM, 90 mM, and 100 mM, respectively. The yield was increased from 39% to consistently more than 74% 2-iodo-L-phenylalanine. Structure confirmation and quality control was performed by 1H-NMR, mass spectroscopy (MS), and high-performance liquid chromatography (HPLC) (reverse phase [RP] and chiral). No phenylalanine-related impurities or racemization was detected. Subsequent radioiodination of the obtained 2-iodo-L-phenylalanine was performed in kit conditions with n.c.a. Na123/125I, resulting in a labeling yield of > 98%. After Ag-membrane filtration, a radiochemical purity of > 99% was obtained. The Cu1+ -assisted nucleophilic exchange reaction allows both routine kit preparation and "cold" synthesis of 2-iodo-L-phenylalanine from 2-bromo-L-phenylalanine. The reaction presents an interesting alternative for a cumbersome multistep, stereo-specific synthesis.

Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.

OBJECTIVE: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA. METHODS: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. RESULTS: The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. CONCLUSION: These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.

Valine-based biphenylsulphonamide matrix metalloproteinase inhibitors as tumor imaging agents.

Among matrix metalloproteinases (MMPs), the subfamily of gelatinases (MMP-2, MMP-9) is of particular interest due to their ability to degrade type IV collagen and other non-fibrillar collagen domains and proteins such as fibronectin and laminin. Whilst malignant cells often over-express various MMPs, the gelatinases have been most consistently detected in malignant tissues and associated with tumor growth, metastatic potential and angiogenesis. Radiosynthesis of carboxylic (1') and hydroxamic (2') MMPIs resulted in radiochemical yields of 70 +/- 5% (n = 6) and 60 +/- 5% (n = 4), respectively. Evaluation in A549-inoculated athymic mice showed a tumor uptake of 2. 0+/- 0.7%ID/g (3 h p.i.), a tumor/blood ratio of 0.5 and a tumor/muscle ratio of 4.6 at 48 h p.i. for 1'. For compound 2' a tumor uptake of 0.7 +/- 0.2%ID/g (3 h p.i.), a tumor/blood ratio of 1.2 and a tumor/muscle ratio of 1.8 at 24 h p.i. were observed. HPLC analysis of the blood (plasma) showed no dehalogenation or other metabolites of 1' 2 h p.i. For compound 2', 65.4% of intact compound was found in the blood (plasma) and one polar metabolite (31%) was detected whereas in the tumor 91.8% of the accumulated activity was caused by intact compound and only 8.1% by the metabolite. Planar imaging, using a Toshiba GCA-9300A/hg SPECT camera, showed that tumor tissue could be visualized and that image quality improved by decreasing specific activity resulting in lower liver uptake, indicating some degree of saturable binding in the liver. In vivo evaluation of these radioiodinated carboxylic and hydroxamic MMP inhibitor tracers revealed that MMP inhibitors could have potential as tumor imaging agents, but that further research is necessary.

In vitro and in vivo evaluation of [123I]-VEGF165 as a potential tumor marker.

One of the research challenges in oncology is to develop new biochemical methods for noninvasive tumor therapy evaluation to determine whether the chemotherapeutics is effective. Vascular endothelial growth factor (VEGF) was labeled with radioiodine and evaluated in vitro as well as in vivo, using A2058, a melanoma cell line overexpressing VEGFR-1 and -2. Saturation binding analysis with [(125)I]-VEGF resulted in a K(d) of 0.1 nM. Internalization assays indicate the preserved ligand induced internalization and metabolization of the tracer. Biodistribution studies with [(123)I]-VEGF in wild type and A2058 tumor-bearing athymic mice showed low background activity and a tumor to reference tissue ratio of maximum 6.12. These results suggest that [(123)I]-VEGF is a potentially suitable tracer for tumor therapy evaluation.

Synthesis, radiosynthesis, in vitro and preliminary in vivo evaluation of biphenyl carboxylic and hydroxamic matrix metalloproteinase (MMP) inhibitors as potential tumor imaging agents.

Excess matrix degradation is one of the hallmarks of cancer and is an important factor in the process of tumor progression. It is implicated in invasion, metastasis, growth, angiogenesis and migration. Many characteristics of matrix metalloproteinases (MMPs) make them attractive therapeutic and diagnostic targets. MMP expression is upregulated at the tumor site, with localization of activity in the tumor or the surrounding stroma, providing a target for medical imaging techniques. Radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'-[123I] iodo-biphenyl-4-sulfonylamino)-3-methyl-butyric acid (9) and 2-(4'-[123I] iodo-biphenyl-4-sulfonylamino)-3-methyl-butyramide (11), their unlabelled standards and precursors were synthesized. Radioiodination was conducted by electrophilic aromatic substitution of the tributylstannyl precursors and resulted in radiochemical yields of 70+/-5% (n=6) and 60+/-5% (n=4), respectively. In vitro zymography and enzyme assays showed for both hydroxamic acid and carboxylic acid compounds a good inhibition activity and a high selectivity for MMP-2. In vivo biodistribution in NMRI mice showed no long-term accumulation in organs and the possibility to accumulate in the tumor in a later phase of this study.

