Validation of an automated dispensing system for subsequent dose dispensing of different radionuclides.

BACKGROUND: Automated dispensing systems (ADSs) for radiopharmaceuticals have been developed to reduce the radiation exposure of personnel, to improve the accuracy of the dispensed dose and to limit the microbiological contamination. However, before implementing such systems, validation according to various applicable guidelines is necessary to ensure safety and quality. Here we present the selection, validation and implementation of the PT459R2 from manufacturer Lynax s.r.o. as a guidance protocol for validation according to GMP and GRPP guidelines. Validation included linearity accuracy and precision of the internal scintillation detector for different isotopes and microbiological validation for aseptic procedures. RESULTS: The ADS can dispense accurate doses in the following linear range: 1000-10,000 MBq for lutetium-177, 20-74 MBq for zirconium-89, 100-1000 MBq for gallium-68 and 100-2000 MBq for fluorine-18. The maximum bias is 2.35% and the maximum coefficient of variation is 3.03% which meets the acceptance criteria of < 5%. Furthermore, the ADS does not affects the GMP class A environment in a laminar airflow cabinet and can dispense aseptically. In addition, radiation exposure is acceptable and data integrity is preserved. CONCLUSION: The PT459R2 ADS met all the requirements from our performance qualification and is therefore suitable for daily routine use in our center. Our approach can be used as a guidance for PQ of an ADS in a Radiopharmacy according to GMP and GRPP guidelines.

The cycle effect quantified: reduced tumour uptake in subsequent cycles of [177Lu]Lu-HA-DOTATATE during peptide receptor radionuclide therapy.

BACKGROUND: Clear evidence regarding the effect of reduced tumour accumulation in later peptide receptor radionuclide therapy (PRRT) cycles is lacking. Therefore, we aimed to quantify potential cycle effects for patients treated with [177Lu]Lu-HA-DOTATATE using a population pharmacokinetic (PK) modelling approach. METHODS: A population PK model was developed using imaging data from 48 patients who received multiple cycles of [177Lu]Lu-HA-DOTATATE. The five-compartment model included a central, kidney, spleen, tumour and lumped rest compartment. Tumour volume and continued use of long-acting somatostatin analogues (SSAs) were tested as covariates in the model. In addition, the presence of a cycle effect was evaluated by relating the uptake rate in a specific cycle as a fraction of the (tumour or organ) uptake rate in the first cycle. RESULTS: The final PK model adequately captured observed radioactivity accumulation in kidney, spleen and tumour. A higher tumour volume was identified to increase the tumour uptake rate, where a twofold increase in tumour volume resulted in a 2.3-fold higher uptake rate. Also, continued use of long-acting SSAs significantly reduced the spleen uptake rate (68.4% uptake compared to SSA withdrawal (10.5% RSE)). Lastly, a cycle effect was significantly identified, where tumour uptake rate decreased to 86.9% (5.3% RSE) in the second cycle and even further to 79.7% (5.6% RSE) and 77.6% (6.2% RSE) in the third and fourth cycle, respectively, compared to cycle one. CONCLUSIONS: Using a population PK modelling approach, the cycle effect of reduced tumour uptake in subsequent PRRT cycles was quantified. Our findings implied that downregulation of target receptors is probably not the major cause of the cycle effect, due to a plateau in the decrease of tumour uptake in the fourth cycle.

A physiologically based pharmacokinetic (PBPK) model to describe organ distribution of 68Ga-DOTATATE in patients without neuroendocrine tumors.

BACKGROUND: Physiologically based pharmacokinetic (PBPK) models combine drug-specific information with prior knowledge on the physiology and biology at the organism level. Whole-body PBPK models contain an explicit representation of the organs and tissue and are a tool to predict pharmacokinetic behavior of drugs. The aim of this study was to develop a PBPK model to describe organ distribution of 68Ga-DOTATATE in a population of patients without detectable neuroendocrine tumors (NETs). METHODS: Clinical 68Ga-DOTATATE PET/CT data from 41 patients without any detectable somatostatin receptor (SSTR) overexpressing tumors were included. Scans were performed at 45 min (range 30-60 min) after intravenous bolus injection of 68Ga-DOTATATE. Organ (spleen, liver, thyroid) and blood activity levels were derived from PET scans, and corresponding DOTATATE concentrations were calculated. A whole-body PBPK model was developed, including an internalization reaction, receptor recycling, enzymatic reaction for intracellular degradation and renal clearance. SSTR2 expression was added for several organs. Input parameters were fixed or estimated using a built-in Monte Carlo algorithm for parameter identification. RESULTS: 68Ga-DOTATATE was administered with a median peptide amount of 12.3 µg (range 8.05-16.9 µg) labeled with 92.7 MBq (range 43.4-129.9 MBq). SSTR2 amounts for spleen, liver and thyroid were estimated at 4.40, 7.80 and 0.0108 nmol, respectively. Variability in observed organ concentrations was best described by variability in SSTR2 expression and differences in administered peptide amounts. CONCLUSIONS: To conclude, biodistribution of 68Ga-DOTATATE was described with a whole-body PBPK model, where tissue distribution was mainly determined by variability in SSTR2 organ expression and differences in administered peptide amounts.

