Ernest K. J. Pauwels

Large scale production of recombinant human lactoferrin in the milk of transgenic cows

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.

Technetium-99m labelled antimicrobial peptides discriminate between bacterial infections and sterile inflammations.

Abstract. The aim of this study was to select technetium-99m labelled peptides that can discriminate between bacterial infections and sterile inflammations. For this purpose, we first assessed the binding of various 99mTc-labelled natural or synthetic peptides, which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI) or human lactoferrin (hLF), to bacteria and to leucocytes in vitro. In order to select peptides that preferentially bind to bacteria over host cells, radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the bacteria and with the leucocytes was quantitated. The next phase focussed on discrimination between bacterial infections and sterile inflammatory processes using 99mTc-labelled peptides in mice intramuscularly infected with various bacteria (e.g. multi-drug-resistant Staphylococcus aureus) and in animals that had been injected with lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory process. Also, we studied the distribution of 99mTc-labelled UBI 29-41 and UBI 18-35 in rabbits having an experimental thigh muscle infection with K. pneumoniae and in rabbits injected with LPS. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI 29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The radiolabelled peptides can detect infections with both gram-positive and gram-negative bacteria in mice as early as 5–30 min after injection, with a target-to-non-target (T/NT) ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection. In rabbits, high T/NT ratios (>5) for 99mTc-labelled UBI 29-41 were observed from 1 h after injection. No accumulation of the selected 99mTc-labelled UBI-derived peptides was observed in thighs of mice and rabbits previously injected with LPS. Scintigraphic investigation into the biodistribution of 99mTc-labelled UBI peptides revealed that these peptides were rapidly removed from the circulation by renal excretion. Similar data were observed for 99mTc-labelled defensin 1-3. Our data for 99mTc-labelled hLF and related peptides indicate that these compounds are less favourable for infection detection. Taken together, 99mTc-labelled UBI 18-35 and UBI 29-41 enable discrimination between bacterial infections and sterile inflammatory processes in both mice and rabbits. Based on their characteristics, we consider these peptides the candidates of preference for detection of bacterial infections in man.

Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria.

ABSTRACT Since human lactoferrin (hLF) binds to bacterial products through its highly positively charged N terminus, we investigated which of the two cationic domains is involved in its bactericidal activity. The results revealed that hLF lacking the first three residues (hLF−3N) was less efficient than hLF in killing of antibiotic-resistant Staphylococcus aureus, Listeria monocytogenes, and Klebsiella pneumoniae. Both hLF preparations failed to kill Escherichia coli O54. In addition, hLF−3N was less effective than hLF in reducing the number of viable bacteria in mice infected with antibiotic-resistant S. aureus and K. pneumoniae. Studies with synthetic peptides corresponding to the first 11 N-terminal amino acids, designated hLF(1–11), and fragments thereof demonstrated that peptides lacking the first three N-terminal residues are less effective than hLF(1–11) in killing of bacteria. Furthermore, a peptide corresponding to residues 21 to 31, which comprises the second cationic domain, was less effective than hLF(1–11) in killing of bacteria in vitro and in mice having an infection with antibiotic-resistant S. aureus or K. pneumoniae. Using fluorescent probes, we found that bactericidal hLF peptides, but not nonbactericidal peptides, caused an increase of the membrane permeability. In addition, hLF killed the various bacteria, most probably by inducing intracellular changes in these bacteria without affecting the membrane permeability. Together, hLF and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant S. aureus and K. pneumoniae, and the first two arginines play an essential role in this activity.

Comparison of gated single-photon emission computed tomography with magnetic resonance imaging for evaluation of left ventricular function in ischemic cardiomyopathy.

