Pharmacodynamic evaluation and safety assessment of treatment with antibodies to serum amyloid P component in patients with cardiac amyloidosis: an open-label Phase 2 study and an adjunctive immuno-PET imaging study.

BACKGROUND: In a Phase I study treatment with the serum amyloid P component (SAP) depleter miridesap followed by monoclonal antibody to SAP (dezamizumab) showed removal of amyloid from liver, spleen and kidney in patients with systemic amyloidosis. We report results from a Phase 2 study and concurrent immuno-positron emission tomography (PET) study assessing efficacy, pharmacodynamics, pharmacokinetics, safety and cardiac uptake (of dezamizumab) following the same intervention in patients with cardiac amyloidosis. METHODS: Both were uncontrolled open-label studies. After SAP depletion with miridesap, patients received ≤ 6 monthly doses of dezamizumab in the Phase 2 trial (n = 7), ≤ 2 doses of non-radiolabelled dezamizumab plus [89Zr]Zr-dezamizumab (total mass dose of 80 mg at session 1 and 500 mg at session 2) in the immuno-PET study (n = 2). Primary endpoints of the Phase 2 study were changed from baseline to follow-up (at 8 weeks) in left ventricular mass (LVM) by cardiac magnetic resonance imaging and safety. Primary endpoint of the immuno-PET study was [89Zr]Zr-dezamizumab cardiac uptake assessed via PET. RESULTS: Dezamizumab produced no appreciable or consistent reduction in LVM nor improvement in cardiac function in the Phase 2 study. In the immuno-PET study, measurable cardiac uptake of [89Zr]Zr-dezamizumab, although seen in both patients, was moderate to low. Uptake was notably lower in the patient with higher LVM. Treatment-associated rash with cutaneous small-vessel vasculitis was observed in both studies. Abdominal large-vessel vasculitis after initial dezamizumab dosing (300 mg) occurred in the first patient with immunoglobulin light chain amyloidosis enrolled in the Phase 2 study. Symptom resolution was nearly complete within 24 h of intravenous methylprednisolone and dezamizumab discontinuation; abdominal computed tomography imaging showed vasculitis resolution by 8 weeks. CONCLUSIONS: Unlike previous observations of visceral amyloid reduction, there was no appreciable evidence of amyloid removal in patients with cardiac amyloidosis in this Phase 2 trial, potentially related to limited cardiac uptake of dezamizumab as demonstrated in the immuno-PET study. The benefit-risk assessment for dezamizumab in cardiac amyloidosis was considered unfavourable after the incidence of large-vessel vasculitis and development for this indication was terminated. Trial registration NCT03044353 (2 February 2017) and NCT03417830 (25 January 2018).

Validation of an automated UPLC-MS/MS method for methylmalonic acid in serum/plasma and its application on clinical samples.

Vitamin B12 is essential for cell function and only accessible in food for mammals. To monitor vitamin B12 deficiency, methylmalonic acid (MMA) is used. Since MMA in serum/plasma is a frequently requested analyte at clinical laboratories the analytical method was improved and validated on a 96 well plate. Using a Tecan robot a working solution of acetonitrile containing MMA-D3 was added to plasma/serum samples. The solution was shaken for 1 min and then centrifuged for 10min. The supernatant was transferred to another plate and evaporated with nitrogen gas. The residual was redissolved with 0.2% formic acid in MilliQ-water and the plate was shaken for 1 min prior to LC-MS/MS analysis. The total analysis time was 3 min, retention time for MMA was 1.1 min and it was well separated from the interfering succinic acid. The calibrator curve was 0.044 - 1.63 µmol/L, which was also the linear range and LLOQ was 0.044 µmol/L. The within- and between-run CV:s were 3-7%. Age dependent clinical cut-offs at 0.28 (age <50 years) and 0.36 µmol/L (age ≥50 years) were applied. In 404 clinical routine samples 10% were >0.28, 7% > 0.4, and only 1% were >0.7 µmol/L. The method has been successfully implemented in the laboratory for routine MMA analysis.

