SDZ PSC 833, a non-immunosuppressive cyclosporine: its potency in overcoming P-glycoprotein-mediated multidrug resistance of murine leukemia.

Cyclosporin A (CsA, Sandimmune) is known to reverse P-glycoprotein (P-gp170)-mediated multidrug resistance as efficiently as other prototype compounds of resistance modifiers. The immunosuppressive activity and nephrotoxicity of CsA, however, may limit its clinical use. PSC-833, a new cyclosporine, exerts a similar resistance-modifying activity but lacks toxicity or immunosuppressive activity. We have tested its potency in vitro and in vivo on the L1210 leukemia cell line transfected with a full-length cDNA copy of the human mdr I gene, which showed a stable 30-fold resistance towards adriamycin as compared to the parental cell line. In vitro growth of the transfected cell was unchanged. In vivo growth was less aggressive; the survival time of inoculated mice was prolonged. In vitro, PSC-833 was at least as potent as CsA or verapamil in reversing multidrug resistance. In vivo, the drug-resistant L1210 leukemia was completely unresponsive to i.v. monotherapy with adriamycin at its maximum tolerated dose (MTD). PSC-833 enhanced the activity and toxicity of adriamycin. The MTD of adriamycin was about 3 times lower than when given alone. On this basis, the MTD of i.v. adriamycin in combination with oral PSC-833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred.

In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.

The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier. In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines. In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice). Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o. were otherwise ineffective doses of anticancer drugs given i.p. This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o. 4 h before a doxorubicin i.p. injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times. Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.

Inhibition of interleukin-1 release by IX 207-887.

Compound IX 207-887 is a novel antiarthritic agent which inhibits the release of interleukin-1 (IL-1) from human monocytes and mouse peritoneal macrophages in vitro at concentrations which are achieved therapeutically in human rheumatoid arthritis and in animal models of arthritis. In the present studies IL-1 activity in conditioned media, homogenates or lysates was monitored using four independent assay systems. Biologically active IL-1 was determined by, a) the induction of latent metalloproteinase-release from rabbit articular chondrocytes, which is relatively specific for IL-1 and b) by a sensitive thymocyte proliferation assay. Immunoreactive IL-1-beta was assayed by RIA and ELISA. In all test systems IX 207-887 significantly reduced both biologically active and immunoreactive IL-1 in culture media, whereas the levels of IL-1 in homogenates or lysates were either unaffected or only marginally reduced. The release of other monokines tested, such as interleukin-6 and tumour necrosis factor-alpha, and the secretion of lysozyme were only marginally influenced. IX 207-887 neither affected the adherence of human monocytes nor markedly inhibited IL-1 or IL-2-induced thymocyte proliferation. In the chondrocyte test no IL-1 antagonistic activity of IX 207-887 could be observed. All of these data indicate that IX 207-887 has the novel property of being an inhibitor of IL-1 release.

Overcoming multidrug resistance in Chinese hamster ovary cells in vitro by cyclosporin A (Sandimmune) and non-immunosuppressive derivatives.

Cyclosporin A (Sandimmune) increased the in vitro susceptibility of 'parental' and 'multidrug-resistant' (MDR) chinese hamster ovary (CHO) cell lines to three anti-tumour drugs: colchicine, daunomycin, and vincristine. Several immunosuppressive or non-immunosuppressive derivatives of cyclosporin (Cs) were compared for their ability to sensitise both parental and MDR cells to chemotherapeutic agents. Although 5-10-fold increases of sensitivity to anti-tumour drugs could be obtained for cells of the parental line with several Cs-derivatives, the largest 'gains' of sensitivity (chemosensitisation) were obtained for the cells of the MDR line and with only some of the Cs derivatives. The MDR cells employed displayed the typical MDR phenotype. However, we found no correlation between the immunosuppressive activity of Cs derivatives and their capacity to reverse MDR and all four possible combinations of these two activities could indeed be shown among the tested Cs derivatives. This study demonstrates for the first time that some immunosuppressive Cs can be devoid of chemosensitising activity.