To perform a head-to-head comparison between magnetic resonance imaging (MRI) and gated single-photon emission computed tomography (SPECT) for the evaluation of left ventricular (LV) function (LV ejection fraction [LVEF], LV volumes, and regional wall motion) in patients with ischemic cardiomyopathy, we studied 22 patients with chronic coronary artery disease and LV dysfunction. Multislice, multiphase echoplanar MRI was performed with Philips Gyroscan ACS-NT15. Image analysis was performed using the MASS software package to determine LV end-systolic volume, LV end-diastolic volume, and LVEF. The same parameters were calculated using quantitative gated SPECT software (QGS, Cedars-Sinai Medical Center). The different parameters were compared using linear regression, and correlation coefficients were calculated. Regional wall motion was also determined from both techniques, according to a 13-segment model and a 3-point scoring system (from 1 = normokinesia to 3 = akinesia or dyskinesia). A summed wall motion score was also calculated for MRI and gated SPECT. Good correlations were found between MRI and gated SPECT for all parameters: (1) summed wall motion scoreMRI versus summed wall motion scoreSPECT: y = 0.74x + 8.0, r = 0.88, p <0.01; (2) LV end-systolic volumeMRI versus LV end-systolic volumeSPECT: y = 0.94x - 12.3, r = 0.87, p <0.01; (3) LV end-diastolic volumeMRI versus LV end-diastolic volumeSPECT: y = 0.93x - 18.4, r = 0.84, p <0.01; and (4) LVEFMRI versus LVEFSPECT: y = 0.97x + 0.68, r = 0.90, p <0.01. For regional wall motion, an exact agreement of 83% was found, with a kappa statistic of 0.77 (95% confidence intervals 0.71 to 0.83, SE 0.03), indicating essentially excellent agreement. Thus, close and significant correlations were observed for assessment of LVEF, LV volumes, and regional wall motion by MRI and gated SPECT in patients with ischemic cardiomyopathy.

Radiochemical and biological characteristics of 99mTc-UBI 29-41 for imaging of bacterial infections

A technetium-99m-labeled peptide derived from ubiquicidine, further referred to as 99mTc-UBI 29-41, targets bacterial and fungal infections, but not sterile inflammatory processes, in experimental animals. This paper reports on the radiochemical and biological features of this radioactive agent and the importance of the amino acid sequence of UBI 29-41 for imaging of infections. Radiochemical analyses of 99mTc-UBI 29-41 and a radiolabeled scrambled version of this peptide, i.e. 99mTc-Sc-UBI 29-41, revealed that both peptides were labeled rapidly (within 10 min) and effectively with little colloid formation (less than 5% of the total radioactivity) and very little free pertechnetate (or radioactive intermediates) in the preparations containing radiolabeled peptide. Furthermore, association of the peptides with bacteria could be competed with excess unlabeled peptide and this association proved to be temperature-dependent. Based on this in vitro data we concluded that labeling of peptides with 99mTc by this direct method is rapid, efficient, and safe. Scintigraphy demonstrated that radioactivity is rapidly removed from the circulation (half-lifes of UBI 29-41 and Sc-UBI 29-41 were 16 and 21 min, respectively) mainly by renal clearance. Analysis of murine blood revealed that only a small proportion of the intravenously injected 99mTc-peptides is associated with blood cells. Although both radiolabeled peptides accumulated rapidly at sites of infection, the values for 99mTc-UBI 29-41 were higher (P < 0.05) than for 99mTc-Sc-UBI 29-41. Moreover, injection of excess unlabeled UBI 29-41, but not Sc-UBI 29-41, into Staphylococcus aureus-infected mice prior to injection of 99mTc-UBI 29-41 significantly (P < 0.05) reduced the accumulation of this radiopharmaceutical at the site of infection. In addition, we observed significantly (P < 0.01) higher amounts of 99mTc-UBI 29-41 at the site of infection in mice using a carrier-free radiolabeled UBI 29-41 as compared with unpurified preparations containing radiolabeled UBI 29-41. This in vivo data indicates that the amino acid sequence of 99mTc-UBI 29-41 contributes to its accumulation at the site of infection.

Impact of viability, ischemia, scar tissue, and revascularization on outcome after aborted sudden death.

Background—Survivors of aborted sudden death attributable to ventricular arrhythmias in the presence of coronary artery disease are at risk for recurrences. The substrate underlying these arrhythmias is not clear, and therefore the relation between ischemia, viability, scar tissue (and revascularization), and the incidence of ventricular arrhythmias (and survival) was studied over up to 3 years. Methods and Results—One hundred fifty-three survivors of sudden death underwent stress-rest perfusion imaging. Patients with ischemic/viable myocardium (n=73) were revascularized if possible. Final antiarrhythmic therapy was based on the outcome of electrophysiological testing or left ventricular ejection fraction (LVEF). Implantation of a defibrillator was performed in 112 (72%) patients. During 3-year follow-up, 15 cardiac deaths occurred and 42 (29%) patients had recurrent ventricular arrhythmias. Patients with events (death or recurrence) exhibited more often a severely depressed LVEF (≤30%), more extensive scar tissue, and less ischemic/viable myocardium on perfusion imaging and less frequently underwent revascularization. Multivariate analysis identified extensive scar tissue and LVEF ≤30% as the only predictors of death/recurrent ventricular arrhythmias. Conclusions—In patients with aborted sudden death, extensive scar tissue and severely depressed LVEF are the only predictors of death or recurrent ventricular arrhythmias. These patients should be considered for implantation of a defibrillator.