Muscarinic inhibition of salivary glands with glycopyrronium bromide does not reduce the uptake of PSMA-ligands or radioiodine.

RATIONALE: Salivary glands are highly perfused and express the prostate-specific membrane antigen (PSMA) receptor as well as the sodium-iodide symporter. As a consequence, treatment with 177Lu/225Ac-PSMA for prostate cancer or 131I for thyroid cancer leads to a high radiation dose in the salivary glands, and patients can be confronted with persistent xerostomia and reduced quality of life. Salivation can be inhibited using an antimuscarinic pharmaceutical, such as glycopyrronium bromide (GPB), which may also reduce perfusion. The primary objective of this work was to determine if inhibition with GPB could provide a considerable (> 30%) reduction in the accumulation of administered 123I or 68Ga-PSMA-11 in salivary glands. METHODS: Ten patients who already received a whole-body 68Ga-PSMA-11 PET/CT scan for (re)staging of prostate cancer underwent a repeat PET/CT scan with tracer administration at 90 min after intravenous injection of 0.2 mg GPB. Four patients in follow-up after thyroid cancer, who had been treated with one round of ablative 131I therapy with curative intent and had no signs of recurrence, received 123I planar scintigraphy at 4 h after tracer administration without GPB and a repeated scan at least one week later, with tracer administration at 30 min after intramuscular injection of 0.4 mg GPB. Tracer uptake in the salivary glands was quantified on PET and scintigraphy, respectively, and values with and without GPB were compared. RESULTS: No significant difference in PSMA uptake in the salivary glands was seen without or with GPB (Mean SULmean parotid glands control 5.57, intervention 5.72, p = 0.50. Mean SULmean submandibular glands control 6.25, intervention 5.89, p = 0.12). Three out of 4 patients showed increased 123I uptake in the salivary glands after GPB (Mean counts per pixel control 8.60, intervention 11.46). CONCLUSION: Muscarinic inhibition of salivation with GPB did not significantly reduce the uptake of PSMA-ligands or radioiodine in salivary glands, and can be dismissed as a potential strategy to reduce toxicity from radionuclide therapies.

The development and validation of a high performance liquid chromatography method to determine the radiochemical purity of [177Lu]Lu-HA-DOTA-TATE in pharmaceutical preparations.

Lutetium-177 [177Lu] tetra-azacyclododecanetetra-acetic acid [DOTA]-(Tyr3)-octreotate [TATE] ([177Lu]Lu-DOTA-TATE) is a radiopeptide used for peptide receptor radionuclide therapy in patients with neuroendocrine tumours (NETs). This radiopeptide is made by labelling the ligand octreotate with Lutetium-177 using the linker DOTA. After labelling, and before clinical application quality control of the radiopeptide is needed and the radiochemical purity is assessed. Acceptance limits for radiochemical purity should be within 90-110% of the label claim for radiopharmaceuticals for diagnostic use and within 95-105% of the label claim for radiopharmaceuticals for therapeutic use. Moreover, the amount of unlabelled [177Lu]LuCl3 cannot exceed 2% of the radioactive dose. Since no monograph is available for [177Lu]Lu-DOTA-TATE in the European Pharmacopeia (Ph Eur), this article describes the development and validation of a high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection and radiodetection. A Waters Acquity Arc UHPLC system equipped with a Waters 2998 photodiode array (PDA) detector was used coupled to a Berthold Lb 514 Flowstar detector equipped with a BGO-X gamma measuring cell. A reversed phase Symmetry Shield C18 column (4.6 mm × 250 mm, 5 µm) was used for chromatographic separation. A flow of 1.5 mL/min was maintained during analysis, using 0.1% TFA in water as mobile phase A and 0.1% TFA in ACN as mobile phase B. The retention time was around 1.7 min and 13.5 min for [177Lu]LuCl3 and [177Lu]Lu-HA-DOTA-TATE, respectively. Stock solutions of [177Lu]LuCl3 were made by serial dilution and were injected to test for linearity, accuracy and precision, carry over and signal-to-noise ratio. A [177Lu]Lu-HA-DOTA-TATE sample was prepared and injected to determine the carry over. The results showed that the method is linear over a range of 0.300-130 MBq/mL, which covers the range for clinical samples, provided that the clinical sample is diluted ten times before analysis. The LLOQ can be measured accurately even after dilution, with a signal-to-noise ratio of at least 5. In short, the method is accurate, precise and sensitive and can be implemented as part of the quality control of [177Lu]Lu-HA-DOTA-TATE.

