Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

The Production, Quality Control, and Characterization of ZED8, a CD8-Specific 89Zr-Labeled Immuno-PET Clinical Imaging Agent.

Immuno-PET is a molecular imaging technique utilizing positron emission tomography (PET) to measure the biodistribution of an antibody species labeled with a radioactive isotope. When applied as a clinical imaging technique, an immuno-PET imaging agent must be manufactured with quality standards appropriate for regulatory approval. This paper describes methods relevant to the chemistry, manufacturing, and controls component of an immuno-PET regulatory filing, such as an investigational new drug application. Namely, the production, quality control, and characterization of the immuno-PET clinical imaging agent, ZED8, an 89Zr-labeled CD8-specific monovalent antibody as well as its desferrioxamine-conjugated precursor, CED8, is described and evaluated. PET imaging data in a human CD8-expressing tumor murine model is presented as a proof of concept that the imaging agent exhibits target specificity and comparable biodistribution across a range of desferrioxamine conjugate loads.

Optimization of 89Zr PET Imaging for Improved Multisite Quantification and Lesion Detection Using an Anthropomorphic Phantom.

Our objective was to harmonize multicenter 89Zr PET imaging for oncology trials and to evaluate lesion detection. Methods: Seven PET scanners were evaluated using a custom chest oncology phantom with 9 spheric lesions 7-20 mm in diameter. A 4:1 signal-to-background ratio simulated a patient dose of 92.5 MBq. Various image reconstructions were evaluated. Images were assessed for lesion detection, and recovery coefficients and background signal variance were measured. Results: Two scanners failed to provide acceptable images and data. Optimal reconstruction algorithms enabling adequate lesion detection and reliable quantification across the other 5 scanners were determined without compromising the data quality. On average, 95% of the 10-mm lesions were detected, and the 7-mm lesion was visualized by only 1 scanner. Background variance was 8.6%-16%. Conclusion: We established multicenter harmonization procedures for 89Zr PET imaging in oncology, optimizing small-lesion (≥10 mm) detectability and accurate quantification.

[18F]GTP1 (Genentech Tau Probe 1), a radioligand for detecting neurofibrillary tangle tau pathology in Alzheimer's disease.

OBJECTIVE: Neurofibrillary tangles (NFTs), consisting of intracellular aggregates of the tau protein, are a pathological hallmark of Alzheimer's disease (AD). Here we report the identification and initial characterization of Genentech Tau Probe 1 ([18F]GTP1), a small-molecule PET probe for imaging tau pathology in AD patients. METHODS: Autoradiography using human brain tissues from AD donors and protein binding panels were used to determine [18F]GTP1 binding characteristics. Stability was evaluated in vitro and in vivo in mice and rhesus monkey. In the clinic, whole-body imaging was performed to assess biodistribution and dosimetry. Dynamic [18F]GTP1 brain imaging and input function measurement were performed on two separate days in 5 ß-amyloid plaque positive (Aß+) AD and 5 ß-amyloid plaque negative (Aß-) cognitive normal (CN) participants. Tracer kinetic modeling was applied and reproducibility was evaluated. SUVR was calculated and compared to [18F]GTP1-specific binding parameters derived from the kinetic modeling. [18F]GTP1 performance in a larger cross-sectional group of 60 Aß+ AD participants and ten (Aß- or Aß+) CN was evaluated with images acquired 60 to 90 min post tracer administration. RESULTS: [18F]GTP1 exhibited high affinity and selectivity for tau pathology with no measurable binding to ß-amyloid plaques or MAO-B in AD tissues, or binding to other tested proteins at an affinity predicted to impede image data interpretation. In human, [18F]GTP1 exhibited favorable dosimetry and brain kinetics, and no evidence of defluorination. [18F]GTP1-specific binding was observed in cortical regions of the brain predicted to contain tau pathology in AD and exhibited low (< 4%) test-retest variability. SUVR measured in the 60 to 90-min interval post injection correlated with tracer-specific binding (slope = 1.36, r2 = 0.98). Furthermore, in a cross-sectional population, the degree of [18F]GTP1-specific binding increased with AD severity and could differentiate diagnostic cohorts. CONCLUSIONS: [18F]GTP1 is a promising PET probe for the study of tau pathology in AD.

89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer.

