A proof-of-principle pharmacokinetic, pharmacodynamic, and clinical study with purine nucleoside phosphorylase inhibitor immucillin-H (BCX-1777, forodesine).

The discovery of purine nucleoside phosphorylase (PNP) deficiency and T lymphocytopenia suggested that inhibition of this enzyme could serve as a therapeutic target. Inhibitors of PNP failed until structure-based synthesis of immucillin-H (BCX-1777, forodesine), a transition-state analog of PNP. The picomolar potency for PNP, T cell-selective cytotoxicity, and animal studies provided the rationale for use of forodesine in T-cell malignancies. Five patients were treated with an intravenous infusion of forodesine (40 mg/m2) on day 1; treatment continued on day 2; forodesine was administered every 12 hours for an additional 8 doses. Plasma and cellular pharmacokinetics and pharmaco-dynamics were investigated. Median peak level of forodesine (5.4 microM) was achieved at the end of infusion. This level was sufficient to increase plasma 2'-deoxyguanosine (dGuo) concentrations in all patients. Intracellular deoxyguanosine triphosphate (dGTP) increased by 2- to 40-fold in 4 of 5 patients (8 of 9 courses) and correlated with antileukemia activity in 4 patients. However, objective responses were not observed. This was the first clinical study in humans to demonstrate the plasma pharmacokinetics and the pharmacodynamic effectiveness of the PNP inhibitor, forodesine; however, regrowth of leukemia cells in the blood and marrow after course 1 suggested that a different therapeutic schedule should be considered for future studies.

Aggressive chemotherapy for acute leukaemia frequently causes intestinal protein leakage.

Cytostatic drugs are known to produce disturbances in intestinal absorption of carbohydrates. To further explore the gastrointestinal (GI) toxicity of cytostatic therapy, 37 patients with acute leukemia were investigated during and/or after remission induction courses by the use of the differential sugar absorption test (DSAT) and the intestinal clearance of alpha-1-antitrypsin (ClAAT). The ratio of the lactulose to the mannitol excretion in the urine was found abnormal in 44% of the tests. The ClAAT was increased in 74% of tests. The tests results differed considerably from patient to patient and depended on the chemotherapy course; correlation between the tests was low, probably indicating the unrelated pathophysiological processes were measured. After haematological regeneration, abnormal test results normalised. It is concluded that aggressive chemotherapy not only causes a reduction in the absorption of sugars, but commonly also protein leakage. These GI side-effects are reversible, and the application of both tests in combination provides a practical and reproducible method for investigation of GI toxicity in patients treated with cytostatic drugs.

Richter's syndrome with two different B-cell clones.

The diagnoses of chronic lymphocytic leukemia (CLL) in prolymphocytic transformation, and diffuse large cell lymphoma (DLC), were made simultaneously in a 71-year-old man. The DLC showed mu lambda surface immunoglobulin. The CLL in a lymph node and in the peripheral blood showed mu kappa. Immunoglobulin gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of the CLL and DLC. Other cases reported of Richter's syndrome are discussed, and it is concluded that there may be two types of Richter's syndrome, those arising from transformation of a single clone, and those occurring from expansion of two morphologically and immunologically distinct clones, as, it is believed, is the case in this patient.

Purine excretion during tumor lysis in children with acute lymphocytic leukemia receiving allopurinol: relationship to acute renal failure.

We measured serial urine levels of hypoxanthine, xanthine, and uric acid in 19 children with acute lymphocytic leukemia (ALL) receiving allopurinol therapy during tumor lysis; four of these children developed acute renal failure. The urinary excretion of uric acid rose moderately from 447 +/- 251 micrograms/dl glomerular filtrate before chemotherapy to 778 +/- 463 micrograms/dl glomerular filtrate during tumor lysis (P less than 0.05) whereas the urinary excretion of hypoxanthine (17.9 +/- 15 to 292 +/- 213 micrograms/dl glomerular filtrate) and xanthine (74 +/- 62 to 1091 +/- 1085 micrograms/dl glomerular filtrate) rose dramatically (P less than 0.001). The urinary excretion of uric acid, hypoxanthine, and xanthine per deciliter of filtrate was significantly higher (P less than 0.001) in those who developed acute renal failure than in those who did not, but the highest urine concentration of these purine metabolites did not differ in the two groups. In all 19 children, the highest urine concentration of uric acid and hypoxanthine during tumor lysis did not exceed the solubility limit of each in an alkaline urine specimen. In contrast, the peak urine concentration of xanthine exceeded its solubility limit in an alkaline urine specimen in 16 of 19 children. The urine sediment during the period of tumor lysis was examined by diffuse reflectance infrared spectroscopy; precipitated xanthine was found in sediment from eight of the 19 children, was significantly (P less than 0.001) associated with a urine xanthine level greater than 350 mg/dL, and occurred with equal frequency in those who did or did not develop acute renal failure. We conclude that urinary excretion of hypoxanthine and xanthine increases dramatically whereas uric acid excretion rises moderately in children undergoing tumor lysis while receiving allopurinol, that acute renal failure occurs in children with a higher purine load per deciliter of glomerular filtrate, but that factors other than tubular precipitation of purine metabolites are likely to be involved in the pathogenesis of renal failure during tumor lysis.

