Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable ...plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications. This study assessed 111In-DANBIRT as an in vivo, noninvasive SPECT/CT imaging probe targeting an inflammatory marker, Lymphocyte Function Associated Antigen-1 (LFA-1), in atherosclerotic plaques. Methods. Selective binding of 111In-DANBIRT was assessed using Sprague-Dawley rats exposed to filtered air and ozone (1 ppm) by inhalation for 4 hours to induce a circulating leukocytosis and neutrophilia in peripheral blood. After 24 hours, whole blood was collected and incubated with radiolabeled DANBIRT (68Ga-DANBIRT and 111In-DANBIRT). Isolated cell component smeared slides using cytospin technique were stained with Wright-Giemsa stain. Apolipoprotein E-deficient (apoE−/−) mice were fed either a normal diet or a high-fat diet (HFD) for 8 weeks. Longitudinal SPECT/CT imaging was performed 3 hours after administration at baseline, 4, and 8 weeks of HFD diet, followed by tissue harvesting for biodistribution, serum lipid analysis, and histology. 3D autoradiography was performed in both groups 24 hours after administration of 111In-DANBIRT. Results. Increased specific uptake of radiolabeled DANBIRT by neutrophils in the ozone-exposed group was evidenced by the acute immune response due to 4-hour ozone exposure. Molecular imaging performed at 3 hours using SPECT/CT imaging evidenced an exponential longitudinal increase in 111In-DANBIRT uptake in atherosclerosis lesions in HFD-fed mice compared to normal-diet-fed mice. Such results were consistent with increased immune response to vascular injury in cardiovascular and also immune tissues, correlated by 24 hours after administration of 3D autoradiography. Histologic analysis confirmed atherosclerotic disease progression with an increased vascular lesion area in HFD-fed mice compared to normal-diet-fed mice. Conclusion. 111In-DANBIRT is a promising molecular imaging probe to assess inflammation in evolving atheroma and atherosclerotic plaque.
The majority of preclinical studies investigating aberrant glial-neuroimmune actions underlying neuropathic pain have focused on male rodent models. Recently, studies have shown peripheral immune ...cells play a more prominent role than glial cells in mediating pathological pain in females. Here, we compared the onset and duration of allodynia in males and females, and the anti-allodynic action of a potentially novel therapeutic drug (BIRT377) that not only antagonizes the action of lymphocyte function-associated antigen-1 (LFA-1) to reduce cell migration in the periphery, but may also directly alter the cellular inflammatory bias.
Male and female mice were subjected to peripheral nerve injury chronic constriction injury (CCI) applying two methods, using either 4-0 or 5-0 chromic gut suture material, to examine potential sex differences in the onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined using flow cytometry.
Sex differences in proinflammatory CCL2 and IL-1β and the anti-inflammatory IL-10 were observed from a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1β and TNF, and increased IL-10 and transforming growth factor (TGF)-β1, also an anti-inflammatory cytokine, in both sexes. However, female-derived T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A production with concurrent increases in IL-10, TGF-β1 and the anti-inflammatory regulatory T cell-related factor, FOXP3.
This study supports that divergent peripheral immune and neuroimmune responses during neuropathy exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are predominantly specific to females. These data highlight the necessity of including both sexes for studying drug efficacy and mechanisms of action in preclinical studies and clinical trials.