Flow cytochemical patterns of white blood cells in human hematopoietic malignancies: III. Miscellaneous hemopoietic diseases.

Peripheral blood samples from 48 untreated and 20 treated patients with disease entities that directly or indirectly affect hematopoiesis [dys-myelopoietic syndrome (DMS), refractory anemia with excess blasts (RAEB) or in transformation (RAEBIT), lymphoma, myeloma, acquired immunodeficiency syndrome (AIDS), and solid tumors with uninvolved bone marrow] were measured with the Technicon H-6000 automated hematology analyzer; this instrument provides a differential count on 10(4) white blood cells (WBC) effected by means of flow cytochemistry (peroxidase content) and volume (light scatter) discrimination. Cases with DMS and RAEB showed statistically significantly lower WBC counts than normal, whereas cases with lymphoma showed significantly higher values. No disease entity demonstrated changes in mean peroxidase activity (MPA) that were significantly different from normal, although all disease entities, including cases with solid tumors, showed significantly higher (two to severalfold) proportions of cells with high peroxidase (HPX) content, probably as a reflection of a disturbance of normal hemopoiesis with the emergence of younger granulocytic forms. All cases with paraleukemia (DMS, RAEB, and RAEBIT) showed significantly higher values of large unstained cells (LUC), whereas cases with lymphoma showed significantly lower LUC values. There were no statistically significant differences for any parameter (WBC counts, MPA, HPX, or LUC) among the paraleukemia subtypes. However, based on the displayed trends, a case presenting with dyserythropoiesis, relatively low WBC counts, abnormal HPX values, and LUC below 10% should be suspected for RAEB, whereas the presence of greater than 10% LUC and almost normal or even slightly elevated WBC counts should suggest a more accelerated phase of RAEB. Unless complicated by a leukemic phase, cases of lymphoma or myeloma did not display changes in any of the parameters analyzed by the H-6000. Similarly, patients with AIDS had no overt changes other than a trend to lower WBC counts with occasionally higher or lower absolute lymphocyte counts than normal. The peripheral blood of patients with solid tumors displayed a slight increase in HPX, suggesting an indirect effect on hemopoiesis since careful workup failed to demonstrate bone marrow involvement. Our data demonstrates that an H-6000 analysis has a role in the evaluation and follow-up of all these entities particularly to document leukemic transformation of either lymphoma, myeloma, or RAEB.

Flow cytochemical patterns of white blood cells in human haematopoietic malignancies. II. Chronic leukaemias.

Peripheral blood samples from 73 patients with chronic leukaemia were measured with the Technicon H-6000 automated haematology analyser to provide flow cytochemical (peroxidase content) and volume (light scatter) discriminated scattergram patterns. For chronic granulocytic leukaemia (CGL), these patterns were so reproducible and distinct that they allowed an immediate diagnosis even without the benefit of microscopic examination. Relative and absolute basophilia was an invariable feature, and remained detected by the H-6000 even when the patient was in haematologic and cytogenetic remission or progressed into blast crisis (BC). Most patients in BC also demonstrated an inordinately high number of large unstained cells (LUC) and high proportions of 'lymphocytes' (small blasts with no peroxidase content by visual inspection). Thus, for patients with CGL, LUC values above 10%, and/or steady increments in the proportion of 'lymphocytes', merit concern as these changes may herald an accelerated phase of disease. The scattergram pattern of untreated chronic lymphocytic leukaemia (CLL) showed a dense accumulation of data points within the lymphocytic 'box' with a small cluster of granulocytic elements. Most patients also had a frankly abnormal proportion of LUC. Sixteen patients with CLL were compared for ratios of LUC to lymphocytes and stage of disease; patients with the most advanced stage (IV) had the highest, statistically significant values, than the patients with more benign disease. Thus, it is possible that follow up with this instrument of patients with CLL will also allow early detection of an impending prolymphocytoid transformation (accelerated phase) of this disease.

