Methotrexate and its polyglutamate derivatives in erythrocytes during and after weekly low-dose oral methotrexate therapy of children with acute lymphoblastic leukemia

Summary Methotrexate and methotrexate polyglutamates were quantitatively determined in red blood cells from 12 children with acute lymphoblastic leukemia who were treated with MTX (15–20 mg/m² per week) and daily 6-mercaptopurine orally during the steady-state period of erythrocyte MTX concentration (ery-MTX). The terminal decline of the ery-MTX and its polyglutamate metabolites were determined for up to 15 weeks after cessation of MTX treatment. Methotrexate polyglutamates with 2–4 extra glutamyl derivatives (MTX-glu_2-5) constituted 75% of the MTX in the entire red blood cell population. MTX-glu₃ was the principal metabolite; no MTX-glu_6-7 was identified. After discontinuation of MTX therapy, the ery-MTX declined in a non-linear manner because of different half-lives for the individual polyglutamates. From about 5 weeks until 13–15 weeks after the last MTX dose, the erythrocyte MTX elimination curve was linear. The approximate half-life of MTX-glu₁ was 2–3 days; for MTX-glu₂ it was 4–15 days. The concentration of MTX-glu_3-5 remained constant in the erythrocyte throughout its life span and declined only with age-dependent destruction of the red blood cell. It was calculated that 80%–90% of MTX in newly formed reticulocytes was MTX-glu₁₊₂, the remainder being MTX-glu_3-5. Mature red blood cells did not form methotrexate polyglutamates to any significant degree. There was a significant correlation between the amount of MTX-glu_3-5 and the steady-state ery-MTX, which to some extent explained the interindividual variation of the ery-MTX in children with ALL in maintenance therapy.     

Anal abscess imaged with 99mTc-labeled leukocytes. Case report

Abscess imaging with leukocyte scintigraphy is described in a case report. Autologous leukocytes were separated from whole blood in a patient with localized inflammation in the perineal region and labeled with 99mTc, an ideal radionuclide for clinical examination with gamma camera. Scintigraphic investigations demonstrated a deep-lying anal abscess. The procedure may prove useful for detection of occult infections and may provide a new diagnostic approach in fever of unknown origin.     

Targeted alpha therapy: part I

The possibility of pinpointing biological targets, and thereby potentially targeting and eradicating small tumors or even single cancer cells, is a tantalizing concept that has been discussed since the magic-bullet concept was first presented by Paul Erlich in the beginning of the 20th century in connection with his work on tissue staining for histological examinations and the work by Kohler and Milstein on antibody production published in 1975. This concept now seems feasible through the use of highly specific targeting constructs, chemical labeling of radioactive substances to these targeting constructs that results in high specific activities, radioimmunocomplexes with good stability even after injection, and the use of radionuclides emitting alpha( α)-particles having exceedingly high ionizing density and, therefore, a high probability of killing cells along its track in tissue. The short range of the emitted α-particles makes them even more interesting by minimizing unwanted irradiation of normal tissue surrounding the targeted cancer cells of interest, assuming high specificity of the targeting construct and good stability of the chemical bonds between the targeting construct and the α-particle emitter. Targeted Alpha Therapy (TAT), in which an α-particle emitting radionuclide is specifically directed to the biological target, is gaining more attention as new targets, targeting constructs, chemical labeling techniques, and α-particle emitters are, respectively, identified, constructed, developed, and made available. Results and improvements are now being published at an increasing rate and the number of conceivable applications is expanding, especially in the field of cancer treatment. Therefore, it is of utmost importance to provide an overview of the overall progress in the research field of TAT on a regular basis. However, problems such as limited or delayed diffusion of the α-radioimmunocomplex and inhomogeneous activity distributions in the targeted tumors, resulting in inhomogeneous absorbed dose distributions, are challenges that need to be addressed. These challenges need to be overcome before TAT becomes a standard treatment for diseases such as micrometastatic cancer. Hopefully, when enough funding will be provided and, hence, more treatment strategies of TAT will reach the clinical level the importance to conduct controlled, randomized trials with sufficient patient numbers, enabling statistical significance to occur must be emphasized in order to be able to properly compare and evaluate different approaches. In this issue, of the two hot-topic issues for targeted alpha therapy, articles discuss the recent developments in radionuclide availability, biomolecular targeting, labeling chemistry, and dosimetry for the most promising α-particle emitters. In the first article, Zalutsky et al. discuss the possibilities and limitations of using the promising α-particle emitter, 211At, and emphasize the need for funding new cyclotrons and prioritizing beam-times of already existing cyclotrons to improve the availability of 211At. Haddad et al. describe the status of the ARRONAX project through which a number of important nuclear medicine radionuclides will be produced, including some of those suitable for TAT. Relevant targeting constructs and their associated antigens used today and candidates for use in the future are discussed by Olafsen et al. in the third article. The next article, by Scott Wilbur, discusses chemical and radiochemical issues of radiolabeling using α-particle emitting radionuclides, e.g. factors that are important in selecting chelation or bonding reagents during the development of α-particle emitting radiopharmaceuticals. Lindegren at al. continue the discussion of chemical considerations in the following article, but focuses on pre-targeting techniques, which will hopefully enhance both the activity distribution in the targeted tumor and the tumor-to-normal tissue absorbed dose ratio. The two final articles discuss different aspects of the dosimetry related to α-particles. The article by Sgouros et al. discusses how knowledge of the microscopic distribution of α-particle emitters is necessary to perform correct dosimetry, as well as the importance of the translation of activity distributions obtained in pre-clinical studies to the human situation, which requires micro-scale models of the source-target geometry at human dimensions according to the authors. Chouin et al. focus in the following article on the microdosimetry of α-particles. The authors present basic concepts and some applications of the microdosimetry for TAT, and conclude microdosimetry should only be considered when alternative approaches fail to provide an account of a given biological endpoint. The intention of this particular hot-topic issue is to present an up-to-date overview of key areas in the research field of TAT, i.e. radionuclides available, targeting constructs, labeling chemistry, and dosimetry. This issue will hopefully be followed by similar ones jointly produced by contributions from the research community active in the field, of which most researchers are participating in these two particular issues, i.e. Targeted Alpha Therapy - Part I and II.     

