Comprehensive Quality Control of the ITG 68Ge/68Ga Generator and Synthesis of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for Clinical Imaging.

UNLABELLED: A good-manufacturing-practices (GMP) (68)Ge/(68)Ga generator that uses modified dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a octadecyl silica resin (C-18) as an adsorbent has been developed that allows for dilute HCl (0.05N) to efficiently elute metal-impurity-free (68)Ga(3+) ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of (68)Ga-labeled DOTATOC and Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (PSMA-HBED-CC) intended for clinical studies and established protocols for batch release. METHODS: A 2,040-MBq self-shielded (68)Ge/(68)Ga generator provided metal-free (68)GaCl3 ready for peptide labeling in the fluidic labeling module after elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. (68)Ga labeling of peptides using GMP kits was performed in 15-20 min, and the total production time was 45-50 min. Batch release quality control specifications were established to meet investigational new drug submission and institutional review board approval standards. RESULTS: Over a period of 12 mo, (68)Ga elution yields from the generator averaged 80% (range, 72.0%-95.1%), and (68)Ge breakthrough was less than 0.006%, initially decreasing with time to 0.001% (expressed as percentage of (68)Ge activity present in the generator at the time of elution), a unique characteristic of this generator. The radiochemical purity of both (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC determined by high-performance liquid chromatography analysis was greater than 98%, with a minimum specific activity of 12.6 and 42 GBq/µmol, respectively. The radionuclidic ((68)Ge) impurity was 0.00001% or less (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiologic saline with 5%-7% ethanol. CONCLUSION: The GMP-certified (68)Ge/(68)Ga generator system was studied for a year. The generator system is contained within the fluidic labeling module, and it is compact, self-shielded, and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The (68)Ge levels in the final drug products were under the detection limits at all times. (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under investigational new drug (IND) applications submitted to the U.S. Food and Drug Administration.

Unboosted atazanavir plus co-formulated lamivudine/abacavir as a ritonavir-sparing simplification strategy in routine clinical practice.

OBJECTIVE: To assess the effectiveness and safety of antiretroviral therapy with unboosted atazanavir (400 mg once daily) plus co-formulated abacavir/lamivudine as a treatment simplification strategy in HIV-infected patients with sustained viral suppression in routine clinical practice. METHODS: We performed a retrospective study including patients who were switched to unboosted atazanavir plus abacavir/lamivudine and whose HIV-1 RNA was <50 copies/mL. The primary endpoint was the percentage of subjects who maintained viral suppression after 48 weeks of follow-up. Secondary endpoints included the percentage of subjects who maintained viral suppression after 96 weeks of follow-up, the incidence of adverse events, changes in CD4+ T-cell count and in lipid profile, and the percentage of patients with subtherapeutic atazanavir trough concentrations during follow-up. RESULTS: Forty-six patients were included. None had a prior history of resistance to protease inhibitors or to lamivudine or abacavir. The percentage of patients with viral suppression at Week 48 was 73.9% when all the included patients were considered (full dataset analysis) and 85.0% when only subjects on treatment were considered. There was a continuous immune recovery and an improvement in lipid profile during follow-up. Two thirds of the patients had subtherapeutic atazanavir trough concentrations in plasma in at least one determination during follow-up. CONCLUSION: Antiretroviral therapy with unboosted atazanavir plus abacavir/lamivudine is safe and effective in the long term as a treatment simplification strategy in HIV-infected patients with sustained virological suppression in routine clinical practice.

Establishment of reference values for endocrine tests. Part VII: growth hormone deficiency.

BACKGROUND: Plasma insulin-like growth factor (IGF-I) concentration can be used as a rough indicator of the growth-hormone status. However, for the diagnosis of growth hormone deficiency, dynamic tests are required. The growth hormone (GH) response in the insulin tolerance test (ITT) is considered to be the gold standard in this respect. An alternative for the ITT is the GHRH/ GHRP-6 test, which has fewer side effects. In this study we established reference values for IGF-I levels and for the GH response in both dynamic tests. METHODS: We studied 296 subjects recruited from the general population, equally distributed according to sex and aged between 20 and 70 years. Serum IGF-I level was measured in all subjects and an insulin tolerance test (0.15 U/kg Actrapid iv) and GHRH/GHRP-6 test (1 microg GHRH/kg and 1 microg GHRP-6/kg) were performed in 49 subjects. RESULTS: In multivariate analyses both IGF-I and the GH response in the ITT were significantly influenced by age, whereas the GH response in the GHRH/GHRP-6 test was significantly affected by BMI. There was no sex difference in IGF-I and in the GHRH/GHRP-6 test, but in the ITT males had a higher GH peak. There was a significant correlation between the GH responses in both tests, and the GH response was significantly higher in the GHRH/GHRP-6 test than in the ITT. Age-adjusted reference values were established for each test. CONCLUSION: We have established age-adjusted reference values for serum IGF-I and for the GH response in the ITT and GHRH/GHRP-6 test.

Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27-peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60-80%, depending on the application) on the separation of both mixtures of peptides. The greater the charge on the peptides, the better the separation achievable by HILIC/CEX. In addition, HILIC/CEX separation of both the peptide mixtures used in the present study was shown to be superior to that of the more commonly applied RP-HPLC mode. Our results highlight again the efficacy of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC.

Integration of atazanavir into an existing liquid chromatography UV method for protease inhibitors: validation and application.

Atazanavir (ATV) is a widely used human immunodeficiency virus (HIV)-1 protease inhibitor (PI) that, like other approved PIs, has been considered as a candidate for therapeutic drug monitoring (TDM). To provide ATV assay results that can be applied to patient management through TDM, the assay would need to perform in a manner consistent with Clinical Laboratory Improvement Amendments (CLIA) standards. To quantitate ATV concentrations in human plasma, the authors added ATV to a previously published reversed-phase high-performance liquid chromatography (HPLC) method from their laboratory. Detection was effected with use of a photodiode-array detector (PDA) collecting spectra at 248 nm. This method allows for detection of ATV to a lower limit of quantitation of 0.05 microg/mL, with an intra-assay coefficient of variation (CV%) of 8.9% or less over 5 days of testing and an interassay CV% ranging from 1.4 to 6.4%. The assay has met passing requirements for interlaboratory proficiency testing for 2 years nationally and internationally, with accuracy within +/-15% over all test samples. During 2 years, more than 100 batches of analyses have been performed and have proved the method is rugged, specific, and accurate. This assay method is currently used in the authors' clinical research program in TDM.

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain.

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.

Batch-type chemiluminescence detection cell for sensitization and simplification of capillary electrophoresis.

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10(-10)-6.5 x 10(-7) M (correlation coefficient, 0.999), with a detection limit (S/N = 3) of 2.5 x 10(-10) M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylgycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to postcolumn detection in capillary electrophoresis. When acetonitrile stacking was used under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about 1 order of magnitude higher than those obtained without stacking. The detection limit for luminol was 1.5 x 10(-11) M (S/N = 3), representing the highest sensitivity of luminol yet reported. Finally, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was approximately 2 orders of magnitude higher than that in the unenhanced reaction. A preliminary immunoassay using horseradish peroxidase-labeled anti-mouse IgG was also developed.

Synthesis of paucidisperse poly(gamma-benzyl-alpha,L-glutamate) oligomers and star polymers with rigid arms.

The synthesis of highly uniform gamma-benzyl-alpha,L-glutamate (BLG) oligomers via a convergent solution phase approach is reported. BLG oligomers were produced with designed lengths of 4, 8, 12, and 16 as a first step to production of BLG-4-mer and BLG-8-mer rod stars. The star oligomers were purified by size-exclusion chromatography and reversed phase HPLC, and characterized by MALDI-TOF mass spectrometry and reversed phase HPLC. These star-shaped BLG oligomers could be used as initiators for growing larger stars.

Levels of biogenic amines and peptides in individual corn earworm moths, Helicoverpa zea, using high performance liquid chromatography with electrochemical detection.

Quantitative levels of octopamine, serotonin, and dopamine were measured from brain and corpus cardiacum in individual male and female Helicoverpa zea, using high performance liquid chromatography (HPLC) with electrochemical detection. By increasing the proportion of organic modifiers in the mobile phase we were also able to quantify two peptides, adipokinetic hormone and hypertrehalosemic hormone. Levels of amines in both tissues were similar in males and females, but with greater quantities in the brain than the corpus cardiacum. In contrast, the two hormones were found predominantly in the corpus cardiacum, with the level of adipokinetic hormone three times greater than that of the hypertrehalosemic hormone, a finding in agreement with other studies. The described methodology demonstrates the power of HPLC with electrochemical detection for analysis of amines and peptides found in small quantities within the nervous tissue of individual insects.

An investigation of factors that might influence the radiochemical purity and stability of 99mTc-MAG3.

Technetium 99m mercaptoacetyltriglycine (99mTc-MAG3) was prepared from a commercial kit by various techniques to assess the effect of a number of variables on radiochemical purity and stability. Its radiochemical purity was determined by high performance liquid chromatography at 0 and 6 h after preparation and was found to be consistently greater than 95%. It has been demonstrated that the radiochemical purity of 99mTc-MAG3 prepared according to the manufacturer's instructions is not influenced by the volume of 99mTc generator eluate used, agitation, the presence of air in the reaction vial or the use of a 99mTc generator eluate with a 99Tc:99mTc ratio of 16:1. A modified method of preparation in which the MAG3 kit is reconstituted with saline before addition of 99mTc-pertechnetate has been shown to yield a satisfactory product and should help to minimise the radiation dose to the fingers of radiopharmacy staff.