Advanced imaging for quantification of abnormalities in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVES: To assess non-invasive imaging for detection and quantification of gland structure, inflammation and function in patients with primary Sjogren's syndrome (pSS) using PET-CT with 11C-Methionine (11C-MET; radiolabelled amino acid), and 18F-fluorodeoxyglucose (18F-FDG; glucose uptake marker), to assess protein synthesis and inflammation, respectively; multiparametric MRI evaluated salivary gland structural and physiological changes. METHODS: In this imaging/clinical/histology comparative study (GSK study 203818; NCT02899377) patients with pSS and age- and sex-matched healthy volunteers underwent MRI of the salivary glands and 11C-MET PET-CT. Patients also underwent 18F-FDG PET-CT and labial salivary gland biopsies. Clinical and biomarker assessments were performed. Primary endpoints were semi-quantitative parameters of 11C-MET and 18F-FDG uptake in submandibular and parotid salivary glands and quantitative MRI measures of structure and inflammation. Clinical and minor salivary gland histological parameter correlations were explored. RESULTS: Twelve patients with pSS and 13 healthy volunteers were included. Lower 11C-MET uptake in parotid, submandibular and lacrimal glands, lower submandibular gland volume, higher MRI fat fraction, and lower pure diffusion in parotid and submandibular glands were observed in patients vs healthy volunteer, consistent with reduced synthetic function. Disease duration correlated positively with fat fraction and negatively with 11C-MET and 18F-FDG uptake, consistent with impaired function, inflammation and fatty replacement over time. Lacrimal gland 11C-MET uptake positively correlated with tear flow in patients, and parotid gland 18F-FDG uptake positively correlated with salivary gland CD20+ B-cell infiltration. CONCLUSION: Molecular imaging and MRI may be useful tools to non-invasively assess loss of glandular function, increased glandular inflammation and fat accumulation in pSS.

Phase 0, including microdosing approaches: Applying the Three Rs and increasing the efficiency of human drug development.

Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of 'kill-early-kill-cheap' to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.

Evaluation of the Meritas® BNP Test for Point-of-Care Testing.

BACKGROUND: BNP and NT-proBNP are widely used as rule-out tests for heart failure (HF) and they are frequently requested by primary care doctors. A point-of-care (POC) test would reduce the time to diagnosis for patients with suspected HF. The aim of the study was to evaluate a POC BNP test. METHODS: Plasma BNP results obtained with the Meritas® POC instrument (n = 82) were compared with the corresponding plasma BNP results analyzed on an Advia Centaur analyzer (Siemens Healthcare, Erlangen, Germany). RESULTS: The two methods showed concordant results with a Passing-Bablok correlation between the two methods: BNP(Meritas) = 1.00 x BNP(Siemens) + 1.09; r = 0.9773. CONCLUSIONS: The study show that the Meritas® BNP assay could be used in primary care permitting rapid BNP testing to rule out heart failure.

Detection of anabolic androgenic steroids in serum samples.

When testing for anabolic androgenic steroids (AAS) outside sports communities, for example, in healthcare and forensic medicine, urine is the matrix of choice. However, there are drawbacks with urinary sampling, and serum might be useful as a complementary matrix. The aim was to develop an LC-MS/MS method for serum measuring AAS frequently used outside of sport, including testosterone (T), steroid esters, and eight other synthetic AAS. The sample pretreatment included sample precipitation and evaporation. Limit of quantification for the AAS was 0.05-0.5 ng/mL, and linearity was 0.05-20 ng/mL for most of the substances. Generally, the within- and between-day CV results, matrix effect, and process efficiency were <15%. The AAS were stable for at least 6 months at -20°C. Serum samples were obtained from previous studies. A novel finding from an administration study was that T enanthate was present in serum even after 5 years of storage at -20°C. Serum samples from self-reporting AAS individuals, where T esters were detected, were positive for testosterone using the urinary testosterone/epitestosterone criterion >10. Of those identified as positive in traditional urinary doping tests (n = 15), AAS in serum were found in 80% of the subjects. Our results show that serum may be a valid complementary matrix to urine samples for AAS testing.

CD64 as novel molecular imaging marker for the characterization of synovitis in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. OBJECTIVES: We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. METHODS: First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for 111In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. RESULTS: We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A/CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [111In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. CONCLUSION: Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.

Preclinical evaluation of [11C]GW457427 as a tracer for neutrophil elastase.

