Correction: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fibrinolytic therapy in subacute bacterial endocarditis: an experimental study.

The effect of adding fibrinolytic to penicillin therapy in experimentally induced streptococcal bacterial endocarditis has been studied in the rabbit. In 9 day old infected lesions, the vegetations could be substantially reduced in size after a 3 day course of intravenous streptokinase. Quantitative microbiological techniques demonstrated that the addition of streptokinase to standard intravenous penicillin treatment led to more rapid sterilisation of the vegetations. Embolism to lungs and kidneys was assessed in treated and untreated rabbits. Penicillin reduced the rate of embolism but the addition of streptokinase reversed this effect and gave values similar to those recorded in untreated animals.

Macrophage infiltration of breast tumours: a prospective study.

In 50 cases of infiltrating breast cancer investigated in a prospective study the number of macrophages within each tumour was assessed. The macrophages were identified by their cytoplasmic acid phosphatase activity. The number of lymphocytes and plasma cells within the tumours were graded by a scoring technique. Significantly fewer cases with metastases were found among those with high macrophage and plasma cell scores. There was no correlation between lymphoreticular infiltration and the degree of tumour differentiation, but in cases without metastases the lymphoreticular infiltration between tumour cells was nearly always only slight when the macrophage score was low.

Pathological changes in virus infections of the lower respiratory tract in children.

The pathological changes are described in 22 children with proven or suspected virus infection of the lower respiratory tract. Two main patterns of disease were found: acute bronchiolitis and interstitial pneumonia. Particular viruses were not specifically associated with particular histological changes. The prime importance of the respiratory syncytial virus (RSV) as a cause of disease and death in young infants is again shown. Structural lesions and clinical dysfunction correlate fairly well; in acute bronchiolitis the main lesion is epithelial necrosis when a dense plug is formed in the bronchiolar lumen leading to trapping air and other mechanical interference with ventilation: in interstitial pneumonia there is widespread inflammation and necrosis of lung parenchyma, and severe lesions of the bronchial and bronchiolar mucosa as well. The implications of these structural changes for clinical management are discussed. The possibility of a hypersensitivity reaction in the cot death syndrome is raised, mediated by a serum antibody-antigen-complement reaction.

Metabolism, excretion and pharmacokinetics of a single dose of [14C]-raltitrexed in cancer patients.

Raltitrexed (Tomudex) is a specific inhibitor of thymidylate synthase and has recently been licensed in Europe for use in the treatment of advanced colorectal carcinoma. This study evaluated the metabolism, excretion and pharmacokinetics after a single dose of 3.0 mg/m2 [14C]-raltitrexed in patients with advanced solid malignancies not amenable to curative therapy. From April 1994 to July 1995, nine patients (six men and three women) were recruited into the study. Pharmacokinetics analysis was performed during the first cycle of treatment in all patients and, in two patients, limited sampling was done prior to and during the second cycle of treatment. The mean observed peak plasma concentration (Cmax) was 700.6 ng/ml and the median time (tmax) to reach maximal raltitrexed concentrations was 15 min after the initiation of the infusion. After reaching Cmax the drug declined in a triexponential manner with a terminal half-life of 257 h. The AUC0-infinity as predicted by the pharmacokinetic model was 2341.7 ng h ml(-1). Clearance was 41.3 ml/min, of which renal clearance accounted for 50-60%. Urinary collection for the measurement of radiolabeled drug revealed that renal excretion extrapolated to infinity accounted for 40% of the total radioactive dose. Faecal excretion accounted for only 3% of the dose when samples were collected to day 5 in the first six patients. Collection was extended to 10 days in the last three patients and faecal elimination accounted for 14% in these patients. Raltitrexed measurements prior to subsequent doses suggest that there was no accumulation of the drug with repeated administration. Low levels of radioactivity measured in the red cell pellets on days 15, 22 and 29 are likely to represent drug retained by newly forming red cells at the time of dosing. Examination of the urine revealed that the drug was excreted unchanged. The toxicities seen were in line with those encountered in previous studies. Grade 3 and 4 thrombocytopenia occurred in three patients and grade 3 neutropenia occurred in two patients.