Urinary nucleosides in leukemia: laboratory and clinical applications.

Urinary nucleosides offer a number of useful laboratory and clinical applications in the study and analysis of leukemia. There are significant differences in the excretion of modified nucleosides between normal individuals and individuals with various forms of leukemia, as well as between leukemia patients at opposite ends of the clinical spectrum, i.e., those with active disease and those in remission. The nucleoside excretion levels correlate to bone marrow tumor burden in certain forms of leukemia, and limited serial data indicate the potential value of the nucleosides for predicting relapse before the disease deterioration can be recognized clinically. In addition, it may be feasible to assess the effectiveness of chemotherapy used in the treatment of leukemia much more rapidly with the urinary nucleoside markers than with conventional invasive methods.

Monoclonal Bence Jones proteinuria in chronic lymphocytic leukaemia.

A series of 52 consecutive patients with newly diagnosed chronic lymphocytic leukaemia (CLL) was investigated for the presence of serum and urine monoclonal immunoglobulins. The overall incidence of monoclonal gammopathy (MG) was 69%. Serum M components belonging to the IgG and IgM classes were found in 27% of cases with a concentration ranging from 1.7 to 40.3 g/l (mean 10 g/l). A monoclonal free light chain, i.e. Bence Jones protein (BJP), was documented in the urine of 65% of cases. In 42% of the 52 patients the urinary excretion of BJP represented the sole detectable monoclonal immunoglobulin abnormality. The occurrence of serum monoclonal immunoglobulins was not found to correlate with the stage or progression of the disease. Conversely, the isolated urinary excretion of BJP showed a relationship with the tumour load, as evaluated in terms of clinical enlargement of lymphoid areas. The presence of BJP alone was also a major distinctive feature of patients with more aggressive disease. These preliminary data would suggest that the isolated urinary excretion of BJP may represent a parameter of clinical significance in evaluating patients with CLL.

Transient idiopathic hyperammonaemia in adults.

Transient severe hyperammonaemia developed in the absence of serious liver dysfunction in three patients being treated for acute leukaemia. The onset of the biochemical disturbance was abrupt and led rapidly to acute encephalopathy, fatal in two cases. In the third patient, prompt initiation of aggressive haemodialysis and intravenous sodium benzoate and sodium phenylacetate infusion successfully controlled plasma ammonium levels until they spontaneously resolved. The cause of the disorder remains to be determined, but urinary nitrogen partition studies suggest temporary impairment of ureagenesis in a catabolic setting as a major pathophysiological feature of this disorder. The absence of liver disease, the normal mitochondrial ultrastructure seen in two cases, and the plasma aminoacid profiles observed serve to distinguish this disorder from others such as Reye's syndrome.

Pharmacokinetics of elimination of 6-mercaptopurine in the urine of children with acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP)-induced sodium azide--iodine reaction was adapted for determination of 6-MP in urine of four children treated with single oral doses of the drug in tablets. Open one-compartment body model was assumed, and first-order elimination rate constants (K) and biological half-life times (t0,5) were calculated, and their precision was determined by statistical treatment.

High incidence of monoclonal proteins in the serum and urine of chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is generally considered a nonsecretory B cell immunoproliferative disorder. Conventional electrophoretic and immunoelectrophoretic methods have revealed serum monoclonal proteins in less than 10% of these patients. However, there is increasing experimental evidence from in vitro studies demonstrating that CLL cells may secrete immunoglobulins, particularly free light chains. We examined the serum and urine of 36 consecutive CLL patients for monoclonal proteins using sensitive immunochemical methods (high resolution agarose gel electrophoresis combined with immunofixation). The results obtained were correlated with the Rai stage, quantitative immunoglobulin levels, and lymphocyte membrane immunoglobulin phenotype of the leukemic cells. Twenty-three monoclonal proteins were identified in the serum or urine of 22 patients, an incidence of 61%. Six patients had serum monoclonal proteins, seven had only urinary monoclonal proteins, and nine had monoclonal proteins in serum and urine. In every instance the monoclonal protein was the same light chain type as expressed on the leukemic cells. Our findings suggest that the monoclonal proteins observed in the serum or urine of CLL patients are secretory products of the tumor cells and that their discovery is a function of the sensitivity of the method used for their detection.