INTRODUCTION: Neutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, [11C]GW457427, as an in vivo biomarker for the study of NE, now available for human studies. METHODS: [11C]GW457427 was produced by methylation of GW447631 using [11C]methyl triflate and GMP validated production and quality control methods were developed. Chemical purity was high with no traces of the precursor GW611437 or other uv-absorbing compounds. A method for the determination of intact [11C]GW457427 in plasma was developed and the binding characteristics were evaluated in vitro and in vivo. An animal model for lung inflammation was used to investigate the specificity and sensitivity of the [11C]GW457427 tracer for neutrophil elastase (NE) in pulmonary inflammation, verified by blockade using two structurally different elastase inhibitors. RESULTS: [11C]GW457427 was obtained in approximately 45% radiochemical yield and with a radiochemical purity higher than 98%. Molar activity was in the range 130-360 GBq/µmol. Binding to NE was shown to be highly specific both in vitro and in vivo and a significantly higher uptake of tracer was found in a lipopolysaccharide mouse model of pulmonary inflammation compared with control animals. The uptake in lung tissue measured as standardized uptake value (SUV) strongly correlated with tissue NE content as measured by ELISA. In vitro studies also showed specific tracer binding in aortic tissue of patients with abdominal aorta aneurysm (AAA). The rate of metabolism in rats was appropriate considering the critical balance between available tracer for binding and requirement for blood clearance with about 40% and 20% intact [11C]GW457427 in plasma at 5 and 40 min, respectively. Radioactivity was cleared from blood and organs in control animals with mainly hepatobiliary excretion with distribution in the intestines and the urinary bladder; but without retention of the tracer in healthy organs of interests such as the lung, liver, kidneys or in the cardiovascular system. A dosimetry study in rat indicated that the whole-body effective dose was 2.2 µSv/MBq with bone marrow as the limiting organ. It is estimated that up to five PET-CT investigations could be performed in humans without exceeding a total dose of 10 mSv. CONCLUSION: [11C]GW457427 is a promising in vivo PET-biomarker for NE with high specific binding demonstrated both in vitro and in vivo. A GMP validated production method including quality control has been developed and a microdosing toxicity study performed with no adverse signs. [11C]GW457427 is currently being evaluated in a First-In-Man PET study.

Multimodal imaging distribution assessment of a liposomal antibiotic in an infectious disease model.

Liposomes are promising targeted drug delivery systems with the potential to improve the efficacy and safety profile of certain classes of drugs. Though attractive, there are unique analytical challenges associated with the development of liposomal drugs including human dose prediction given these are multi-component drug delivery systems. In this study, we developed a multimodal imaging approach to provide a comprehensive distribution assessment for an antibacterial drug, GSK2485680, delivered as a liposomal formulation (Lipo680) in a mouse thigh model of bacterial infection to support human dose prediction. Positron emission tomography (PET) imaging was used to track the in vivo biodistribution of Lipo680 over 48 h post-injection providing a clear assessment of the uptake in various tissues and, importantly, the selective accumulation at the site of infection. In addition, a pharmacokinetic model was created to evaluate the kinetics of Lipo680 in different tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was then used to quantify the distribution of GSK2485680 and to qualitatively assess the distribution of a liposomal lipid throughout sections of infected and non-infected hindlimb tissues at high spatial resolution. Through the combination of both PET and MALDI IMS, we observed excellent correlation between the Lipo680-radionuclide signal detected by PET with the GSK2485680 and lipid component signals detected by MALDI IMS. This multimodal translational method can reduce drug attrition by generating comprehensive biodistribution profiles of drug delivery systems to provide mechanistic insight and elucidate safety concerns. Liposomal formulations have potential to deliver therapeutics across a broad array of different indications, and this work serves as a template to aid in delivering future liposomal drugs to the clinic.

Reporting and incidence trends of hydatidiform mole in Sweden 1973-2004.