Quantification of taxanes in biological matrices: a review of bioanalytical assays and recommendations for development of new assays.

Since the isolation of paclitaxel and its approval for the treatment of breast cancer, various taxanes and taxane formulations have been developed. To date, almost 100 bioanalytical assays have been published with the method development and optimization often extensively discussed by the authors. This Review presents an overview of assays published between January 1970 and September 2013 that described method development and validation of assays used to quantify taxanes in biological matrices such as plasma, urine, feces and tissue samples. For liquid chromatography assays, sample pretreatment, chromatographic separation and assay performance are compared. Since this Review discusses the limitations of previously developed liquid chromatography assays and gives recommendations for future assay development, it can be used as a reference for future development of liquid chromatography assays for the quantification of taxanes in various biological matrices to support preclinical and clinical studies.

Oral co-administration of elacridar and ritonavir enhances plasma levels of oral paclitaxel and docetaxel without affecting relative brain accumulation.

BACKGROUND: The intestinal uptake of the taxanes paclitaxel and docetaxel is seriously hampered by drug efflux through P-glycoprotein (P-gp) and drug metabolism via cytochrome P450 (CYP) 3A. The resulting low oral bioavailability can be boosted by co-administration of P-gp or CYP3A4 inhibitors. METHODS: Paclitaxel or docetaxel (10 mg/kg) was administered to CYP3A4-humanised mice after administration of the P-gp inhibitor elacridar (25 mg kg(-1)) and the CYP3A inhibitor ritonavir (12.5 mg kg(-1)). Plasma and brain concentrations of the taxanes were measured. RESULTS: Oral co-administration of the taxanes with elacridar increased plasma concentrations of paclitaxel (10.7-fold, P<0.001) and docetaxel (four-fold, P<0.001). Co-administration with ritonavir resulted in 2.5-fold (paclitaxel, P<0.001) and 7.3-fold (docetaxel, P<0.001) increases in plasma concentrations. Co-administration with both inhibitors simultaneously resulted in further increased plasma concentrations of paclitaxel (31.9-fold, P<0.001) and docetaxel (37.4-fold, P<0.001). Although boosting of orally applied taxanes with elacridar and ritonavir potentially increases brain accumulation of taxanes, we found that only brain concentrations, but not brain-to-plasma ratios, were increased after co-administration with both inhibitors. CONCLUSIONS: The oral availability of taxanes can be enhanced by co-administration with oral elacridar and ritonavir, without increasing the brain penetration of the taxanes.

Quantification of docetaxel and its metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

RATIONALE: During drug development accurate quantification of metabolites in biological samples using mass spectrometry is often hampered by the lack of metabolites of chemically pure quality. However, quantification of metabolites can be useful for assessment and interpretation of (pre)clinical data. We now describe an approach to quantify docetaxel metabolites in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using docetaxel calibration standards. METHODS: Metabolites (M1/M3, M2 and M4) were generated using microsomal incubations. Retention times of docetaxel and its metabolites were assessed using an LC/UV assay and peak identification was performed by LC/MS(n). Samples containing isolated metabolites from human faeces were quantified by LC/UV and used as references for spiking human plasma samples. LC/MS/MS was applied to sensitively quantify docetaxel and its metabolites in human plasma using docetaxel calibration standards in a range of 0.25-500 ng/mL. RESULTS: Because ionisation of docetaxel and its metabolites differed, correction factors were established to quantify the metabolites using docetaxel calibration samples. During method validation, accuracy and precision of the metabolites were within ±7.7% and ≤17.6%, respectively, and within ±14.3% and ≤10.1%, respectively, for docetaxel. Metabolites were found to be unstable in human plasma at ambient temperature. After storage up to 1 year at -20 °C, recovered metabolite concentrations were within ±25%. CONCLUSIONS: Development and validation of an LC/MS/MS assay for the quantification of docetaxel and its metabolites M1/M3, M2 and M4 using docetaxel calibration standards is described. The same approach may be used for quantification of metabolites of other drugs by LC/MS/MS when chemically pure reference substances are unavailable.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount-BD and ISHAGE protocols.