Programmed cell death protein-1/ligand-1 (PD-1/PD-L1) blockade is effective in a subset of patients with several tumor types, but predicting patient benefit using approved diagnostics is inexact, as some patients with PD-L1-negative tumors also show clinical benefit1,2. Moreover, all biopsy-based tests are subject to the errors and limitations of invasive tissue collection3-11. Preclinical studies of positron-emission tomography (PET) imaging with antibodies to PD-L1 suggested that this imaging method might be an approach to selecting patients12,13. Such a technique, however, requires substantial clinical development and validation. Here we present the initial results from a first-in-human study to assess the feasibility of imaging with zirconium-89-labeled atezolizumab (anti-PD-L1), including biodistribution, and secondly test its potential to predict response to PD-L1 blockade (ClinicalTrials.gov identifiers NCT02453984 and NCT02478099). We imaged 22 patients across three tumor types before the start of atezolizumab therapy. The PET signal, a function of tracer exposure and target expression, was high in lymphoid tissues and at sites of inflammation. In tumors, uptake was generally high but heterogeneous, varying within and among lesions, patients, and tumor types. Intriguingly, clinical responses in our patients were better correlated with pretreatment PET signal than with immunohistochemistry- or RNA-sequencing-based predictive biomarkers, encouraging further development of molecular PET imaging for assessment of PD-L1 status and clinical response prediction.

Preparation and evaluation of L- and D-5-[18F]fluorotryptophan as PET imaging probes for indoleamine and tryptophan 2,3-dioxygenases.

Indoleamine and tryptophan 2,3-dioxygenases (IDO1 and TDO2) are pyrrolases catalyzing the oxidative cleavage of the 2,3-double bond of L-tryptophan in kynurenine pathway. In the tumor microenvironment, their increased activity prevents normal immune function, i.e. tumor cell recognition and elimination by cytotoxic T-cells. Consequently, inhibition of the kynurenine pathway may enhance the activity of cancer immunotherapeutics by reversing immune dysfunction. We sought to investigate the properties of radiolabeled 5-[18F]fluorotryptophan with respect to its ability for measuring IDO1 and TDO2 activity by positron emission tomography (PET). RESULTS: L-5-[18F]fluorotryptophan and D-5-[18F]fluorotryptophan were synthesized by Cu(I) catalyzed [18F]fluorodeboronylation of Boc/tBu protected precursors in moderate yields (1.5±0.6%) sufficient for pre-clinical studies. The specific activity of the product was 407-740GBq/µmol, radiochemical purity >99% and enantiomeric excess 90-99%. Enzymatic assay confirmed that L-5-fluorotryptophan is an IDO1 and TDO2 substrate whereas the D-isomer is not. In-vitro cell uptake experiments using CT26 cells with doxycycline-induced overexpression of human-IDO1 and human-TDO2 revealed an elevated cell uptake of L-5-[18F]fluorotryptophan upon induction of IDO1 or TDO2 enzymes compared to baseline; however, the uptake was observed only in the presence of low L-tryptophan levels in media. PET imaging experiments performed using tumor bearing mouse models expressing IDO1 at various levels (CT26, CT26-hIDO1, 17082A, 17095A) showed tumor uptake of the tracer elevated up to 8%ID/g; however, the observed tumor uptake could not be attributed to IDO1 activity in the tumor tissue. The metabolism of L- and D- isomers was markedly different in vivo, the D-isomer was excreted by a combination of hepatobiliary and renal routes, the L-isomer underwent extensive metabolism to [18F]fluoride. CONCLUSION: The observed in vivo tumor uptake of the tracer could not be attributed to IDO1 or TDO2 enzyme activity in the tumor, presumably due to competition with endogenous tryptophan as well as rapid tracer metabolism.

Preclinical Efficacy of an Antibody-Drug Conjugate Targeting Mesothelin Correlates with Quantitative 89Zr-ImmunoPET.

Antibody-drug conjugates (ADC) use monoclonal antibodies (mAb) as vehicles to deliver potent cytotoxic drugs selectively to tumor cells expressing the target. Molecular imaging with zirconium-89 (89Zr)-labeled mAbs recapitulates similar targeting biology and might help predict the efficacy of these ADCs. An anti-mesothelin antibody (AMA, MMOT0530A) was used to make comparisons between its efficacy as an ADC and its tumor uptake as measured by 89Zr immunoPET imaging. Mesothelin-targeted tumor growth inhibition by monomethyl auristatin E (MMAE), ADC AMA-MMAE (DMOT4039A), was measured in mice bearing xenografts of ovarian cancer OVCAR-3×2.1, pancreatic cancers Capan-2, HPAC, AsPC-1, and HPAF-II, or mesothelioma MSTO-211H. Ex vivo analysis of mesothelin expression was performed using immunohistochemistry. AMA-MMAE showed the greatest growth inhibition in OVCAR-3×2.1, Capan-2, and HPAC tumors, which showed target-specific tumor uptake of 89Zr-AMA. The less responsive xenografts (AsPC-1, HPAF-II, and MSTO-211H) did not show 89Zr-AMA uptake despite confirmed mesothelin expression. ImmunoPET can demonstrate the necessary delivery, binding, and internalization of an ADC antibody in vivo and this correlates with the efficacy of mesothelin-targeted ADC in tumors vulnerable to the cytotoxic drug delivered. Mol Cancer Ther; 16(1); 134-42. ©2016 AACR.