Chronopharmacology.

The time of day that drugs are given can have a profound effect upon both their pharmacodynamics, i.e. their effectiveness and toxicity, and their pharmacokinetics, i.e. the way they themselves behave in the body. The chronopharmacokinetics of relatively few drugs have been investigated and are mostly those used for the treatment of cancer. We have shown that the toxicity and chronopharmacokinetics of drugs can be altered by pretreatment with steroids or melatonin.

Some morphometric methods for the central nervous system.

It is the quantitative study of the central nervous system to enumerate neurone populations. Since neurones are generally arranged in non-random patterns the conventional method of counting with a square lattice graticule in the ocular of the microscope is severely restricted. Appropriately tailored methods of (a) counting, or (b) estimating the density of a neurone population must therefore be used. Four types of neuronal distribution pattern are discussed and morphometric methods adapted to them are presented, as follows: (1) Where the whole population is circumscribed, as in the anterior horn motor neurones of the cord, direct counting is feasible. Only those cells whose nucleoli are visible should be included; this diminishes the bias in favour of large cells, and if one corrects for the fact that larger neurones have larger nucleoli the bias can be eliminated completely. (2) Where neurones are situated on an "interface", as Purkinje cells are, the length of its profile (in this instance the boundary between the molecular and granular zones) is estimated by superimposing parallel lines on the microscopical image and counting intersections as these lines cross the interface. The number of Purkinje cells in each particular field is counted at the same time. A "linear dinsity" (PL) is then calculated from the formula pl = 2n/pid where n is the quotient of (a) the number of cells counted along (b) an estimated length of profile, and d is the distance between the parallel lines of the graticule. For comparative studies between cerebella of different sizes a correction factor is easily introduced. Those who prefer to work on photomicrographs can use a mapping wheel to measure a length of profile and then proceed as before. (3) Where neurones are organised in a curving band, as in the dentate nucleus, another form of cell density can be established. The microscope is focused on a neurone at random, and the distance from this cell to its nearest neighbour is measured, preferably by means of a screw-micrometer eyepiece. The mean value r of a number of such measurements is substituted in a formula which gives the "areal density" of the neurone population: pa = 1/4r2. This method can be applied to photomicrographs by using a pair of dividers and/or an accurate rule to measure the set of r values. (4) In large collections of neurones, e.g., in the thalamus, three methods are available: (a) the nearest neighbour method; (b) a conventional squared graticule count, and (c) a count of cells intersected by a line probe as in Haug's (1972) technique (fig. 5), or a modified form of Strong's (1966) transect method.

Radioimmunoassay of methotrexate: use of 75Se-labelled methotrexate.

75Se-labelled methotrexate has been used as the radiolabel in a radioimmunoassay for methotrexate and its use compared with that of 3H-methotrexate. The gamma-labelled drug has proved to be extremely reliable and easier to use than 3H-methotrexate. Results obtained with it compare well with those obtained with tritiated drug. There are practical advantages in using a gamma emitter, and it is suggested that the modified radioimmunoassay is invaluable for routine monitoring of methotrexate in patients undergoing chemotherapy with the drug.

The measurement of deoxynucleotide (dNTP) pools by radioimmunoassay (RIA).

Radioimmunoassay provides an alternative, sensitive and reproducible high throughput method for the measurement of dNTP pools. The extent and duration of inhibition of TS can be investigated by determination of the TTP and "dUMP" pools and the effects of D1694 and ZD9331 have confirmed their biochemical profiles. The RIAs will be useful in providing information for the design of treatment protocols for TS inhibitors and with the specific assay of dUTP, on mechanisms of cell death in different cell lines.

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

A prospective, blinded analysis of thymidylate synthase and p53 expression as prognostic markers in the adjuvant treatment of colorectal cancer.