Reply: A study of finger lengths, semen quality and sex hormones in 360 young men from the general Danish population

Sir, We thank Drs Voracek and Dressler for their interest in ourpublication ‘A study of finger lengths, semen qualityand sex hormones in 360 young men from the general Danish population’(Bang et al., 2005). We agree that our findings are interesting.It is somewhat peculiar that the average second to fourth digitratio (2D:4D) found in the Danish men is higher than that     

Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 μL) and different volumes of venous blood (50 μL, 200 μL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 μL, 200 μL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ∼100-fold lower than 5 μL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 μL DBS method missed 27% of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.     

A systematic review and meta-analysis of 130,000 individuals shows smoking does not modify the association of APOE genotype on risk of coronary heart disease

Background

Conflicting evidence exists on whether smoking acts as an effect modifier of the association between APOE genotype and risk of coronary heart disease (CHD).

Methods and results

We searched PubMed and EMBASE to June 11, 2013 for published studies reporting APOE genotype, smoking status and CHD events and added unpublished data from population cohorts. We tested for presence of effect modification by smoking status in the relationship between APOE genotype and risk of CHD using likelihood ratio test.In total 13 studies (including unpublished data from eight cohorts) with 10,134 CHD events in 130,004 individuals of European descent were identified. The odds ratio (OR) for CHD risk from APOE genotype (ε4 carriers versus non-carriers) was 1.06 (95% confidence interval (CI): 1.01, 1.12) and for smoking (present vs. past/never smokers) was OR 2.05 (95%CI: 1.95, 2.14). When the association between APOE genotype and CHD was stratified by smoking status, compared to non-ε4 carriers, ε4 carriers had an OR of 1.11 (95%CI: 1.02, 1.21) in 28,789 present smokers and an OR of 1.04 (95%CI 0.98, 1.10) in 101,215 previous/never smokers, with no evidence of effect modification (P-value for heterogeneity = 0.19). Analysis of pack years in individual participant data of >60,000 with adjustment for cardiovascular traits also failed to identify evidence of effect modification.

Conclusions

In the largest analysis to date, we identified no evidence for effect modification by smoking status in the association between APOE genotype and risk of CHD.     

