Therapeutic potential of CAMPATH-1H in skeletal tumours

Fritsche‐Guenther R, Gruetzkau A, Noske A, Melcher I, Schaser K‐D, Schlag P M, Kasper H‐U, Krenn V & Sers C
(2010) Histopathology 57, 851–861 Therapeutic potential of CAMPATH‐1H in skeletal tumours Aims: CD 52 is a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein that is expressed abundantly on all lymphocytes, monocytes, macrophages, eosinophils and in the male genital tract. To date, the physiological role of CD52 on lymphocytes has not been elucidated. However, an antibody directed to CD52 called CAMPATH‐1H has been shown to be capable of depleting lymphocytes. The aim of this study was to analyse tissue and cell lines of non‐neoplastic bone, cartilage and skeletal tumours for CD52 expression. Methods and results: The expression of CD52 mRNA and protein both in vivo and in vitro was detected. Malignant tumours showed higher CD52 expression compared to benign tumours, suggesting a role in the development and progression of bone tumours. Interestingly, immunohistochemistry and flow cytometry revealed that CD52 was expressed not only on the surface of tumour cells, but also in the cytoplasm. The results obtained in osteosarcoma cells showed that CAMPATH‐1H leads to a complement‐independent reduction of viable cells. Conclusion: CD52 is expressed in a variety of bone tumours and the in vitro studies presented herein suggest that CAMPATH‐1H treatment might have therapeutic potential for osteosarcoma patients.     

The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo.The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.     

Pharmacokinetics of cephalosporins in the neonate: a review

The aim of this work was to review the published data on the pharmacokinetics of cephalosporins in neonates to provide a critical analysis of the literature as a useful tool for physicians. The bibliographic search was performed for articles published up to December 3, 2010, using PubMed. In addition, the book Neofax: A Manual of Drugs Used in Neonatal Care by Young and Mangum was consulted. The cephalosporins are mainly eliminated by the kidneys, and their elimination rates are reduced at birth. As a consequence, clearance is reduced and t1/2 is more prolonged in the neonate than in more mature infants. The neonate's substantial body water content creates a large volume of distribution (Vd) of cephalosporins, as these drugs are fairly water soluble. Postnatal development is an important factor in the maturation of the neonate, and as postnatal age proceeds, the clearance of cephalosporins increases. The maturation of the kidney governs the pharmacokinetics of cephalosporins in the infant. Clearance and t1/2 are influenced by development, and this must be taken into consideration when planning a cephalosporin dosage regimen for the neonate.     

Epitope analysis for human sperm-immobilizing monoclonal antibodies,MAb H6-3C4, 1G12 and campath-1

Human monoclonal antibody, MAb H6-3C4, possesses strong sperm immobilizing activity. MAb H6-3C4 has been suggested by several research groups to react with a carbohydrate moiety of male reproductive tract CD52 (mrtCD52). In the present study, we analysed the epitope on mrtCD52 for MAb H6-3C4 and found that it was polymorphic in Western blot analysis and disappeared after enzymatic removal of the N-linked carbohydrate moiety. Two other monoclonal antibodies (1G12, campath-1) with sperm-immobilizing activity recognized mrtCD52 in a polymorphic manner similar to MAb H6-3C4. Further analysis showed that 1G12 recognized a structure formed by the peptide and/or a glycosylphosphatidylinositol (GPI) anchor portion as does campath-1. Results of a lectin binding assay suggested the presence of O-linked carbohydrates on mrtCD52. Our results also indicated that the peptide portion of CD52 could serve as an epitope for sperm-immobilizing antibodies. It was concluded that the epitope of MAb H6-3C4 is similar to, but distinct from, those of 1G12 and campath-1, and that mrtCD52 contains different antigenic epitopes.     

Pharmacokinetics and absolute bioavailability of diltiazem in humans

Summary Six healthy male volunteers received single doses of diltiazem hydrochloride on three occasions separated by at least 10 days. Modes of administration were: 10-minute intravenous infusion of a 20-mg dose; oral administration of 120 mg in solution form; and oral administration of 120 mg as two 60-mg sustained-release tablets. Diltiazem concentrations were measured by electroncapture gas chromatography in multiple plasma samples drawn during the 36 hours after dosage. Following intravenous administration, mean (±S.E.) pharmacokinetic variables were: elimination half-life, 11.2 (±2.1) hours; volume of distribution, 11.1 (±3.0) liters/kg; and total clearance, 11.5 (±0.7) ml/min/kg. Oral diltiazem in solution form was rapidly absorbed, with peak plasma levels attained at 38 (±6) minutes after the dose. Absolute systemic availability averaged 44% (±4%). Oral administration of sustained-release tablets yielded, as predicted, slower absorption, with peak plasma concentrations attained at an average of 165 (±22) minutes after dosage. Thus, oral diltiazem is incompletely bioavailable after oral administration, mainly because of first-pass hepatic extraction.Supported in part by Grant Oc 10/6-3 from Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, and by a Grant from Gödecke AG, Freiburg, West GermanyIn part presented at Frühjahrstagung der Deutschen Gesellschaft für Herz- und Kreislaufforschung, Bad Nauheim 1982 [7]     

