Plasma pharmacokinetics and cerebrospinal fluid penetration of thioguanine in children with acute lymphoblastic leukemia: a collaborative Pediatric Oncology Branch, NCI, and Children's Cancer Group study.

PURPOSE: In preclinical studies, thioguanine (TG) has been shown to be more potent than the standard acute lymphoblastic leukemia (ALL) maintenance agent, mercaptopurine (MP), suggesting that TG may be more efficacious than MP in the treatment of childhood ALL. As part of a pilot trial in which TG was used in place of MP, we studied the plasma pharmacokinetics of oral TG and measured steady-state plasma and CSF TG concentrations during a continuous intravenous infusion (CIVI) in children with newly diagnosed standard-risk ALL. METHODS: Nine plasma samples were collected after each patient's first 60 mg/m2 oral TG dose during maintenance. CIVI TG (20 mg/m2/h over 24 h) was administered during the consolidation phase of therapy, and simultaneous plasma and CSF samples were collected near the end of the infusion. TG was measured by reverse-phase HPLC with ultraviolet detection. Erythrocyte TG nucleotide (TGN) concentrations were measured 7 days after a course of CIVI TG and prior to the start of each maintenance cycle. RESULTS: After oral TG (n = 35), the mean (+/- SD) peak plasma concentration was 0.46 +/- 0.68 microM and the AUC ranged from 0.18 to 9.5 microM.h (mean 1.5 microM.h). Mean steady-state plasma and CSF TG concentrations during CIVI (n = 33) were 2.7 and 0.5 microM, respectively. The mean (+/- SD) TG clearance was 935 +/- 463 ml/min per m2. Plasma TG concentrations did not correlate with erythrocyte TGN concentrations after oral or CIVI TG. The 8-OH-TG metabolite was detected in plasma and CSF. CONCLUSIONS: TG concentrations that are cytotoxic to human leukemia cell lines can be achieved in plasma after a 60 mg/m2 oral dose of TG and in plasma and CSF during CIVI of TG.

Thioguanine administered as a continuous intravenous infusion to pediatric patients is metabolized to the novel metabolite 8-hydroxy-thioguanine.

Thiopurine antimetabolites have been in clinical use for more than 40 years, yet the metabolism of thiopurines remains only partially understood. Data from our previous pediatric phase 1 trial of continuous i.v. infusion of thioguanine (CIVI-TG) suggested that TG was eliminated by saturable mechanism, with conversion of the drug to an unknown metabolite. In this study we have identified this metabolite as 8-hydroxy-thioguanine (8-OH-TG). The metabolite coeluted with the 8-OH-TG standard on HPLC and had an identical UV spectrum, with a lambda(max) of 350 nm. On mass spectroscopy, the positive ion, single quad scan of 8-OH-TG yielded a protonated molecular ion at 184 Da and contained diagnostic ions at m/z 167, 156, 142, and 125 Da. Incubation of TG in vitro with partially purified aldehyde oxidase resulted in 8-OH-TG formation. 8-OH-TG is the predominant circulating metabolite found in patients receiving CIVI-TG and is likely generated by the action of aldehyde oxidase.

A pediatric phase I trial and pharmacokinetic study of thioguanine administered by continuous i.v. infusion.