OBJECTIVE: To examine temporal trends in the incidence of hydatidiform mole (HM) in relation to maternal age, the occurrence of choriocarcinoma in women with a history of HM and the extent of underreporting of HM to the Swedish Cancer Register (SCR). METHODS: Women registered with a diagnosis of HM were identified in the Swedish Cancer Register and the Swedish Inpatient Register (IPR). Record linkage to the Medical Birth Register provided information to assess the incidence of HM. RESULTS: We identified 3 844 unique cases of HM in the SCR and in the IPR combined between 1973 and 2004, yielding an incidence of 1.2 per 1 000 deliveries. The incidence of HM increased during the period under study. The highest incidence was observed in women below 20 and above 39 years of age. Of all registered cases of choriocarcinoma, 37% occurred in women with a previous history of HM. The risk of choriocarcinoma following HM was 1.3%, compared to 0.005% in women without a previous molar diagnosis. The records of the Cancer Register included 83.2% of all identified cases of HM. CONCLUSION: The incidence of HM in Sweden has increased over time, and is characterized by a bimodal pattern with distinctive peaks in the youngest and oldest women of reproductive age. More than one third of all women registered with choriocarcinoma had a previous diagnosis of HM. Despite mandatory reporting, there was evidence of underreporting of HM to the SCR that remained virtually unchanged over calendar time.

Detection of Drugs in Oral Fluid Samples Using a Commercially Available Collection Device: Agreement with Urine Testing and Evaluation of A and B Samples Obtained from Employees at Different Workplace Settings with Uncontrolled Sampling Procedures.

The use of oral fluid tests to detect drugs is of growing interest in various areas, including treatment centers, roadside and workplace testing. In this study, we investigated drug detection in oral fluid samples collected using a commercially available device, Oral Eze. Drug detection in oral fluid was compared to paired urine samples, which were simultaneously collected. We also evaluated the collection device by comparing A and B oral fluid samples. Finally, we studied the stability of various drugs in samples stored for at least 1 year. The drug profile was investigated by comparing the drugs detected in oral fluid samples with paired urine samples collected in a treatment center. A total of 113 paired oral fluid and urine samples were investigated for the presence of drugs in the following groups: amphetamines, benzodiazepines, opiates and opioids, cocaine and cannabis. A and B samples were collected from different workplaces through an uncontrolled sampling procedure (n = 76). The stability of drugs in A samples was assessed after storage at -20°C for 1 year. Generally, there was a good correlation between drugs detected in oral fluid samples and urine samples. The heroin metabolite, 6-MAM, was more frequently detected in oral fluid samples than in urine samples, while cannabis was better detected in urine samples. Drugs in oral fluid samples were stable when stored at -20°C for at least 1 year. However, in many positive A and B oral fluid samples, there was significant variation in the concentrations obtained. Hence, the collection device may need to be further standardized and improved.

Cross-species/cross-modality physiologically based pharmacokinetics for biologics: 89Zr-labelled albumin-binding domain antibody GSK3128349 in humans.

Two-pore physiologically-based pharmacokinetics (PBPK) for biologics describes the tissue distribution and elimination kinetics of soluble proteins as a function of their hydrodynamic radius and the physiological properties of the organs. Whilst many studies have been performed in rodents to parameterize the PBPK framework in terms of organ-specific lymph flow rates, similar validation in humans has been limited. This is mainly due to the paucity of the tissue distribution time course data for biologics that is not distorted by target-related binding. Here, we demonstrate that a PBPK model based on rodent data provided good to satisfactory extrapolation to the tissue distribution time course of 89Zr-labeled albumin-binding domain antibody (AlbudAb™) GSK3128349 in healthy human volunteers, including correct prediction of albumin-like plasma half-life, volume of distribution, and extravasation half-life. The AlbudAb™ used only binds albumin, and hence it also provides information about the tissue distribution kinetics and turnover of that ubiquitous and multifunctional plasma protein.

Principal component analysis with pre-normalization improves the signal-to-noise ratio and image quality in positron emission tomography studies of amyloid deposits in Alzheimer's disease.