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration

Osteoporosis is a skeletal disorder attributable to an imbalance in osteoblast and osteoclast activity. NELL-1, a secretory protein that promotes osteogenesis while suppressing osteoclastic activity, holds potential as an osteoporosis therapy. Recently, we demonstrated that PEGylation of NELL-1 significantly improves its thermostability while preserving its bioactivity in vitro. However, the effect of PEGylation on the pharmacokinetics and osteogenic potential of NELL-1 in vivo have yet to be investigated. The present study demonstrated that PEGylation of NELL-1 significantly increases the elimination half-life time of the protein from 5.5 h to 15.5 h while distributing more than 2–3 times the amount of protein to bone tissues (femur, tibia, vertebrae, calvaria) in vivo when compared to naked NELL-1. In addition, microCT and DXA analyses demonstrated that systemic NELL-PEG therapy administered every 4 or 7 days significantly increases not only femoral and lumbar BMD and percent bone volume, but also new bone formation throughout the overall skeleton after four weeks of treatment. Furthermore, immunohistochemistry revealed increased osteocalcin expression, while TRAP staining showed reduced osteoclast numbers in NELL-PEG groups. Our findings suggest that the PEGylation technique presents a viable and promising approach to further develop NELL-1 into an effective systemic therapeutic for the treatment of osteoporosis.     

Pharmacokinetics and pharmacodynamics of intravenous digoxin and digitoxin

Summary Healthy volunteers received single 1.0-mg doses of intravenous digoxin (n=10) or digitoxin (n=12). Glycoside pharmacokinetics were determined from multiple plasma samples drawn over the 48 hours (for digoxin) or 14 days (for digitoxin) after the dose. Electrocardiogram, echocardiogram, and blood pressure were recorded at multiple time points 24 h after the dose. To control for nonspecific cardiovascular changes, pharmacodynamic measurements were repeated on a second occasion for 8 hours after an intravenous injection of saline. Mean (±S.E.) kinetic variables for digoxin were: volume of distribution (Vd), 8.3 (±0.6) l/kg; elimination half-life (t1/2), 49 (±5) h; clearance 2.1 (±0.2) ml/min/kg. Changes in blood pressure, ventricular rate, and corrected QT-interval attributable to digoxin were small. However, echocardiographically-determined mean rate of circumferential fibre shortening (mVcf) and ejection fraction (EF) increased significantly following digoxin when compared to saline infusion. Changes were maximal at 4–6 h after dosage, and were highly correlated with plasma digoxin concentration. mVcf and EF returned to baseline by 24 h post-dosage. Mean kinetic variables for digitoxin were: Vd, 0.63 (±0.03) l/kg; t1/2, 7.3 (±0.4) days; clearance, 0.043 (±0.003) ml/min/kg. Like digoxin, digitoxin infusion produced minimal change in blood pressure, ventricular rate, or QT-interval. However, mVcf and EF increased significantly when compared to saline control. Changes were maximal at 4–8 h after infusion, and were correlated with plasma digitoxin concentration; at 24 h post-dosage, mVcf and EF were still increased over baseline. Thus, digoxin and digitoxin significantly increase myocardial contractility in healthy humans, but without important change in heart rate and blood pressure. Changes in contractility are of slow onset, probably due to slow distribution of glycoside to sites of pharmacologic activity.Supported in part by Grant Oc 10/4 from Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, FRG; and by Grant MH-12279 from the United States Public Health Service     

Pharmacokinetics and dynamics of penbutolol in humans: Evidence for pathway-specific stereoselective clearance