Nanoparticles in Cancer

Nano-engineered particles have been developed to reach specific molecular targets on diseased cells and have been used in various experimental and clinical conditions. The medical application involves diagnostic and therapeutic applications and a large deal of this research concerns malignant disease. Various approaches have been tried to effectively reach the cancer cell and PEGylated liposomes have demonstrated targeting and controlled release of antineoplastic drugs. For cancer diagnostics nanoparticles have been engineered to optimize magnetic resonance imaging, ultrasound imaging and nuclear medicine imaging. Radiolabeled nanoparticles can also be used for therapeutic purposes when tagged with appropriate radionuclides. This article aims to provide an overview how nanomedicine is presently influencing drug design and, more specifically, the development of radiopharmaceuticals for cancer management.

Daily Oral Pamidronate in Women and Men With Osteoporosis: A 3-Year Randomized Placebo-Controlled Clinical Trial With a 2-Year Open Extension

The efficacy and safety of oral pamidronate was examined in a double‐blind, placebo‐controlled trial in women and men with established osteoporosis. Seventy‐eight postmenopausal women and 23 men with at least one prevalent vertebral fracture were randomized separately to 150 mg/day of pamidronate or placebo for 3 years followed by 150 mg/day of pamidronate for an additional 2 years. In addition, all patients received 400 U/day of cholecalciferol and 500 mg/day of elemental calcium. Pamidronate increased significantly bone mineral density of the lumbar spine (LS‐BMD) and of the femoral neck (FN‐BMD). The total increase in BMD of the spine after 5 years of treatment was 14.3%. Lateral spine radiographs were obtained at baseline and after 3 years of treatment. Fractures of previously normal vertebrae occurred in 15 of 45 patients treated with placebo (33.3%) and in 5 of 46 patients treated with pamidronate (11%). The relative risk was 0.33 (95% CI, 0.14‐0.77). Treatment was well tolerated and there was no difference in gastrointestinal toxicity between pamidronate and placebo‐treated patients. One hundred fifty milligrams daily of pamidronate is an effective and safe treatment of women and men with established osteoporosis.

Molecular imaging with SPECT as a tool for drug development.

Molecular imaging techniques are increasingly being used as valuable tools in the drug development process. Radionuclide-based imaging modalities such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) have proven to be useful in phases ranging from preclinical development to the initial stages of clinical testing. The high sensitivity of these imaging modalities makes them particularly suited for exploratory investigational new drug (IND) studies as they have the potential to characterize in vivo pharmacokinetics and biodistribution of the compounds using only a fraction of the intended therapeutic dose (microdosing). This information obtained at an early stage of clinical testing results in a better selection among promising drug candidates, thereby increasing the success rate of agents entering clinical trials and the overall efficiency of the process. In this article, we will review the potential applications of SPECT imaging in the drug development process with an emphasis on its applications in exploratory IND studies.

A comparison between the diagnostic efficacy of 99mTc-MDP, 99mTc-DPD and 99mTc-HDP for the detection of bone metastases.

We have investigated the clinical efficacy for the detection of bone metastases of two recently marketed bone-seeking radiopharmaceuticals, HDP and DPD, compared with traditionally used MDP. Twenty patients received 15 mCi 99mTc-MDP; after assessment ten of these patients later received 15 mCi 99mTc-DPD and ten other patients of this group were injected with 15 mCi 99mTc-HDP. Scintigraphy took place 3 h after tracer injection. Quantitative analysis included the calculation of normal bone to soft tissue ratios, lesion to soft tissue ratios and lesion to normal bone ratios for all three radiopharmaceuticals. Visual inspection of the scintiphotos revealed the same number of bone lesions at the same localisations.Statistical evaluation of our quantitative data showed that the lesion to normal bone ratio was significantly higher for MDP than for DPD. No further significant differences in the uptake in normal bone or in the metastatic lesions were found between all three radiopharmaceuticals.It is concluded that the new bone-seeking agents DPD and HDP do not possess clinical advantages over MDP for the detection of skeletal metastases.