This study introduces a new approach for the application of principal component analysis (PCA) with pre-normalization on dynamic positron emission tomography (PET) images. These images are generated using the amyloid imaging agent N-methyl [(11)C]2-(4'-methylaminophenyl)-6-hydroxy-benzothiazole ([(11)C]PIB) in patients with Alzheimer's disease (AD) and healthy volunteers (HVs). The aim was to introduce a method which, by using the whole dataset and without assuming a specific kinetic model, could generate images with improved signal-to-noise and detect, extract and illustrate changes in kinetic behavior between different regions in the brain. Eight AD patients and eight HVs from a previously published study with [(11)C]PIB were used. The approach includes enhancement of brain regions where the kinetics of the radiotracer are different from what is seen in the reference region, pre-normalization for differences in noise levels and removal of negative values. This is followed by slice-wise application of PCA (SW-PCA) on the dynamic PET images. Results obtained using the new approach were compared with results obtained using reference Patlak and summed images. The new approach generated images with good quality in which cortical brain regions in AD patients showed high uptake, compared to cerebellum and white matter. Cortical structures in HVs showed low uptake as expected and in good agreement with data generated using kinetic modeling. The introduced approach generated images with enhanced contrast and improved signal-to-noise ratio (SNR) and discrimination power (DP) compared to summed images and parametric images. This method is expected to be an important clinical tool in the diagnosis and differential diagnosis of dementia.

Quantifying disease activity in rheumatoid arthritis with the TSPO PET ligand 18F-GE-180 and comparison with 18F-FDG and DCE-MRI.

PURPOSE: While the aetiology of rheumatoid arthritis (RA) remains unclear, many of the inflammatory components are well characterised. For diagnosis and therapy evaluation, in vivo insight into these processes would be valuable. Various imaging probes have shown value including dynamic contrast-enhanced (DCE) MRI and PET/CT using 18F-fluorodeoxyglucose (18F-FDG) or tracers targeting the translocator protein (TSPO). To evaluate 18F-GE-180, a novel TSPO PET tracer, for detecting and quantifying disease activity in RA, we compared 18F-GE-180 uptake with that of 18F-FDG and DCE-MRI measures of inflammation. METHODS: Eight RA patients with moderate-to-high, stable disease activity and active disease in at least one wrist were included in this study (NCT02350426). Participants underwent PET/CT examinations with 18F-GE-180 and 18F-FDG on separate visits, covering the shoulders and from the pelvis to the feet, including hands and wrists. DCE-MRI was performed on one affected hand. Uptake was compared visually between tracers as judged by an experienced radiologist and quantitatively using the maximum standardised uptake value (SUVmax). Uptake for both tracers was correlated with DCE-MRI parameters of inflammation, including the volume transfer coefficient Ktrans using Pearson correlation (r). RESULTS: PET/CT imaging with 18F-GE-180 in RA patients showed marked extra-synovial uptake around the affected joints. Overall sensitivity for detecting clinically affected joints was low (14%). 18F-GE-180 uptake did not or only weakly correlate with DCE-MRI parameters in the wrist (r = 0.09-0.31). 18F-FDG showed higher sensitivity for detecting symptomatic joints (34%), as well as strong positive correlation with DCE-MRI parameters (SUVmax vs. Ktrans: r = 0.92 for wrist; r = 0.68 for metacarpophalangeal joints). CONCLUSIONS: The correlations between DCE-MRI parameters and 18F-FDG uptake support use of this PET tracer for quantification of inflammatory burden in RA. The TSPO tracer 18F-GE-180, however, has shown limited use for the investigation of RA due to its poor sensitivity and ability to quantify disease activity in RA.

Immuno-PET Imaging to Assess Target Engagement: Experience from 89Zr-Anti-HER3 mAb (GSK2849330) in Patients with Solid Tumors.