Although mercaptopurine is the thiopurine antimetabolite predominantly used in the treatment of childhood acute lymphoblastic leukemia (ALL), thioguanine (TG) is more potent than mercaptopurine in in vitro cytotoxicity studies in human leukemic cell lines and leukemic cells from patients with ALL. We conducted a pediatric Phase I trial of TG administered as a continuous i.v. infusion (CIV). A pharmacokinetically guided dose escalation was performed to define the dose rate of TG required to achieve a steady-state plasma concentration (Css) exceeding the target concentration of 1 microM, and then the maximum tolerated duration of infusion of TG at this dose rate was defined. Eighteen patients (median age, 18 years; range, 4-25 years) with refractory malignancies (16 solid tumors and 2 ALL) were enrolled in this study. The starting dose rate of 10 mg/m2/h administered for 24 h achieved an average Css of 0.9 microM (range, 0.7-1.2 microM). Therefore, the dose rate was escalated to 20 mg/m2/h, which achieved an average Css of 4.1 microM (range, 1. 0-8.3 microM). This disproportionate increase in the Css of TG suggested a capacity-limited (saturable) elimination process, and a pharmacokinetic model incorporating two compartments with capacity-limited elimination from the central compartment was developed to describe the disposition of TG. The TG clearances (derived from model parameters) at the 10- and 20-mg/m2/h dose rates were 987 and 608 ml/min/m2, respectively. Dose-limiting myelosuppression (absolute granulocyte count < 500/mm3 and platelet count < 25,000/mm3) was observed in two of three patients treated with a dose rate of 20 mg/m2/h administered for 36 h. Administration of CIV of TG at 20 mg/m2/h for 24 h was well tolerated in nine patients. Nonhematological toxicities included nonneutropenic infections and mild, reversible changes in hepatic function tests. The recommended dose rate and duration for CIV of TG is 20 mg/m2/h for 24 h.

Cytomegalovirus infection in children with human immunodeficiency virus infection.

OBJECTIVES: To determine retrospectively the prevalence of positive cytomegalovirus (CMV) cultures in pediatric patients with human immunodeficiency virus infection. METHODS: We reviewed the records of 273 children with human immunodeficiency virus infection referred to the Pediatric Branch of the National Cancer Institute for whom CMV cultures were performed between January, 1991, and October, 1994. RESULTS: Of this group 189 patients (69%) had negative CMV cultures and 84 (31%) had positive cultures. The prevalence of CMV-related disease was 9% for the entire group, including 4 (2.1%) patients with negative CMV cultures. Among the 84 patients with positive CMV cultures, 21 (25%) had evidence of CMV disease. Patients with positive CMV cultures had a statistically significant decrease in survival in the presence of severe immunocompromise defined as an age-corrected CD4 count of < 21%. Nine of 35 (26%) autopsies performed demonstrated evidence of CMV disease, including 7 patients with disseminated CMV disease. CONCLUSIONS: Although CMV disease appears to be less frequent in children than adults, CMV infection still contributes significantly to morbidity and mortality in this population, especially when combined with severe immunosuppression.

Changes in structure of the bovine milk fat globule membrane on heating whole milk.

The effects of heat-induced interactions between milk fat globule membrane components and skim milk proteins in whole milk on the structure of the membrane were examined by isopycnic sucrose density gradient centrifugation and by using Triton X-100 as a membrane probe. Skim milk components were incorporated into all the lipoprotein fractions separated by density gradient centrifugation. High density complexes, higher in density than those found in the natural milk fat globule membrane, were formed during the heat treatment. Losses of natural membrane polypeptides from the medium and low density lipoproteins were observed on heating. Heating whole milk also altered the rate of release of membrane components by detergent, with decreases in protein released and an increase in phospholipid constituents released. Studies on washed cream indicated that some of the changes in the membrane on heating whole milk occurred due to the heat treatment alone, independent of the interactions with skim milk proteins.

Purification and properties of bovine mammary gland N-acetyl-beta-D-glucosaminidase.

Two forms of N-acetyl-beta-D-glucosaminidase were purified from bovine mammary gland by DEAE-cellulose chromatography, Sephadex G-200 gel filtration, affinity chromatography on Con A-Sepharose and preparative isoelectric focusing. The two forms, designated A and B on the basis of their binding to DEAE-cellulose at pH 7, were glycoproteins with different molecular weights as determined by gel filtration and sedimentation equilibrium analysis. The A form had a molecular weight of 118 000, while the B form had a molecular weight of 234 000. Both A and B forms of the purified enzyme showed the presence of two distinct subunits, having apparent molecular weights of 55 000 and 25 000 as determined by sodium dodecyl sulphate-electrophoresis. Amino acid composition of the purified forms showed that a high degree of similarity existed between the two forms. However, the B form had slightly higher levels of serine and threonine than the A form. The structure and possible interrelationship of these two forms in the bovine mammary gland are discussed in relation to the structure of N-acetyl-beta-D-glucosaminidase from other sources.