Urinary glycoproteins in acute leukemias: a 41 000 dalton glycoprotein follows the kinetic of cytoreduction.

Glycoproteins of leukemic cells and 24-hour urinary proteins were subjected to SDS polyacrylamide gel electrophoresis followed by affinity labelling I125 with Concanavalin A, indicating glycoproteins with mannose and/or glucose carbohydrate residues. Among the cellular glycoproteins a 41 000 dalton glycoprotein appeared under induction therapy in close correlation to the reduction of leukemic cells in ALL as well as in AML.

Excretion of modified nucleosides during development of malignant lymphomas in mice after whole body irradiation.

During x-ray-induced development of malignant lymphomas in mice their urinary excretion of eight modified nucleosides was monitored and the values were compared to the results of the histological examination of the animals at time of their sacrifice. It was found that the pathologically augmented excretion of modified nucleosides begins as much as several weeks before the malignant lymphomas can be diagnosed clinically. Thus some mice had increased levels of modified nucleosides even 10 weeks before sacrifice, though at the time of sacrifice the histological investigation revealed only some small foci of reticulum cell neoplasm in their spleen. It is therefore stressed that the usefulness of the determination of urinary modified nucleosides as an early noninvasive screening test for cancer in man and as an in vivo carcinogenicity test should be evaluated.

Relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia.

The levels of urinary excretion of five modified nucleosides were quantitated by high-performance liquid chromatography for 15 normal children and 24 children with acute lymphoblastic leukemia (ALL). Excretion of each nucleoside decreased linearly with age when quantitation was based on urine creatinine content. Patients with childhood ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine, N2,N2-dimethylguanosine, 1-methylguanosine, and pseudouridine in their urine when compared to the concentrations in either patients in remission (P less than 0.001, P less than 0.001, P less than 0.01, and P less than 0.05, respectively) or normal controls (P less than 0.001, P less than 0.02, P less than 0.01, and P less than 0.001, respectively). Excretion of 2-pyridone-5-carboxamide-N'-ribofuranoside did not show significant differences. Urinary excretion of 1-methylinosine demonstrated a positive linear relationship with the percentage of blast cells in the bone marrow [correlation coefficient (r) = 0.90]; the other nucleosides had lower degrees of correlation. In comparison, the absolute blast cell count in the peripheral blood showed less correlation to the percentage of blast cells in the bone marrow (r = 0.47) than did four of the five nucleosides. The data demonstrate that excretion of modified nucleosides reflects disease activity in childhood ALL and that the urinary nucleosides could be useful clinical markers for this disease.

S-adenosylhomocysteine catabolism and basis for acquired resistance during treatment of T-cell acute lymphoblastic leukemia with 2'-deoxycoformycin alone and in combination with 9-beta-D-arabinofuranosyladenine.

A patient with refractory T-cell acute lymphoblastic leukemia was treated with eight courses of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), over a 5-month period. After developing resistance to dCF, he responded to treatment with the combination of dCF and 9-beta-D-arabinofuranosyladenine (ara-A). We monitored the levels in plasma and urine of adenosine, 2'-deoxyadenosine, and ara-A as well as the accumulation of their nucleotide derivatives in erythrocytes and circulating lymphoblasts. We also monitored the activities of adenosine deaminase and S-adenosylhomocysteine (AdoHcy) hydrolase and the concentrations of AdoHcy and S-adenosylmethionine in lymphoblasts. Production of 2'-deoxyadenosine was related to both the duration of dCF infusion and the magnitude of cytolysis that occurred during treatment: much more 2'-deoxyadenosine was produced by dCF infusion when disease was active than by the same infusion given during remission. Resistance to dCF was associated with a decrease of greater than 90% in the amount of deoxyadenosine 5'-triphosphate accumulated by circulating lymphoblasts. Infusion of dCF resulted in increases of up to 20-fold in the concentration of AdoHcy in circulating lymphoblasts, causing a decrease in the S-adenosylmethionine:AdoHcy ratio (the "methylation index") from a pretreatment value of greater than 40:1 to less than 4:1. This ratio decreased to 2.5:1 during combined treatment with dCF and ara-A, which caused nearly complete inactivation of lymphoblast AdoHcy hydrolase. Decline in the methylation index was accompanied by inhibition of the methylation of newly synthesized lymphoblast RNA. Impaired ability to catabolize AdoHcy may have contributed to the cytolytic responses to dCF and ara-A, as well as to hepatic and central nervous system toxicity associated with their combined use.