Summary The pharmacokinetics and dynamics of thed- andl-isomers of the beta-adrenergic blocking agent penbutolol were investigated in healthy human volunteers. In Study One, subjects received a single 40-mg oral dose ofl-penbutolol (the pharmacologically active stereoisomer), and matching placebo on two occasions. A mean peak serum penbutolol concentration of 268 ng/ml was reached at 0.9 h after dosing. Elimination half-life averaged 1.6 h, and total clearance 16.6 ml/min per kg body weight. Changes in blood pressure, ventricular rate, and rate of circumferential fiber shortening (Vcf) did not differ betweenl-penbutolol and placebo. In Study Two, subjects received 40 mgd-penbutolo,l-penbutolol, and placebo on three occasions. Total clearance ofd-penbutolol was higher than for thel-isomer (43.7 vs 15.9 ml/min/kg;P<0.01); this was reflected in correspondingly increased area under the serum concentration curve for conjugates of the oxidized metabolite 4-hydroxy penbutolol (2.25 vs 0.66 µg/ml×h;P<0.005). In contrast, direct conjugates ofl-penbutolol achieved higher serum concentrations than conjugates ofd-penbutolol. Alterations in blood pressure, ventricular rate, and Vcf ford-penbutolol,l-penbutolol, and placebo were quantitatively small. Thus the clearance of penbutolol after oral administration in humans is stereoselective, but the oxidative pathway is more stereosensitive than the parallel conjugative pathway. Penbutolol causes minimal alterations in parameters of cardiac function after single 40-mg doses in healthy humans.Abbreviations AUC Area under the curve - d Dextro - HPLC High pressure liquid chromatography - l Levo - Vcf Rate of circumferential fiber shortening - t1/2 Elimination half-life Supported in part by Grant Oc/10 6-4 from Deutsche Forschungsgemeinschaft, by Hoechst AG, Frankfurt, Federal Republic of Germany; and by Grant MH-34223 from the United States Public Health ServiceIn part presented at the 50th Jahrestagung der Deutschen Gesellschaft für Herz- und Kreislaufforschung, Mannheim, 1984.     

Pharmacokinetics of hexobarbital in young and old rats

Hexobarbital is a drug widely used to study the capacity of the liver to metabolize drugs. The pharmacokinetics of hexobarbital in 3- and 30-month-old male BN/BiRij rats were studied. The half-life of hexobarbital in 30-month-old rats (34.9 ± 4.1 min) was significantly higher than that of 3-month-old ones (21.3 ± 3.8 min). The volume of distribution (ml·kg⁻¹ body weight) did not change with age. The intrinsic clearance, expressed as ml·min⁻°kg⁻¹ body weight, or hexobarbital in 30-month-old rats (2-.2 ± 6.6) was half that of the 3-month-old ones (39.5± 7.6). Further studies will be performed to investigate the effect of age on the intrinsic clearance of S(+)- and R(−) — hexobarbital.     

中华眼镜蛇毒组份Ⅲ的药代动力学及组织分布研究

目的:观察中华眼镜蛇(Najanajaatra)蛇毒组份Ⅲ在兔体内的药代动力学过程和在小鼠体内的分布状况。方法:用氯胺-T法对眼镜蛇毒组份Ⅲ进行碘化标记,用放射性核素示踪动力法检测血液中的药物浓度,以放射性参与量(脏器与血液放射比)的比值作为组份Ⅲ在组织中分布的依据。结果:兔静脉注射眼镜蛇毒组份Ⅲ75、150和300μg／kg3个剂量后,快分布相半衰期T_1/2α为39.6-42.5min,慢分布相半衰期T_1/2β为16.8-17.3h,消除相半衰期T_1/2γ为21.7-22.1h。小鼠尾静脉注射[¹²⁵Ⅰ]-组份Ⅲ后,2h及4h放射性参与量大于1的器官为肝脏、肾脏、肺脏、心脏和肌肉,其中以肾脏分布最高,且4h放射性参与量高于2h。结论:兔静脉注射组份Ⅲ3个剂量后,血药-时间曲线经3P87药动学程序拟合符合三房室模型特征,3个时相的半衰期各剂量组之间无显著差异,曲线下面积(AUC)与剂量成正比,表明药物在兔体内的分布和消除为一级线性动力学过程。小鼠静注组份Ⅲ后2h,以肾脏分布最高,肝脏与肺脏的放射性参与量也较高,尿中含量很高。     

Tissue microarray technology: validation in colorectal carcinoma and analysis of p53, hMLH1, and hMSH2 immunohistochemical expression

Tissue microarray technology enables the analysis of hundreds of specimens by arranging numerous 0.6-mm tissue core biopsy specimens into a single paraffin block. Validation studies are necessary to evaluate the representativeness of small disks taken from the original tissue. We validated the tissue microarray technology in colorectal carcinoma by analyzing the immunohistochemical expression of proteins involved in the two main pathways of colorectal carcinogenesis: p53 protein for loss of heterozygosity tumors, hMLH1 and hMSH2 proteins for microsatellite instability (MSI) tumors. We compared in 30 colorectal carcinomas (15 MSI^– and 15 MSI⁺), 8 microarrays disks, and the whole section of the block from which they were derived. Tumoral tissue was present in 95.7% of the microarray disks. The analysis of three disks per case was comparable to the analysis of the whole section in 99.6% (p53), 98.8% (hMLH1), and 99.2% (hMSH2) of cases. In the second part we applied the tissue microarray technology to 263 consecutive cases of colorectal carcinoma, sampled by three cores. We showed that 48.5% overexpressed p53 and 8.7% lost hMLH1 or hMSH2. Tissue microarray technology, validated in colorectal carcinoma, appears as a useful research tool for rapid analysis of the clinical interest of molecular alterations.     

Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab

Introduction of biotherapeutics has been a major milestone in the treatment of different chronic diseases. Nevertheless, the immune system can recognize the administered biological as non-self and respond with generation of anti-drug antibodies (ADA), including neutralizing ADA (nADA). Immunogenic responses may result in altered drug dynamics and kinetics leading to changes in safety and efficacy. However, there are several challenges with standard techniques for immunogenicity testing. Ustekinumab (UST), used in different inflammatory diseases, is a therapeutic antibody directed against the shared p40 subunit of interleukin (IL)-12 and IL-23, interfering in the pathogenically crucial T helper type 1 (Th1)/Th17 pathway. We established and validated different approaches for detection and quantitation of UST, UST-specific ADA and nADA. Addressing the obstacle of complex formation of UST with nADA, we developed an acidification assay to approach the total amount of nADA. Validated methods were based on surface plasmon resonance spectroscopy (SPR), enzyme-linked immunosorbent assay (ELISA) and a cell-based approach to characterize neutralizing capacity of nADA. Parameters assessed were determination and quantitation limits, linearity, range, precision, accuracy and selectivity. Quantitation of ADA and UST was feasible at lower concentrations using ELISA, whereas SPR showed a wider linear range for determination of ADA and UST. Accuracy, precision and linearity for quantitation were comparable using ELISA, SPR and the cell-based approach. All validated parameters fulfill the requirements of regulatory agencies. A combination of the testing approaches could address the increasing demand of precision medicine as it can be suitable for capturing the whole spectrum of immunogenicity and is transferable to other biologicals.     

Assessment of baroreflex control from beat-to-beat blood pressure and heart rate changes: A validation study

In this study, we tested the validity of a new method designed to estimate baroreflex control of heart rate from spontaneous changes in systolic pressure and pulse interval. This method was compared with a conventional method of assessing baroreflex control through measuring reflex adjustments in pulse interval associated with pharmacological manipulations of blood pressure. The estimates of baroreflex control derived from the two methods were signficantly correlated; however, only the estimate derived using pharmacological changes in pressure detected significant impairment of baroreflex control in patients with damage to baroafferents produced by radiation for oropharyngeal cancer. Analysis of spontaneous changes in pressure and pulse interval therefore provide a meaningful estimate of baroreflex control of heart rate that is, however, less sensitive than estimates obtained using pharmacological manipulations in pressure.     

Contactless respiratory and heart rate monitoring: validation of an innovative tool

Primary objective:?To assess the accuracy of the EverOn? piezoelectric sensor based contactless heart rate and respiration rate monitoring system.

Methods:?Measurements of the Everon? and reference devices were performed in a sleep lab and an intensive care unit (ICU) setting. One minute measurements by both the reference device and the EverOn? were averaged and compared. Accuracy was defined in accordance with industry criteria.

Results:?Respiration rate (RR) accuracy in the 41 children and 16 adults evaluated in the sleep lab was 93.1% and 90.6% respectively, and heart rate (HR) accuracy was 94.4% and 91.5% respectively. For the 42 ICU patients RR accuracy was 82.0% and 75% (versus end-tidal CO₂ and manual respectively), while accuracy of HR was 94.0%. The EverOn? was found to be superior to the impedance technique in measuring RR.

Conclusions:?The system described was found to be accurate in accordance with regulatory and industry criteria.     

131I-c(RGD)2的药代动力学及急性毒性研究

目的 用131I标记二硫键成环的RGD肽二聚体c(RGD)2,探讨其在裸鼠体内的药代动力学参数、急性毒性反应和死亡情况,评价其应用的安全性.方法 选取5只裸鼠为实验对象,每只经尾静脉注射131I-c(RGD)2(7.4 MBq/200μL),分别于注射后3、6、10、15、30、45、60、120、180及360min断尾取血5μL,测量血液的放射性计数,采用PKSolver软件进行药代动力学分析.另取裸鼠10只随机分为实验组及对照组,实验组每只经尾静脉注射131I-c(RGD)2(7.4 MB/60μL);对照组经尾静脉注射生理盐水60μL,观察注射后72 h内裸鼠的不良反应和死亡情况.结果 血液药-时曲线符合权重系数为1/CC的开放性二房室分布模型,其中分布相半衰期(t1/2α)为15.364 min,消除相半衰期(t1/2β)为123.125 min.注射131I-c(RGD)2后72 h内,裸鼠无不良反应与死亡.结论 c(RGD)2具有理想的药代动力学特点,且无明显毒性作用,这些均有利于其作为新药应用于临床.     