Detection of bovine mastitis by bromothymol blue pH indicator test.

A simple bromothymol blue indicator test was evaluated for farm diagnosis of mastitis. The test required highly absorbent blotting paper impregnated with four spots of bromothymol blue. Indicator color scores (1 to 4) for quarter foremilks increased with somatic cell count and pH, although variability within each color score was large. Sensitivity of the bromothymol blue test ranged from 51 to 56% and specificity from 89 to 90% for most reference criteria used to classify normal and abnormal milk. Predictability of a positive test ranged from 49 to 52% (false positives 51 to 48%) and predictability of a negative test from 90 to 97% (false negatives 10 to 3%) for the same criteria. Overall the bromothymol blue test incorrectly diagnosed 11 to 20% of 3772 quarters. By classifying color score 2 as negative, predictability of a positive result was 70 to 75% and sensitivity was 26 to 30%. The test can be used by dairy producers to screen herds with a relatively high incidence of mastitis or used in combination with cow cell counts to locate abnormal quarters. The bromothymol blue test was less sensitive than the California Mastitis Test but offered several practical advantages for use on farm.

Proteolysis in milk: the significance of proteinases originating from milk leucocytes and a comparison of the action of leucocyte, bacterial and natural milk proteinases on casein.

The caseinolytic activities at pH 6.8 of polymorphonuclear (PMN) and mononuclear leucocyte homogenates (equivalent to a level of 10(6) cells/ml milk) were less than the levels of natural milk proteinase activity found in milk from healthy cows. Bulk milks contained approximately 4 times more milk proteinase activity than the composite milks from individual healthy cows. Isolated blood leucocytes, when added to raw milk of good bacteriological quality and stored at 5 degrees C, did not readily degenerate and had no detectable effect on the milk proteins even when these cells were completely disrupted by homogenization of the milk. Pasteurization of milk which contained leucocytes caused loss of cell vitality. Extracellular proteinases of psychrotrophic bacteria growing in milk were not detected until the early stationary phase of growth. The total viable count at which this occurred varied greatly. Proteinase production by a pure culture of Pseudomonas fluorescens was not detected in milk stored at 5 degrees C until a viable count of approximately 10(9) colony forming units (c.f.u.)/ml was obtained, whilst normal bulk milks stored at 5 degrees C produced detectable levels of extracellular proteinase(s) when the psychrotrophic flora reached 10(7)-10(8) c.f.u./ml. Casein proteolysis by PMN and mononuclear leucocyte homogenates resulted in similar polypeptide maps, but plasmin and bacterial proteinase isolated from a strain of Serratia marcescens resulted in polypeptide maps different from each other and from that produced by the leucocyte proteinase(s). The rate of proteolysis of caseins by the different proteinase sources appeared to be in the order alpha s1- greater than beta- greater than greater than kappa-casein for the leucocyte extracts, beta- greater than alpha s1- greater than greater than greater than kappa-casein for bovine plasmin and beta- approximately kappa- greater than alpha s1-casein for for S. marcescens proteinase.

Glucose levels in normal and mastitic milk.

Glucose levels in 188 quarter for milks from different cows were determined by an enzymic procedure. Mean glucose content was 0.22 mM (standard error +/- 0.009) and results ranged from 0.02-0.57 mM. Abnormal quarters had lower glucose levels (P less than 0.01) than normal quarters but variability within each classification was large. Glucose content was negatively correlated with both somatic cell count (r = 0.49) and N-acetyl-beta-D-glucosaminidase level (r = -0.61). Milk glucose was considered to be of limited value as a diagnostic test for mastitis.

N-acetyl-beta-D-glucosaminidase (NAGase) levels in bulk herd milk.