Monoclonal immunoglobulin light chain in urine of patients with B lymphocytic disease: its source and use as a diagnostic aid.

The presence of tumour-related monoclonal light chain has been sought in urine as an immunochemical aid in the diagnosis of B lymphocytic neoplasms. The technique of isoelectric focusing in agarose followed by immunofixation has been applied to concentrated urines from 41 patients. In chronic lymphocytic leukaemia involving neoplastic B lymphocytes, monoclonal light chain was detected in 14 out of 19 patients investigated. For 2 of the positive cases (one kappa light chain type and one lambda light chain type) the urinary light chains were compared directly with culture fluids obtained after incubation of the corresponding neoplastic cells obtained from the patient's peripheral blood: identity of the light chains from urine and cells was established by isoelectric focusing demonstrating for both patients that the tumour cells were the source of the urinary light chain. In patients with non-Hodgkin's lymphoma involving neoplastic B lymphocytes, urinary monoclonal light chains were found in 7/16 of those studied. Such light chains were not detected in 11 control subjects, in 3 patients with true histiocytic tumours or in 2 patients with enlarged reactive lymph nodes. The technique is simple to perform and provides information for diagnosis and possibly monitoring of B cell neoplasms.

Urinary 6-hydroxymethylpterin levels accurately monitor response to chemotherapy in acute lymphoblastic leukemia.

We have recently shown that the levels of urinary 6-hydroxymethylpterin are highly elevated (3 to 20 fold) in a variety of human malignancies as compared to its urinary excretion in patients with nonmalignant diseases or normal healthy subjects. In the subsequent studies, this parameter has been shown to be a reliable index for accurately monitoring the response of patients with acute myeloblastic leukemia (AML) during chemotherapy. In this study, the excreted urinary levels of 6-hydroxymethylpterin as well as the bone marrow lymphoblast values were measured simultaneously in four patients with acute lymphoblastic leukemia (ALL) during antileukemic therapy. Various drug regimens employed for treatment have also been indicated. A good correlation was seen between urinary pterin levels and % blasts during treatment, thus accurately monitoring remission or relapse of the disease in response to the antileukemic therapy. These results again conclusively show that the simple noninvasive determination of 6-hydroxymethylpterin provides a reliable index of the total tumor load in acute lymphoblastic leukemic cases undergoing treatment.

Differential excretion of modified nucleosides in adult acute leukemia.

Excretion of modified nucleosides in urine was measured in 23 adults with acute leukemia to determine correlation of nucleoside excretion with disease activity. In addition, differences in excretion between patients with acute lymphoblastic leukemia (ALL) and patients with acute myeloid leukemia (AML) were established. Six modified nucleosides were resolved and quantitated by reversed-phase high-performance liquid chromatography (HPLC). Patients with ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine and N2,N2-dimethylguanosine in their urine compared to patients in remission (p less than 0.01, p less than 0.05, respectively). One patient with ALL was followed with serial nucleoside determinations over a period of 18 mo; nucleoside excretion correlated closely with disease activity. Nucleoside excretion in patients with AML did not change significantly with disease activity. Considering only those patients at initial diagnosis or in relapse, excretion of 1-methylinosine and N2,N2-dimethylguanosine was significantly higher in ALL than in AML (p less than 0.01, p less than 0.05, respectively). Thus, urinary excretion of 1-methylinosine and N2,N2-dimethylguanosine by adults with acute leukemia may prove to be valuable clinically in following disease activity in patients with ALL and in distinguishing patients with ALL from those with AML.

Excretion of polyamines by children with leukemia during chemotherapy.

Evidence is presented showing a relation between polyamine concentration and the methylation of tRNA in vitro and in vivo. Polyamine excretion in urine of children with ALL and AML is slightly elevated before the commencement of chemotherapy. Immediately thereafter, excretion of acetylputrescine increases drastically over a period of at least 30-60 days. The elevation seems to be caused by different factors, e.g., destruction of tumor cells, induction of ornithindecarboxylase, and cell recovery after termination of chemotherapy. Acetylspermidine excretion also increases 3-4 days after the beginning of chemotherapy. A positive correlation exists between leukocyte counts and excretion of acetylspermidine. The ratio of acetylspermidine excretion at days 3-4 of the therapy to that before commencement of chemotherapy could be an indicator of response to the therapy.