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 +/- 19.4 CD34+ cells/microL and with the ProCount method we found 36.6 +/- 23.2 CD34+ cells/microL. With the ProCount method, CD34+ bright cell counts were 9.3 +/- 8.2 cells/microL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount™ - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount™ (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/æL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/æL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/æL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8 percent of the bright CD34+ cells are alive, whereas a small part (19.0 percent) is undergoing apoptosis and most of them (79.2 percent) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9 percent CD34+ cells, 2.6 ± 2.1 percent of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25 percent. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29 percent of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture.

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 +/- 0.9% CD34+ cells, 2.6 +/- 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 +/- 1.8-fold, but the number of viable CD34+ cells decreased by 46 +/- 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 +/- 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 +/- 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Alexithymia in Noonan syndrome.

Although Noonan syndrome is quite prevalent, there is a general paucity in the description of psychological and psychiatric aspects. In the present paper a 19-year-old female patient with Noonan syndrome is described who presented with anxiety symptoms. Mutation analysis in PTPN11, the NS1 gene on chromosome 12q24, showed no abnormalities. A diagnosis of panic disorder without agoraphobia was established. Treatment with citalopram resulted in a gradual decline of anxiety symptoms. The psychological profile was characterized by a prominent alexithymia. The main conclusion is that patients with Noonan syndrome might have deficits in emotional adaptative functions. It is hypothesized that alexithymia is a key feature of the behavioural phenotype.

Mice lacking a functional chk gene have no apparent defects in the hematopoietic system.

Non-receptor tyrosine kinase Chk has been implicated in hematopoietic development. To study the function of Chk in vivo, we have generated chk-deficient mice using gene targeting. Overall development of mice homozygous for this mutation was apparently normal. Blood counts, FACS analysis of hematopoietic cell populations, CFU-C and CAFC assays showed no significant difference between wild type and mutant animals. Thus, the dispensability of Chk for mouse development and hematopoiesis suggests that its function may be redundant in vivo, and most likely be compensated by activity of a closely related protein tyrosine kinase Csk.

Interleukin-3 treatment of rhesus monkeys leads to increased production of histamine-releasing cells that express interleukin-3 receptors at high levels.

To understand the hematopoietic and nonhematopoietic responses to interleukin-3 (IL-3), expression of cell-surface IL-3 receptors (IL-3R) was examined on bone marrow (BM) cells and peripheral blood (PB) cells of rhesus monkeys during the course of in vivo IL-3 treatment. Whereas IL-3R expression is low in untreated monkeys, IL-3 administration led to a gradual increase in both low- and high-affinity binding sites for IL-3. This increase reflected the total number of cells expressing IL-3Rs, as detected by flow cytometry using biotinylated IL-3. Most of these IL-3R+ cells in both BM and PB could be characterized as basophilic granulocytes that contained high levels of histamine. In contrast to the effect on these differentiated cells, IL-3 administration did not significantly alter the low level IL-3R expression on immature, CD34+ cells. Further flow cytometric analysis using biotinylated growth factors showed that the IL-3R+ basophils also expressed receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), but not for IL-6 or Kit ligand. These findings indicated that the IL-3R+ cells included neither monocytes, which express GM-CSFRs and IL-6Rs abundantly, nor mast cells, which express c-kit. By combining flow cytometric and Scatchard data, it was calculated that the basophils contain as many as 1 to 2 x 10(3) high-affinity IL-3Rs and 15 to 30 x 10(3) low-affinity sites. The finding that in vivo IL-3 treatment leads to the production of large numbers of cells that express high levels of IL-3R and are capable of producing histamine provides an explanation for the often severe allergic reactions that occur during prolonged IL-3 administration. It also indicates that IL-3, in addition to its direct effects on hematopoietic cells, may also stimulate hematopoiesis through the release of secondary mediators such as histamine by IL-3-responsive mature cells.

The use of local anesthesia in second generation extracorporeal shock wave lithotripsy: eutectic mixture of local anesthetics.

Extracorporeal shock wave lithotripsy (ESWL&) can be painful. Of our population of patients treated with a Siemens Lithostar device 51.4% needed intravenous analgesia. A eutectic mixture of local anesthetics, a hydrophylic cream containing 25 mg. lidocaine and 25 mg. prilocaine per gm., proved to be effective for local analgesia. Therefore, we investigated its effectiveness during ESWL. With randomized, double-blind application the eutectic mixture of local anesthetics and placebo were evaluated in 83 patients according to the percentage of patients who required intravenous analgesia during ESWL. Of 40 patients treated with the eutectic mixture of local anesthetics 12 (30%) needed supplementary fentanyl citrate, compared to 23 of 43 (53%) placebo treated patients. Although there is no statistical significance (p = 0.32), the eutectic mixture of local anesthetics does decrease pain during ESWL and it should be particularly useful for patients in whom intravenous analgesia is contraindicated.