ImmunoPET helps predicting the efficacy of antibody-drug conjugates targeting TENB2 and STEAP1.

The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo. 89Zr-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with 111In-mAbs and gamma counting or with 89Zr-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that 89Zr-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload.

ImmunoPET imaging of phosphatidylserine in pro-apoptotic therapy treated tumor models.

UNLABELLED: An immunoPET imaging probe for the detection of phosphatidylserine was developed and tested in animal models of human cancer treated with pro-apoptotic therapy. We hypothesized that the relatively long plasma half-life of a probe based on a full-length antibody coupled with a residualizing radionuclide would be able to catch the wave of drug-induced apoptosis and lead to a specific accumulation in apoptotic tumor tissue. METHODS: The imaging probe is based on a 89Zr-labeled monoclonal antibody PGN635 targeting phosphatidylserine. The probe was evaluated pre-clinically in four tumor xenograft models: one studied treatment with paclitaxel to trigger the intrinsic apoptotic pathway, and three others interrogated treatment with an agonistic death-receptor monoclonal antibody to engage the extrinsic apoptotic pathway. RESULTS: High accumulation of 89Zr-PGN635 was observed in treated tumors undergoing apoptosis reaching 30 %ID/g and tumor-to-blood ratios up to 13. The tumor uptake in control groups treated with vehicle or imaged with a non-binding antibody probe was significantly lower. CONCLUSIONS: The results demonstrate the ability of 89Zr-PGN635 to image drug-induced apoptosis in animal models and corroborate our hypothesis that radiolabeled antibodies binding to intracellular targets transiently exposed on the cell surface during apoptosis can be employed for detection of tumor response to therapy.

The power of FDG-PET to detect treatment effects is increased by glucose correction using a Michaelis constant.

BACKGROUND: We recently showed improved between-subject variability in our [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) experiments using a Michaelis-Menten transport model to calculate the metabolic tumor glucose uptake rate extrapolated to the hypothetical condition of glucose saturation: MRglucmax=Ki*(KM+[glc]), where Ki is the image-derived FDG uptake rate constant, KM is the half-saturation Michaelis constant, and [glc] is the blood glucose concentration. Compared to measurements of Ki alone, or calculations of the scan-time metabolic glucose uptake rate (MRgluc = Ki * [glc]) or the glucose-normalized uptake rate (MRgluc = Ki*[glc]/(100 mg/dL), we suggested that MRglucmax could offer increased statistical power in treatment studies; here, we confirm this in theory and practice. METHODS: We compared Ki, MRgluc (both with and without glucose normalization), and MRglucmax as FDG-PET measures of treatment-induced changes in tumor glucose uptake independent of any systemic changes in blood glucose caused either by natural variation or by side effects of drug action. Data from three xenograft models with independent evidence of altered tumor cell glucose uptake were studied and generalized with statistical simulations and mathematical derivations. To obtain representative simulation parameters, we studied the distributions of Ki from FDG-PET scans and blood [glucose] values in 66 cohorts of mice (665 individual mice). Treatment effects were simulated by varying MRglucmax and back-calculating the mean Ki under the Michaelis-Menten model with KM = 130 mg/dL. This was repeated to represent cases of low, average, and high variability in Ki (at a given glucose level) observed among the 66 PET cohorts. RESULTS: There was excellent agreement between derivations, simulations, and experiments. Even modestly different (20%) blood glucose levels caused Ki and especially MRgluc to become unreliable through false positive results while MRglucmax remained unbiased. The greatest benefit occurred when Ki measurements (at a given glucose level) had low variability. Even when the power benefit was negligible, the use of MRglucmax carried no statistical penalty. Congruent with theory and simulations, MRglucmax showed in our experiments an average 21% statistical power improvement with respect to MRgluc and 10% with respect to Ki (approximately 20% savings in sample size). The results were robust in the face of imprecise blood glucose measurements and KM values. CONCLUSIONS: When evaluating the direct effects of treatment on tumor tissue with FDG-PET, employing a Michaelis-Menten glucose correction factor gives the most statistically powerful results. The well-known alternative 'correction', multiplying Ki by blood glucose (or normalized blood glucose), appears to be counter-productive in this setting and should be avoided.

FDG-PET is a good biomarker of both early response and acquired resistance in BRAFV600 mutant melanomas treated with vemurafenib and the MEK inhibitor GDC-0973.

BACKGROUND: The BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes. METHODS: 18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken. RESULTS: We demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein. CONCLUSIONS: We have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT01271803.

Tissue distribution studies of protein therapeutics using molecular probes: molecular imaging.