Operation of a Radiopharmacy for a Clinical Trial Norenberg, Jeffrey P., MS, PharmD, BCNP, FASHP, FAPhA; Petry, Neil A., MS, BCNP, FAPhA; Schwarz, Sally, MS, BCNP, FAPhA
Seminars in nuclear medicine,
09/2010, Letnik:
40, Številka:
5
Journal Article
Recenzirano
Clinical investigations of radiopharmaceuticals are undertaken to advance promising compounds toward approval by the Food and Drug Administration (FDA) as “legend drugs.” This FDA approval requires ...that the safety and efficacy of the investigational drug (ID) be demonstrated through clinical trials. The investigational radiopharmaceutical drug service (IRDS) is a pharmacy service that plays a critical role in the acquisition, preparation, accountability, and distribution of radiopharmaceuticals used in clinical research. Due to their radioactive and other unique properties, and their potential role as biomarkers or tools in clinical trials of other therapeutic drugs, radiopharmaceutical drugs must be managed by a qualified IRDS rather than by a typical pharmacy-based investigational drug service (IDS). The IRDS is responsible for establishing study-specific procedures for appropriate radiopharmaceutical drug accountability, billing, procurement, storage, preparation, dispensing and destruction of investigational drugs within the hospital. All drugs, and particularly parenteral drug products, must be safe for administration to human subjects enrolled in clinical trials regardless of their FDA regulatory status as approved or investigational new drug products. The United States Pharmacopeia (USP) sterile compounding requirements provides enforceable minimum practice and quality standards for compounded sterile preparations of drug products based on current scientific information and best sterile compounding practices. Consequently, they apply equally to facilities dedicated to IDS and IRDS operations. The FDA also regulates drug manufacturing through current Good Manufacturing Practices (cGMP). This rule (21CFR Part 212) establishes cGMP regulations specific to positron emission tomography radiopharmaceuticals, separate from the regular drug cGMP rule (Parts 210 and 211). Compliance with regulatory, statutory, and sponsor requirements is a major consideration in the operation of a radiopharmacy for clinical trials. Sponsors conduct audits as part of the quality assurance of clinical trials. Audits of clinical trial sites by the sponsor, sponsor's clinical research organization, institutional review board, or FDA always include a detailed review of drug accountability records. Audits for radiopharmaceutical drug products typically begin by confirming the clinical site is appropriately licensed and authorized to receive, possess, store, handle, prepare and administer radiopharmaceuticals. All procedures for radiopharmaceutical drug accountability must comply with applicable federal regulations and the specific requirements specified by the study sponsor.
Background
213
Bismuth (
213
Bi, T
1/2
= 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide ...therapy. The use of generators containing less than 222 MBq
225
Ac (actinium), due to limited availability and the high cost to produce large-scale
225
Ac/
213
Bi generators, might complicate in vitro and in vivo applications though.
Here we present optimized labelling conditions of a DOTA-peptide with an
225
Ac/
213
Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr
3
-octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with
213
Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L
-1
to 0.34 mol.L
-1
, and ascorbic acid from 0 to 71 mmol.L
-1
in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948.
Results
At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq
213
Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L
-1
TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L
-1
ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.
Under optimized labelling conditions,
213
Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid concentration and cell survival.
Conclusion
213
Bismuth-DOTA-peptide labelling conditions including peptide amount, quencher and pH were optimized to meet the requirements needed for preclinical applications in peptide receptor radionuclide therapy.
During gravidity lizards experience a striking decrease in lung volume as a result of lung compression by eggs growing within the body cavity. In order to understand the effect of this decrease in ...lung volume on the respiratory biology of gravid egg-laying lizards, we measured changes in total lung volume, resting and postexercise expired volume, minute volume, respiratory frequency, and carbon dioxide production rate during reproduction in the Collared Lizard, Crotaphytus collaris, and the Leopard Lizard, Gambelia wislizenii. We found that compression of the lungs by shelled eggs resulted in an average 48% (range: 26â70%) decrease in total lung volume compared to the same postlaying C. collaris females, and an average 38% (range: 29â46%) decrease in G. wislizenii. COâ production rates were altered significantly during reproduction in female C. collaris and were 58% higher in females carrying late-stage follicles, compared to after laying. Despite the remarkable reduction in lung volume in both of these species and the increase in COâ production rates in C. collaris, no ventilation parameters changed over the course of reproduction. The highly distensible body cavities of C. collaris and G. wislizenii appear to be able to accommodate both growing eggs and adequate lung volumes for normal respiratory function during gravidity.
Background
Targeted alpha therapy (TAT) offers advantages over current β-emitting conjugates for peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. PRRT with
177
Lu-DOTATATE or
90
...Y-DOTATOC has shown dose-limiting nephrotoxicity due to radiopeptide retention in the proximal tubules. Pharmacological protection can reduce renal uptake of radiopeptides, e.g., positively charged amino acids, to saturate in the proximal tubules, thereby enabling higher radioactivity to be safely administered. The aim of this preclinical study was to evaluate the therapeutic effect of
213
Bi-DOTATATE with and without renal protection using L-lysine in mice. Tumor uptake and kinetics as a function of injected mass of peptide (range 0.03–3 nmol) were investigated using
111
In-DOTATATE. These results allowed estimation of the mean radiation absorbed tumor dose for
213
Bi-DOTATATE. Pharmacokinetics and dosimetry of
213
Bi-DOTATATE was determined in mice, in combination with renal protection. A dose escalation study with
213
Bi-DOTATATE was performed to determine the maximum tolerated dose (MTD) with and without pre-administration of
l
-lysine as for renal protection. Neutrophil gelatinase-associated lipocalin (NGAL) served as renal biomarker to determine kidney injury.