Flow cytochemical patterns of white blood cells in human haematopoietic malignancies. I. Acute leukaemias.

Peripheral blood samples from 118 patients with acute leukaemia (68 untreated; 50 treated) were measured with the Technicon H-6000 automated haematology analyser. This instrument provides, in addition to measurements of the classical haematology parameters (i.e. cell counts, haemoglobin concentration, etc.), a differential count on 10(4) WBC effected by means of flow cytochemistry (peroxidase content) and volume (light scatter) discrimination. Disregarding RBC and platelet counts and their volume distribution profiles, the most important diagnostic parameters for leukaemic disease were the WBC count, the WBC differential count, and the proportions of large unstained cells (LUC) and high peroxidase (HPX) cells obtained by the automated differential count as well as the mean value of the WBC peroxidase content distribution (MPA). Granulocytic leukaemias had lower MPA than normal and lymphocytic leukaemias had MPA values above normal. M1 leukaemias were also characterized by large proportions of LUC and low fractions of HPX, while M2 leukaemias showed low LUC with high HPX. M3 leukaemias had low LUC and very high HPX. M4 leukaemias had large LUC and 'monocytic' components and a modest fraction of HPX. M5 leukaemias had very large numbers of LUC, 'monocytes' and 'lymphocytes' and a normal HPX. For M1 leukaemia, the presence of less than 7% LUC following induction treatment was related to morphological changes of normal cells induced by chemotherapy while LUC above 10% usually indicated unsuccessful induction associated with the presence of residual blasts. If treatment was successful, M2 and M3 leukaemias characteristically decreased their HPX population. All M4 leukaemias studied by us failed to enter remission and continued to display high proportions of HPX and LUC. Similarly, most M5 leukaemias had a poor response to treatment and always showed a very high proportion of LUC. Untreated lymphocytic leukaemias demonstrated high LUC, normal HPX and a high proportion of 'lymphocytes'. Hairy cell leukaemias showed almost equal proportions of 'lymphocytes' and LUC. Successful chemotherapy of all lymphoid leukaemia entities was associated with rapid decreases in LUC, slower decrements of 'lymphocytes' and moderate and transient increments in HPX. Thus, flow cytochemistry can assist not only in the segregation of acute leukaemias along with FAB classification with nonmorphologic criteria, but also in the follow up of patients with these diseases.

The technicon H*1--an automated hematology analyzer for today and tomorrow. Complete blood count parameters.

The performance of the Technicon H*1 was evaluated in the computerized, high-volume hematology laboratory at M.D. Anderson Hospital and Tumor Institute and compared with that of reference instrumentation used for routine patient care. The precision, linearity, and lack of carry-over of this instrument was excellent for the entire dynamic range of all nine tested parameters (white blood cells [WBCs], red blood cells [RBCs], hemoglobin [Hgb], hematocrit [Hct], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red blood cell distribution width [RDW], and platelets [Plts]). Coefficients of correlation for all directly measured parameters were always 0.98 or greater, with the exception of the MCV and RDW parameters, for which r = 0.89 and 0.93, respectively. Special emphasis was placed on WBC and Plt counts at low ranges. When compared with reference methods the correlation for both parameters was very good, r = 0.99 for WBCs less than 4.0 X 10(3)/microL (4.0 X 10(9)/L) and 0.91 for Plt less than 100 X 10(3)/microL (100 X 10(9)/L). These excellent performance characteristics for the CBC parameters, combined with the ability of the analyzer to perform a full WBC differential (to be described in the next report), make the H*1 a superior advancement in the field of automated hematology.

Comparative sensitivity of different methods to detect and quantify circulating fibrinogen/fibrin split products.