In vivo evaluation of [(123)I]-3-(4-iodobenzyl)-1,2,3,4-tetrahydro-8-hydroxychromeno[3,4-c]pyridin-5-one: a presumed dopamine D4 receptor ligand for SPECT studies.

[(123)I]-3-(4-iodobenzyl)-1,2,3,4-tetrahydro-8-hydroxychromeno[3,4-c]pyridin-5-one ([(123)I]-ITCP), a presumed radioligand for visualization of the dopamine D4 receptor by single photon emission computed tomography, was evaluated in vivo in mice and rabbits. This new radioiodinated tracer exhibited high brain uptake (3.64% injected dose per gram of tissue at 10 min p.i.) in mice. No significant amounts (less than 5%) of labeled metabolites were present in the brain, as demonstrated by a metabolite study. Regional brain distribution in rabbits showed atypical CNS uptake with consistently low values in the cortex and high values in other brain parts including cerebellum. Saturable binding was confirmed by a competition experiment with unlabeled product. Selectivity was assessed by competition experiments with a known dopamine D4 ligand and later with a sigma receptor ligand. Both experiments showed no observable competition. In conclusion, our findings indicate that [(123)I]-ITCP is neither a dopamine D4 receptor ligand nor a sigma receptor ligand. The exact nature of [(123)I]-ITCP binding in the brain remains to be elucidated.

In vivo evaluation of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide: a potential sigma receptor ligand for SPECT studies.

In this study, in vivo evaluation in mice and rabbits of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide ([123I]-BPB), a potential radioligand for visualisation of the sigma receptor by single photon emission computed tomography (SPECT), is reported. The compound possesses appropriate lipophilicity (log P=2.2) and binds sigma-1 and sigma-2 receptors (pKi=6.51 and 6.79, respectively). In mice, this new radioiodinated tracer exhibited high brain uptake (4.99% ID/g tissue at 10 min postinjection) and saturable binding (3.06% ID/g tissue at 10 min postinjection) as determined by pretreatment with unlabeled [123I]-BPB. A metabolite study demonstrated no (less than 5%) labeled metabolites in the brain. In rabbits, regional brain distribution was investigated and the tracer displayed high, homogeneous central nervous system uptake. Selectivity was assessed by competition experiments with known sigma ligands. Metabolite analysis showed no (less than 8%) labeled metabolites in the rabbit brain. In conclusion, our findings indicate that [123I]-BPB is not a suitable tracer for visualisation of D3 receptors while its potential for sigma receptor imaging is severely hampered by its affinity for dopamine receptors.

Tryptophane-based biphenylsulfonamide matrix metalloproteinase inhibitors as tumor imaging agents.

AIM: As a part of our efforts to use small organic matrix metalloproteinase (MMP) inhibitors with improved characteristics for the diagnosis and treatment of different kinds of tumor tissues, biphenylsulfonamide analogues were synthesized. This study reports on the in vivo biodistribution of iodine-123-labeled biphenylsulfonide and analogues in A549 lung carcinoma inoculated into athymic mice and the evaluation of their suitability as imaging agents using a single photon emission computed tomography (SPECT) camera. METHODS: The radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'- [(123)I]iodobiphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionic acid (1') and 2-(4'-[(123)I]iodobiphenyl-4- sulfonylamino)-3-(1H-indol-3-yl)-propionamide (2') were synthesized by electrophilic aromatic substitution of the tributylstannyl derivatives. Planar gamma camera imaging was performed in nu/nu athymic mice bearing an A549 tumor using a Toshiba GCA-9300A/hg SPECT camera in planar mode equipped with a high-resolution, parallel-hole collimator. RESULTS: Radiosynthesis of (1') and (2') resulted in radiochemical yields of 60 +/- 5% (n +/- 3) and 70 +/- 5% (n = 6), respectively. Evaluation of tumors induced in athymic mice by the inoculation of non-small cell lung A549 carcinoma cells, showed a tumor uptake of 0.27-0.01 percent injected dose per gram (%ID/g) (3 hours-48 hours p.i.), a tumor-blood ratio of 0.7, a tumor-muscle ratio of 1.6, and a tumor-fat ratio of 0.5 at 24 hours (p.i.) for compound 1'. For compound 2' a tumor uptake of 0.7-0.04 %ID/g (3 hours-48 hours p.i.), a postinjection tumor-blood ratio of 1.2, a tumor-muscle ratio of 3.2, and a tumor-fat ratio of 2.4 at 48 hours p.i. was observed. SPECT evaluation confirmed the results obtained from biodistribution. CONCLUSION: In vivo evaluation of these radioiodinated carboxylic and hydroxamic MMP inhibitor tracers revealed that they do not appear suitable as tumor-imaging agents.