Molecular imaging techniques for protein therapeutics rely on reporter labels, especially radionuclides or sometimes near-infrared fluorescent moieties, which must be introduced with minimal perturbation of the protein's function in vivo and are detected non-invasively during whole-body imaging. PET is the most sensitive whole-body imaging technique available, making it possible to perform biodistribution studies in humans with as little as 1 mg of injected antibody carrying 1 mCi (37 MBq) of zirconium-89 radiolabel. Different labeling chemistries facilitate a variety of optical and radionuclide methods that offer complementary information from microscopy and autoradiography and offer some trade-offs in whole-body imaging between cost and logistic difficulty and image quality and sensitivity (how much protein needs to be injected). Interpretation of tissue uptake requires consideration of label that has been catabolized and possibly residualized. Image contrast depends as much on background signal as it does on tissue uptake, and so the choice of injected dose and scan timing guides the selection of a suitable label and helps to optimize image quality. Although only recently developed, zirconium-89 PET techniques allow for the most quantitative tomographic imaging at millimeter resolution in small animals and they translate very well into clinical use as exemplified by studies of radiolabeled antibodies, including trastuzumab in breast cancer patients, in The Netherlands.

Quantitation of glucose uptake in tumors by dynamic FDG-PET has less glucose bias and lower variability when adjusted for partial saturation of glucose transport.

BACKGROUND: A retrospective analysis of estimates of tumor glucose uptake from 1,192 dynamic 2-deoxy-2-(18F)fluoro-D-glucose-positron-emission tomography [FDG-PET] scans showed strong correlations between blood glucose and both the uptake rate constant [Ki] and the metabolic rate of glucose [MRGluc], hindering the interpretation of PET scans acquired under conditions of altered blood glucose. We sought a method to reduce this glucose bias without increasing the between-subject or test-retest variability and did this by considering that tissue glucose transport is a saturable yet unsaturated process best described as a nonlinear function of glucose levels. METHODS: Patlak-Gjedde analysis was used to compute Ki from 30-min dynamic PET scans in tumor-bearing mice. MRGluc was calculated by factoring in the blood glucose level and a lumped constant equal to unity. Alternatively, we assumed that glucose consumption is saturable according to Michaelis-Menten kinetics and estimated a hypothetical maximum rate of glucose consumption [MRGlucMAX] by multiplying Ki and (KM + [glucose]), where KM is a half-saturation Michaelis constant for glucose uptake. Results were computed for 112 separate studies of 8 to 12 scans each; test-retest statistics were measured in a suitable subset of 201 mice. RESULTS: A KM value of 130 mg/dL was determined from the data based on minimizing the average correlation between blood glucose and the uptake metric. Using MRGlucMAX resulted in the following benefits compared to using MRGluc: (1) the median correlation with blood glucose was practically zero, and yet (2) the test-retest coefficient of variation [COV] was reduced by 13.4%, and (3) the between-animal COVs were reduced by15.5%. In statistically equivalent terms, achieving the same reduction in between-animal COV while using the traditional MRGluc would require a 40% increase in sample size. CONCLUSIONS: MRGluc appeared to overcorrect tumor FDG data for changing glucose levels. Applying partial saturation correction using MRGlucMAX offered reduced bias, reduced variability, and potentially increased statistical power. We recommend further investigation of MRGlucMAX in quantitative studies of tumor FDG uptake.

New imaging paradigms in drug development: the PET imaging approach.

Molecular imaging is becoming an indispensable part of clinical drug development. The presented review highlights few state-of-the-art examples that serve to illustrate specific points and discuss future directions of the use of positron emission tomography (PET) imaging in various phases of clinical drug development.:

The effects of anesthetic agent and carrier gas on blood glucose and tissue uptake in mice undergoing dynamic FDG-PET imaging: sevoflurane and isoflurane compared in air and in oxygen.

PURPOSE: We sought to identify an anesthetic regime that, unlike isoflurane in air, would maintain glucose homeostasis in mice undergoing Positron emission tomography (PET) imaging with 2-deoxy-2-[18F]fluoro-D: -glucose (FDG). MATERIALS AND METHODS: FDG uptake was also measured in normal and tumor tissues. Athymic and Balb/c nude mice were studied. Blood glucose levels were measured before and after 30 min of FDG PET imaging under isoflurane or sevoflurane carried in air or oxygen. FDG uptake was quantified as a percentage of the injected dose and using Patlak analysis yielding Ki values. RESULTS: Blood glucose levels were more stable under sevoflurane than under isoflurane, especially in the athymic nude mice. Under isoflurane, FDG uptake into myocardium was higher than under sevoflurane and was strongly correlated with the intrascan change in blood glucose. CONCLUSION: Sevoflurane should be preferred for physiologic imaging in mice, minimizing changes in glucose and, for FDG PET, reducing signal spillover from the myocardium.

A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging.

A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.