This study compared the sensitivity of three methods: staphyloccocal clumping test, SCT (Sigma and Calbiochem-Behring, CBC, reagents); Thrombo-Wellcotest (TWT); and Dade fibrinogen degradation products detection set, to quantify fibrinogen/fibrin split products (FSP) in blood samples from 696 patients and 124 normal donors using fibrinogen as the reference value. The Dade method gave quantitative results closely approaching the stated amount of fibrinogen. The SCT using Sigma reagents gave higher "fibrinogen" values, while the CBC reagents gave markedly lower "fibrinogen" values. The TWT detected only 25% of the fibrinogen standard. Detection of FSP following plasmin digestion of fibrinogen varied considerably for each test. The TWT, insensitive to most of the native fibrinogen, detected most of the FSP following only 15 minutes of plasmin digestion. In contrast, both assays relying on the SCT were completely negative after 24 hours of plasmin digestion. All four methods yielded FSP titers of less than 10 micrograms/mL in 97 (78.2%) of 124 blood samples from normal donors. The SCT Sigma reagents consistently gave results of less than 10 micrograms/mL in all normal donors. No instance of an FSP value greater than 40 micrograms/mL was noted for the 124 normal donors. Of the 696 patient blood samples tested, the Dade assay gave the highest or equally highest (with respect to another FSP method) value in 604 (87%) cases; the Sigma SCT did so in 360 (52%); the TWT in 316 (45%); and the CBC assay in 184 (26%) cases. The Dade test classified the largest number of blood samples, 328 (47.1%), in the greater than 10 less than 40 micrograms/mL titer category; however, the proportion of cases (32.2%) in which this test yielded values greater than 40 micrograms/mL was about the same as those produced by the Sigma SCT (29.9%) and TWT products (25.8%). Thus, with the exception of the normal (less than 10 micrograms/mL) and the suspicious (10-40 micrograms/mL) range, all three methods (Dade, Sigma, and TWT) are comparable in their abilities to detect abnormal levels of FSP. In the normal range, the Dade method will yield results that are frequently in the suspicious range. The CBC was noticeably inferior in detecting both suspicious and frankly abnormal values of FSP. Eight patients with acute leukemia were monitored sequentially with FSP and fibrinopeptide A (FpA) assays during their first course of chemotherapy. In all instances, elevated FpA levels correlated with elevated FSP values, as determined by the Dade, Sigma, or TWT assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Eight-parameter automated hematology analyzers: comparison of two flow cytometric systems.

The performance of two high-speed 8-parameter automated hematology analyzers (Coulter Counter S-plus and ELT-8) were compared with that of reference instruments (Coulter Counter S Sr and Technicon Autocounter). The precision, linearity, and lack of carry-over of both instruments were superior over that of existing equipment. The especially noteworthy feature of the instruments was their excellent performance in the range of extreme values of both white blood cells and platelets. This enhanced performance, and the fact that all presently known relevant hematologic parameters can be measured by a single instrument on a single sample, make the contribution of these two flow cytometric-based instruments a significant advance in the field of automated laboratory medicine.

A side-by-side evaluation of four platelet-counting instruments.

The performances of four instruments for counting platelets were evaluated in a side-by-side study: the Haema-Count MK-4/HC, an electronic impedance instrument that counts platelets in platelet-rich plasma; the Ultra-Flo 100, and the Coulter Counter Model S-Plus, electronic impedance instruments that count platelets in the presence of intact erythrocytes; and the AutoCounter, an optical instrument that counts platelets in the presence of lysed erythrocytes. The Ultra-Flo 100 and the S-Plus showed the best within-run precision, and all four instruments were considerably more precise than manual platelet counting, especially at low levels of platelet count. The four instruments were all linear in the ranges tested (5 to 650 x 10(9)/or greater), and sample carry-over was less than 0.7% for each. A noteworthy finding was that the erythrocyte concentration of the blood samples affected the displayed platelet count of the S-Plus and, to a lesser extent, that of the AutoCounter, in a predictable way, whereas it did not greatly affect the displayed count of the Ultra-Flo 100. In addition to differences in quality of performances, the four instruments differed considerably in speed and ease of operation and in cost.