N-acetyl-beta-D-glucosaminidase ( NAGase ) levels and somatic cell counts (SCC) were determined monthly for 6 months in the bulk milk of 181 suppliers (1063 samples). A highly significant correlation (r = 0.74; P less than 0.001) was found between supplier's bulk herd milk geometric mean NAGase activity and SCC. Monthly trends which grouped suppliers into various categories defined by different NAGase and SCC thresholds showed that a similar overall pattern was obtained with both SCC and NAGase . However, further analysis indicated that 18% of the bulk milk which had SCC less than 500 X 10(3)/ml had NAGase levels greater than 25 units. Also, 34% of samples with SCC greater than 500 X 10(3)/ml had NAGase levels less than 25 units. Overall, 24% of all samples did not have corresponding NAGase and SCC results. When the bulk milk of 2 commercial dairy herds was tested monthly over 12-18 months whilst the infection status of all quarters in the herds was regularly monitored, those herds with low incidence of mastitis (5% quarters infected) had significantly lower bulk milk SCC and NAGase levels than those with a high incidence of mastitis (22% of quarters infected). This suggests that NAGase measurement on bulk herd milk would be a simple means of monitoring infection status of dairy herds and of rapidly classifying a supplier's milk as being of low, medium or high SCC status. The possible combined use of SCC and NAGase levels in bulk milk monitoring schemes is discussed.

Relationship between the level of N-acetyl-beta-D-glucosaminidase (NAGase) in bovine milk and the presence of mastitis pathogens.

Changes in the level of the tissue damage marker enzyme, N-acetyl-beta-D-glucosaminidase (NAGase) in quarter fore milks were found to be related to the presence and types of pathogenic bacteria present and to somatic cell counts (SCC). Minor pathogens (coagulase-negative staphylococci, Corynebacterium bovis) elicited a mild SCC increase (from a mean of 243 X 10(3)/ml in healthy quarters to 504 X 10(3)/ml in infected quarters) with marginal tissue damage (mean NAGase activity increased from 21 in healthy quarters to 28 in infected quarters). Major pathogens (i.e. Staphylococcus aureus, Streptococcus agalactiae, Str. dysgalactiae and Str. uberis) caused more severe tissue damage (mean NAGase of 48) and SCC increases (mean, 2803 X 10(3)/ml). The NAGase test could also be used effectively on composite milk samples where regular monthly NAGase analysis was able to identify correctly 74% of animals having infected quarters. The possibility of combining SCC and NAGase data in order to give a more definite diagnosis of bovine mastitis is discussed.

The relationship between the levels of free fatty acids, lipoprotein lipase, carboxylesterase, N-acetyl-beta-D-glucosaminidase, somatic cell count and other mastitis indices in bovine milk.

A large-scale survey of milks from healthy and mastitic bovine quarters was undertaken to establish the influence of mastitic infection on milk lipase activity and free fatty acid (FFA) level. Mastitic milks tended to have higher FFA levels, but lower lipoprotein lipase activities compared with milk from healthy quarters. These effects became significant at relatively severe levels of infection. The elevated FFA was attributable to higher FFA levels on secretion and to greater lipolysis during storage. Levels of carboxylesterase activity increased with severity of mastitis and showed high positive correlation with mastitis indices. Marked increases in carboxylesterase, N-acetyl-beta-D-glucosaminidase and phospholipase occurred following the induction of mastitis by intramammary infusion of Escherichia coli endotoxin, in parallel with changes in somatic cell count and other mastitis indices. Relativity little change in lipoprotein lipase activity was observed.

Mastitis diagnostic tests to estimate mammary gland epithelial cell damage.