Effect of Citalopram on Alcohol Intake in Heavy Drinkers

The effect of the selective serotonin reuptake inhibitor citalopram (40 mg daily dose) on alcohol intake was investigated in a doubleblind, placebo-controlled cross-over study. Thirty men with heavy alcohol consumption (mean daily alcohol intake 111 ± 51 g pure alcohol) completed the study. After a 2-week baseline period, subjects were randomly allocated to treatment with either citalopram or placebo for 5 weeks. In the total sample of heavy drinkers, no difference was found between citalopram and placebo treatment in alcohol consumption or days of abstinence. However, the response to citalopram was negatively correlated ( r _a=–0.67, p < 0.01) with baseline levels of mean daily alcohol intake. Therefore, we divided the total sample into two subgroups with baseline mean daily alcohol intake above and below median (107 g pure alcohol), respectively. In the group with the higher baseline values (138 ± 25 g pure alcohol), citalopram was not different from placebo in reducing the daily alcohol intake, but in subjects with the lower baseline values (85 ± 15 g pure alcohol), citalopram was significantly ( p < 0.01) superior to placebo. Consequently, citalopram at the present dose appears capable of reducing alcohol intake only in a subgroup of heavy drinkers with a mean daily consumption of between 60 and 100 g pure alcohol.     

The ratio between serum levels of insulin-like growth factor (IGF)-I and the IGF binding proteins (IGFBP-1, 2 and 3) decreases with age in healthy adults and is increased in acromegalic patients

OBJECTIVE Several in-vitro studies have suggested that the biological actions of IGF-I can be modified by the presence of specific IGF binding proteins. In man, the 24-hour serum levels of IGF-I and IGFBP-3 remain constant, but short-term changes in the IGF-l/IGFBP-3 ratio have been described following GH administration. Serum levels of IGF-I and IGFBP-3 decrease with age in normal adults and are elevated In active acromegaly due to excessive GH secretion. However, the Individual ratios between serum levels of IGF-I and IGFBP-3 in acromegalic and healthy adults have not been described previously. METHODS AND MATERIALS We studied this ratio In 198 healthy adults and In 56 acromegalic patients, grouped according to their serum GH levels (group I GH < 2mLU/l II GH 2–10mLU/l; III GH > 10mLU/l). In all subjects a single blood sample was drawn for IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and GH measurements by specific RIAs. In 38 of the patients a 24-hour urinary collection was performed for GH determination. RESULTS In healthy adults serum levels of IGF-I and IGFBP-3 decreased with Increasing age (r =?0.52 and r=?0.34, respectively, P< 0.0001). In addition, the molar IGF-l/IGFBP-3 ratio declined with increasing age (r =?0.44, P – 0.0001). In patients with acromegaly and high serum GH levels (group III), circulating IGF-I was increased 7–97 standard deviations (SDS) and IGFBP-3 was increased 4.20 SOS (P < 0.0001). Serum levels of IGF-II were normal in all three groups (588 ± 240μ/l) whereas IGFBP-1 and IGFBP-2 levels were low and IGFBP-2 levels decreased significantly with increasing serum GH levels (P < 0.0001). The molar IGF-l/IGFBP-3 ratio in the acromegalic patients was significantly higher than in the controls (P < 0.0001) and correlated significantly with urinary GH excretion (r = 0.67, P < 0.0001) as well as with serum GH levels (r = 0.73, P < 0.0001). CONCLUSION We demonstrated a decreasing molar IGF-l/IGFBP-3 ratio with increasing age in healthy adults and an increased ratio between serum IGF-I and IGFBP-3 levels in acromegalic patients. As IGF-II is normal and IGFBP-1 and IGFBP-2 are inversely correlated to the serum GH levels In the acromegalic patients, we speculate that the molar ratio between IGF-I and IGFBP-3 reflects free (biologically active) IGF-I and Is dependent on GH levels.     

One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.

One hundred and twenty-seven cultured human tumor cell lines produced tumors after sc inoculation of 1-20 million cells into nude mice. They included 56 carcinoma lines, 14 sarcoma lines, and 57 lines from miscellaneous tumors and were all glucose-6-phosphate dehydrogenase type B. Twenty-nine percent of the lines produced tumors of 1 cm3 size within 1 month and 41% in the second month after inoculation. The histopathology correlated with the human tumor of origin in all cases.