PET imaging with radiolabeled drugs provides information on tumor uptake and dose-dependent target interaction to support selection of an optimal dose for future efficacy testing. In this immuno-PET study of the anti-human epidermal growth factor receptor (HER3) mAb GSK2849330, we investigated the biodistribution and tumor uptake of 89Zr-labeled GSK2849330 and evaluated target engagement as a function of antibody mass dose. Methods:89Zr-GSK2849330 distribution was monitored in 6 patients with HER3-positive tumors not amenable to standard treatment. Patients received 2 administrations of 89Zr-GSK2849330. Imaging after tracer only was performed at baseline; dose-dependent inhibition of 89Zr-GSK2849330 uptake in tumor tissues was evaluated 2 wk later using increasing doses of unlabeled GSK2849330 in combination with the tracer. Up to 3 PET scans (2 hours post infusion [p.i.] and days 2 and 5 p.i.) were performed after tracer administration. Biodistribution and tumor targeting were assessed visually and quantitatively using SUV. The 50% and 90% inhibitory mass doses (ID50 and ID90) of target-mediated antibody uptake were calculated using a Patlak transformation. Results: At baseline, imaging with tracer showed good tumor uptake in all evaluable patients. Predosing with unlabeled mAb reduced the tumor uptake rate in a dose-dependent manner. Saturation of 89Zr-mAb uptake by tumors was seen at the highest dose (30 mg/kg). Despite the limited number of patients, an exploratory ID50 of 2 mg/kg and ID90 of 18 mg/kg have been determined. Conclusion: In this immuno-PET study, dose-dependent inhibition of tumor uptake of 89Zr-GSK2849330 by unlabeled mAb confirmed target engagement of mAb to the HER3 receptor. This study further validates the use of immuno-PET to directly visualize tissue drug disposition in patients with a noninvasive approach and to measure target engagement at the site of action, offering the potential for dose selection.

The biodistribution and clearance of AlbudAb, a novel biopharmaceutical medicine platform, assessed via PET imaging in humans.

Conjugation or fusion to AlbudAbs™ (albumin-binding domain antibodies) is a novel approach to extend the half-life and alter the tissue distribution of biological and small molecule therapeutics. To understand extravasation kinetics and extravascular organ concentrations of AlbudAbs in humans, we studied tissue distribution and elimination of a non-conjugated 89Zr-labeled AlbudAb in healthy volunteers using positron emission tomography/computed tomography (PET/CT). METHODS: A non-conjugated AlbudAb (GSK3128349) was radiolabeled with 89Zr and a single 1 mg (~ 15 MBq) dose intravenously administered to eight healthy males. 89Zr-AlbudAb tissue distribution was followed for up to 7 days with four whole-body PET/CT scans. 89Zr-AlbudAb tissue concentrations were quantified in organs of therapeutic significance, measuring standardized uptake value and tissue/plasma ratios. Plasma pharmacokinetics were assessed by gamma counting and LC-MS/MS of blood samples. RESULTS: 89Zr-AlbudAb administration and PET/CT procedures were well tolerated, with no drug-related immunogenicity or adverse events. 89Zr-AlbudAb rapidly distributed throughout the vasculature, with tissue/plasma ratios in the liver, lungs, and heart relatively stable over 7 days post-dose, ranging between 0.1 and 0.5. The brain tissue/plasma ratio of 0.025 suggested minimal AlbudAb blood-brain barrier penetration. Slowly increasing ratios in muscle, testis, pancreas, and spleen reflected either slow AlbudAb penetration and/or 89Zr residualization in these organs. Across all tissues evaluated, the kidney tissue/plasma ratio was highest (0.5-1.5 range) with highest concentration in the renal cortex. The terminal half-life of the 89Zr-AlbudAb was 18 days. CONCLUSION: Evaluating the biodistribution of 89Zr-AlbudAb in healthy volunteers using a low radioactivity dose was successful (total subject exposure ~ 10 mSv). Results indicated rapid formation of reversible, but stable, complexes between AlbudAb and albumin upon dosing. 89Zr-AlbudAb demonstrated albumin-like pharmacokinetics, including limited renal elimination. This novel organ-specific distribution data for AlbudAbs in humans will facilitate a better selection of drug targets to prosecute using the AlbudAb platform and significantly contribute to modeling work optimizing dosing of therapeutic AlbudAbs in the clinic.

Modeling spheroid growth, PET tracer uptake, and treatment effects of the Hsp90 inhibitor NVP-AUY922.