Quarter fore-milk samples, blood serum, and mammary gland tissue extracts were tested for N-acetyl-B-D-glucosaminidae, lactate dehydrogenase, glutamate--oxaloacetate transmaninase, arylesterase, bovine serum albumin, sodium, conductivity, and somatic cell count. Bovine serum albumin, arylesterase, and sodium were high in blood serum while the first three listed were high in mammary gland tissue. All of these components in milk increased as somatic cell count increased. Correlation coefficients between somatic cell count and each component in order were .84, .53, .55, .81, .74, and .75. Some of these procedures (e.g., serum albumin, dehydrogenase, and transaminae) for estimating the extent of udder epithelial cell damage were less valuable because of lengthy assay times, tedious sample preparation, and unsatisfactory assay procedures. The most suitable approach for measuring udder tissue epithelial damage was the glucosaminidase test which was simple and rapid with high sample throughput. It should be a useful diagnostic aid in mastitis monitoring programs.

Bovine milk N-acetyl-beta-D-glucosaminidase and its significance in the detection of abnormal udder secretions.

A new spectrofluorimetric assay procedure for bovine milk N-acetyl-beta-D-glucosaminidase (NAGase) is described for use as a routine screening test for the detection of abnormal udder secretions. This procedure uses 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as substrate. On the basis of the greater sample throughput, increased product sensitivity detection of NAGase and the absence of turbidity problems, it is considered to be superior to a previously reported spectrophotometric procedure (Kitchen, 1976). The correlation coefficient between the somatic cell count and the fluorimetric procedure using 243 quarter fore-milk samples was 0.86. Distribution studies on bovine milk and mammary gland homogenates indicated that this enzyme activity was located predominantly in the soluble whey protein fraction and the post-microsomal supernatant. Mammary gland secretory cells contained high levels of NAGase and appeared to be the major source of the enzyme in milk whilst NAGase from other sources (white blood cells, blood serum) contributed only a minor proportion (5-15%) of the total activity in milk. The implications of these findings on the value of the NAGase test as a means of mastitis diagnosis are discussed.

Enzymic methods for estimation of the somatic cell count in bovine milk. 1. Development of assay techniques and a study of their usefulness in evaluating the somatic cell content of milk.

Assay procedures were developed for a number of enzymes in milk which apparently originate from leucocytes. The enzymes studied were acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, arylsulphatase, alpha-mannosidase, and catalase. Quarter-milk samples were analysed for enzyme activity and results compared with the electronic cell count and the Wisconsin Mastitis Test. All enzymes measured except acid phosphatase and alpha-mannosidase showed good correlation with the electronic cell count. Of the other 4 enzymes tested, beta-glucuronidase and arylsulphatase were unsuitable as diagnostic aids owing to the lengthy incubation periods required in their assay procedures. The assay of catalase, which involved the measurement of the initial rate of release of O2 using an O2 analyser apparatus, was rapid, sensitive and reasonably reliable, if fresh milk samples were used. The assay procedure for N-acetyl-beta-D-glucosaminidase was considered to be the most reliable, simple and rapid enzymic method for estimating the number of somatic cells in milk.

Kinetic studies on the effect of uridine diphosphate galactose and manganous ions on the reaction between lactose synthetase A protein from human milk and p-hydroxymercuribenzoate.

The inhibition of lactose synthetase A protein by p-hydroxymercuribenzoate at pH7.5 and 25 degrees C, which involves the reaction of one molecule of inhibitor with each molecule of enzyme, was decreased in rate by UDP-galactose, especially in the presence of Mn(2+). Pseudo-first-order rate constants for the reaction between 0.1mm-p-hydroxymercuribenzoate and free enzyme, the enzyme-UDP-galactose complex and the enzyme-Mn(2+)-UDP-galactose complex were 4.4x10(-2), 1.9x10(-2) and 0.3x10(-2)min(-1) respectively. The results also indicated that dissociation constants for UDP-galactose in the enzyme-UDP-galactose and enzyme-Mn(2+)-UDP-galactose complexes were 313 and 16mum respectively, the latter value being similar to the K(m) for UDP-galactose in the lactose synthetase reaction. The protective effect of UDP-galactose and the role of Mn(2+) ions in lactose synthetase are discussed.