Development and validation of the high blood pressure-focused health literacy scale

Objective

While the role of health literacy in chronic disease management is well documented, few intervention studies have been reported. A major barrier to designing and implementing such interventions is the lack of valid health literacy tools. This study developed and tested a novel health literacy scale for individuals with high blood pressure (HBP).

Methods

A two-step design process was used: In the construction phase, focus group studies and a literature review were conducted to generate a pool of items. The testing phase involved a psychometric evaluation and pilot-testing of the scale on hypertensive Korean Americans (n = 386). The end product was a HBP-health literacy scale (HBP-HLS) with two essential domains, print literacy and functional health literacy.

Results

Psychometric testing indicated that the scale was reliable (Kuder–Richardson-20 coefficient = 0.98), valid (content validity index ≥0.8), and significantly correlated with theoretically selected variables (education, r = 0.67, p < 0.01; HBP knowledge, r = 0.33, p < 0.01).

Conclusion

The HBP-HLS demonstrated its utility for evaluating HBP management interventions in the community setting.

Practice implications

Utilizing the HBP-HLS should be considered as a potential tool for improving health literacy and evaluating intervention studies in the context of HBP management.     

小鼠海马CA1区锥体神经元树突棘的发育

目的 对小鼠海马CA1区锥体神经元正常发育中树突棘密度及各种形态变化进行分析测定,为深入研究突触发生及突触可塑性提供直接的形态学依据.方法 分别取出生后0、5、10、20及30d 5个年龄段的C57BL/6小鼠各10只,采用基因枪对小鼠海马CA1区锥体神经元树突棘进行亲脂性荧光染料DiI标记,通过激光共焦显微镜对其进行观察分析;同时利用透射电镜技术对树突棘的超微结构进行分析.结果 树突棘的形态、大小及其密度随小鼠发育而变化,成熟树突棘内部存在滑面内质网与棘器,可能参与了突触后膜结合蛋白及其转运体的合成.结论 树突棘的发育过程与突触连接的形成以及突触可塑性密切相关.     

Pharmacokinetics of oxaliplatin during chronomodulated infusion in metastatic gastrointestinal cancer patients: A pilot investigation with preliminary results

Several clinical trials have demonstrated that the three-drug combination of oxaliplatin, 5-fluorouracil (5-FU) and leucovorin (LV) administered chronomodulated improved antitumour efficacy in the treatment of metastatic colorectal cancer and was better tolerated than constant-rate infusion. However, only a few pharmacokinetic data of 5-FU during chronomodulated infusion are available but up to now not for oxaliplatin. In this pilot study, the platinum levels of plasma ultrafiltrate (PUF) and total plasma were monitored during chronomodulated infusion of oxaliplatin, 5-FU and LV in 7 patients with metastatic gastrointestinal cancer. A cycle of the 4-day chemotherapeutic regimen consisted of 12-h infusions with sinusoidal drug delivery rate of: oxaliplatin (25 mg/m²/d, peak at 16:00 hours), 5-FU and LV (750 mg/m²/d and 150 mg/m²/d, respectively, peak at 4:00 hours), the same scheme was reinitiated on day 15. Blood samples were collected on day 1 and day 4 during different cycles. Concentration–time profiles of ultrafilterable and total platinum in plasma during chronomodulated infusion were characterised. As expected, we found residual platinum levels in total plasma but not in PUF prior next cycle. Comparing day 1 with day 4, C_max of platinum in PUF was significantly increased (84 ± 13 ng/ml vs. 131 ± 22 ng/ml, P = 0.012) as well as AUC_0–24h of platinum in PUF (0.97 ± 0.29 μg · h/ml vs. 1.90 ± μ0.44 g · h/ml, P = 0.018). The same effect was observed for total plasma platinum suggesting an accumulation within the cycle. The observed interindividual variability of C_max, t_max, AUC_0–24h, t_1/2 was moderate. Because of the small sample size in this pilot investigation, the findings need to be confirmed in larger pharmacokinetic studies. In a next step individual pharmacokinetic parameters should be associated with patient specific parameters and treatment-induced toxicity.