Results
The maximum mean radiation absorbed tumor dose occurred at 0.03 nmol and the minimum at 3 nmol. Similar mean radiation absorbed tumor doses were determined for 0.1 and 0.3 nmol with a mean radiation absorbed dose of approximately 0.5 Gy/MBq
213
Bi-DOTATATE. The optimal mass of injected peptide was found to be 0.3 nmol. Tumor uptake was similar for
111
In-DOTATATE and
213
Bi-DOTATATE at 0.3 nmol peptide. Lysine reduced the renal uptake of
213
Bi-DOTATATE by 50% with no effect on the tumor uptake. The MTD was <13.0 ± 1.6 MBq in absence of
l
-lysine and 21.7 ± 1.9 MBq with
l
-lysine renal protection, both imparting an LD
50
mean renal radiation absorbed dose of 20 Gy. A correlation was found between the amount of injected radioactivity and NGAL levels.
Conclusions
The therapeutic potential of
213
Bi-DOTATATE was illustrated by significantly decreased tumor burden and improved overall survival. Renal protection with
l
-lysine immediately prior to TAT with
213
Bi-DOTATATE prolonged survival providing substantial evidence for pharmacological nephron blockade to mitigate nephrotoxicity.
Clinical studies of patients treated with somatostatin-receptor (sstr)-targeted DOTA(0)-Tyr(3)-octreotide (DOTATOC) labeled with (177)Lu and (90)Y have shown overall response rates in the range of ...9-33%. This study evaluates the potential for combination therapy with gemcitabine in an effort to improve clinical outcomes.
Human pancreatic adenocarcinoma Capan-2, rat pancreatic cancer AR42J and human small cell lung cancer NCI-H69 cells were each treated with 1 microg/ml gemcitabine for 4 days followed by replacement of the medium alone for four additional days. Cell cycle and direct receptor-uptake studies were performed with (177)Lu-DOTATOC after the total 8-day treatment as described. Cell viability and apoptosis experiments were performed to study the effects of gemcitabine pretreatment and (177)Lu-DOTATOC radionuclide therapy. Parallel control studies were performed with receptor-non-targeted (177)Lu-DOTA and DOTATOC.
Cells treated with gemcitabine for 4 days showed a down-regulation of sstr expression as determined by (177)Lu-DOTATOC uptake. However, after 4 days of additional growth in absence of gemcitabine, the uptake of (177)Lu-DOTATOC was 1.5-3 times greater than that of the untreated control cells. In gemcitabine-pretreated Capan-2 cells, 84% of the cell population was in the G(2)M phase of the cell cycle. Due to sstr up-regulation and cell cycle modulations, synergistic effects of gemcitabine pretreatment were observed in cell viability and apoptosis assays. (177)Lu-DOTATOC resulted in two to three times greater apoptosis in gemcitabine-pretreated Capan-2 cells compared to the untreated cells.
Gemcitabine pretreatment up-regulates sstr expression and acts as a radiosensitizer through cell cycle modulation. The rational combination of gemcitabine and sstr-targeted radiopharmaceuticals represents a promising chemoradiation therapeutic tool with great potential to improve clinical outcomes and, thus, merits further study.
Molecular imaging is the visualization, characterization, and measurement of biologic processes at the molecular and cellular levels in humans and other living systems (1). It comprises an emerging ...set of technologies that builds on advances in imaging procedures (e.g., PET, SPECT, MRI, ultrasound, optical, and photoacoustic), improved understanding of biology, and the development of molecularly targeted agents. These continuously expanding sets of imaging methods are often used in combination, and advances in data acquisition and analyses facilitate a more complete understanding of biology. Molecular imaging aims to improve our understanding of mammalian biology and lead to advances in patient care by providing targeted therapies that will enable personalized medicine and the imaging tools to assess outcome. Implementation of these new technologies in clinical care has many educational, technical, and regulatory challenges that must be overcome before molecular imaging reaches its full potential. The impact of molecular imaging has been significant in several disciplines, because it represents a paradigm shift in how scientists and clinicians can observe biology in real time and in a relatively noninvasive manner to enable the power of repeated measures in living organisms.
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