BACKGROUND: Despite previous studies, uncertainty has persisted about the role of thymidylate synthase (TS) and p53 status as markers of prognosis in colorectal cancer (CRC). PATIENTS AND METHODS: A total of 967 patients accrued to a large adjuvant trial in CRC were included in a prospectively planned molecular substudy, and of them, 59% had rectal cancer and about 90% received adjuvant chemotherapy (either systemically or randomly allocated to intraportal 5-fluorouracil infusion or both). TS and p53 status were determined, blinded to any clinical data, by immunohistochemistry using a validated polyclonal antibody or the DO-7 clone, respectively, and their relationships with overall survival were examined. RESULTS: High TS expression was observed in 58% and overexpression of p53 in 60% of tumours. TS expression correlated with tumour stage, and p53 overexpression, with rectal cancers. There was no evidence that either marker was significantly associated with survival by either univariate (TS hazard ratio (HR) = 0.94, 95% CI 0.76-1.18 and P = 0.6 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.9) or multivariate analyses (TS HR = 0.99, 95% CI 0.79-1.25 and P = 0.9 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.8). CONCLUSIONS: Neither TS nor p53 expression has significant prognostic value in the adjuvant setting of CRC.

A phase I pharmacokinetic and pharmacodynamic study of BGC9331 and carboplatin in relapsed gynaecological malignancies.

BGC9331 is a rationally designed, specific nonpolyglutamatable thymidylate synthase (TS) inhibitor that is active in gynaecological malignancies. In the light of the sensitivity of human ovarian tumour cell lines to BGC9331 and non-cross resistance to platinum drugs, we studied the combination BGC9331/carboplatin (BCA) in a phase I (PI) pharmacokinetic (PK) and pharmacodynamic (PD) study in platinum pretreated gynaecological malignancies. Patients were >or=18 years or over, with a histologically confirmed gynaecological malignancy, radiological evidence of relapse, and a platinum treatment free interval of at least 6 months. Up to three prior lines of chemotherapy were permitted. Carboplatin (AUC5) and BGC9331 were administered on day 1, and BGC9331 was also given on day 8 of a 21-day cycle. In total, 14 patients were enrolled, and treated with BGC9331 at four dose levels, 40, 65, 85 and 100 mg m-2. The principal grade 3 and 4 haematological toxicity was neutropaenia. The principal nonhaematological toxicities were lethargy and nausea. Dose-limiting toxicities were seen in two patients at 100 mg m-2 BGC9331 (grade 4 neutropaenia>7 days, and grade 4 fatigue>7 days). Plasma BGC9331 was measured by an ELISA that was adapted for use in humans. Carboplatin was assayed by flameless atomic absorption spectrometry. There was no PK interaction between the two drugs. Plasma deoxyuridine was elevated indicating TS inhibition to at least day 12. Antitumour activity was observed in four out of 14 (28%) of patients. In conclusion, the combination of BGC9331 and carboplatin is well tolerated with no significant PK interaction between the two drugs. There is evidence of TS inhibition with the combination. We have demonstrated antitumour activity in platinum pretreated gynaecological malignancy. Further exploration of this combination in this disease is warranted.

A Phase I study of the angiogenesis inhibitor SU5416 (semaxanib) in solid tumours, incorporating dynamic contrast MR pharmacodynamic end points.

SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone; semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor receptor (VEGFR2). A Phase I dose escalation study was performed. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used as a pharmacodynamic assessment tool. In all, 27 patients were recruited. SU5416 was administered twice weekly by fixed rate intravenous infusion. Patients were treated in sequential cohorts of three patients at 48, 65, 85 110 and 145 mg m-2. A further dose level of 190 mg m-2 after a 2-week lead in period at a lower dose was completed; thereafter, the cohort at 145 mg m-2 was expanded. SU5416 showed linear pharmacokinetics to 145 mg m-2 with a large volume of distribution and rapid clearance. A significant degree of interpatient variability was seen. SU5416 was well tolerated, by definition a maximum-tolerated dose was not defined. No reproducible changes were seen in DCE-MRI end points. Serial assessments of VEGF in a cohort of patients treated at 145 mg m-2 did not show a statistically significant treatment-related change. Parallel assessments of the impact of SU5416 on coagulation profiles in six patients showed a transient effect within the fibrinolytic pathway. Clinical experience showed that patients who had breaks of therapy longer than a week could not have treatment reinitiated at a dose of 190 mg m-2 without unacceptable toxicity. The 145 mg m-2 dose level is thus the recommended dose for future study.