UNLABELLED: For a PET agent to be successful as a biomarker in early clinical trials of new anticancer agents, some conditions need to be fulfilled: the selected tracer should show a response that is related to the antitumoral effects, the quantitative value of this response should be interpretable to the antitumoral action, and the timing of the PET scan should be optimized to action of the drug. These conditions are not necessarily known at the start of a drug-development program and need to be explored. We proposed a translational imaging activity in which experiments in spheroids and later in xenografts are coupled to modeling of growth inhibition and to the related changes in the kinetics of PET tracers and other biomarkers. In addition, we demonstrated how this information can be used for planning clinical trials. METHODS: The first part of this concept is illustrated in a spheroid model with BT474 breast cancer cells treated with the heat shock protein 90 (Hsp90) inhibitor NVP-AUY922. The growth-inhibitory effect after a pulse treatment with the drug was measured with digital image analysis to determine effects on volume with high accuracy. The growth-inhibitory effect was described mathematically by a combined E(max) and time course model fitted to the data. The model was then used to simulate a once-per-week treatment; in these experiments the uptake of the PET tracers (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) was determined at different doses and different time points. RESULTS: A drug exposure of 2 h followed by washout of the drug from the culture medium generated growth inhibition that was maximal at the earliest time point of 1 d and decreased exponentially with time during 10-12 d. The uptake of (18)F-FDG per viable tumor volume was minimally affected by the treatment, whereas the (18)F-FLT uptake decreased in correlation with the growth inhibition. CONCLUSION: The study suggests a prolonged action of the Hsp90 inhibitor that supports a once-per-week schedule. (18)F-FLT is a suitable tracer for the monitoring of effect, and the (18)F-FLT PET study might be performed within 3 d after dosing.

Biodistribution of 68Ga-labeled LNA-DNA mixmer antisense oligonucleotides for rat chromogranin-A.

In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with 68Ga and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2--differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched--differing only in two nucleotides from the two Antisenses--was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after 68Ga-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.

Alteration in [11C]vorozole binding to aromatase in neuronal cells of rat brain induced by anabolic androgenic steroids and flutamide.

In a previous study, we demonstrated that androgenic-anabolic steroids increased aromatase expression in the bed nucleus of stria terminalis and preoptic area in rat brain, as evaluated using autoradiography with [11C]vorozole, a potential positron emission tomography tracer for aromatase. In this study, we explored whether the increase in aromatase binding is mediated via androgen receptors and whether this increase occurs in neurons or glial cells. Rats were given nandrolone decanoate (15 mg/kg body weight once every 3 days) and flutamide (20 mg/kg/day) alone or in combination for 20 days. Results indicated a significant increase of [11C]vorozole binding by nandrolone decanoate in the bed nucleus of the stria terminalis and preoptic area, as in our previous study. Flutamide treatment, on the other hand, decreased [11C]vorozole binding in the bed nucleus of the stria terminalis, preoptic area, and medial amygdala. Immunohistochemical examination demonstrated that upregulation of aromatase expression occurred in neurons. Our findings suggest that aromatase is regulated through an androgen receptor-mediated system. This aromatase-specific tracer and the positron emission tomography technique could be useful for exploring the role of aromatase in anabolic androgenic steroids abusers.

Positron emission tomography and target-controlled infusion for precise modulation of brain drug concentration.

INTRODUCTION: There are several instances when it is desirable to control brain concentration of pharmaceuticals, e.g., to modulate the concentration of anesthetic agents to different desired levels fitting to different needs during the course of surgery. This has so far only been possible using indirect estimates of drug concentration such as assuming constant relation between tissue and blood including extrapolations from animals. METHODS: A system for controlling target tissue concentration (UIPump) was used to regulate whole-brain concentrations of a central benzodiazepine receptor antagonist at therapeutic levels with input from brain kinetics as determined with PET. The system was tested by using pharmacological doses of flumazenil mixed with tracer amounts of [11C]flumazenil. Flumazenil was used as a model compound for anesthesia. An infusion scheme to produce three different steady-state levels in sequence was designed based on kinetic curves obtained after bolus injection. The subjects (Sprague-Dawley rats, n=6) were monitored in a microPET scanner during the whole experiment to verify resulting brain kinetic curves. RESULTS: A steady-state brain concentration was rapidly achieved corresponding to a whole-brain concentration of 118+/-6 ng/ml. As the infusion rate decreased to lower the exposure by a factor of 2, the brain concentration decreased to 56+/-4 ng/ml. A third increased steady-state level of anesthesia corresponding to a whole-brain concentration of 107+/-7 ng/ml was rapidly achieved. CONCLUSION: The experimental setup with computerized pump infusion and PET supervision enables accurate setting of target tissue drug concentration.