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Area under the inhibition time curve of mixed lymphocyte cultures
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CHARACTERIZATION OF ADENYLATE CYCLASE-LINKED CELL SURFACE RECEPTORS PRESENT ON SPECIFIC POPULATIONS OF NORMAL AND NEOPLASTIC LYMPHOID CELLS S.W. Burchiell, 2 and N.L. Warner 2 The University of New Mexico College of Pharmacy I and School of Medicine 2 Albuquerque, NM 87131, USA. In previous studies, we have shown that various murine tumors within lymphoid lineages can be characterized on the basis of cell surface antigens thought to be indicative of specific subsets of normal lymphoid cells. Because such tumors seem to represent discrete stages of differentiation of normal lymphoid cells, they appear to be in a state of differentiation arrest. Therefore, we have utilized such tumor cells as a model system for studying lymphocyte differentiation. In this particular study, we have characterized drug/hormone receptors present on the surface of normal and neoplastic lymphoid cells by examining pharmacologic agents that can stimulate adenylate cyclase-linked receptors. By monitoring intracellular levels of cyclic AMP via radioimmunoassay, we can identify functional receptors that are present on the surface of cells. The lack of cyclic AMP elevation, however, does not preclude the possibility that hormone receptors are present on the surface of cells in an uncoupled fashion. Therefore, ligand binding assays were employed to examine those murine tumors that were incapable of responding to pharmacologic agents, as measured by cyclic AMP alterations. In agreement with past investigators, we have found that normal B cells and T cells can respond to beta-adrenergic agonists (isoproterenol), PGE2, cholera toxin, and histamine, as determined by cyclic AMP elevation. Our work with murine tumors shows that T lymphomas are, in general, far more responsive to cyclic AMP-induced alterations produced by isoproterenol than are B lymphomas or macrophage-monocytic tumors. Interestingly, T lymphomas differentially respond to histamine, yet have good responses to isoproterenol, PGE2, and cholera toxin. This result suggests that there may be distinct populations of T cells that can be identified on the basis of whether they possess histamine receptors. All of the B lymphomas tested were very responsive to PGE2 and cholera toxin, but these cells showed less sensitivity to histamine and isoproterenol. As a group, macrophage tumors were far less sensitive to alterations in cyclic AMP levels produced via these agents, a result that is analagous to that observed with highly purified normal macrophage populations.
17
AREA UNDER B.M. Frey~ Schools of CA 94143,
THE INHIBITION TIME CURVE OF MIXED LYMPHOCYTE CULTURES F.J. Frey~ F.C. Losada~ K.C. Cochrum and L.Z. Benet Medicine, Dentistry, and Pharmacy, University of California, USA
San Francisco,
With the ultimate goal of developing rational individualized dosage regimens for prednisolone, we examined the relation between the pharmacokinetics of prednisolone and an in vitro measure of immunosuppressive activity. Eight to 15 plasma samples were obtained from 9 patients with oral autoimmune mucocutaneous diseases on chronic corticosteroid treatment following both iv and oral dosing (30-50 mg/day). Total and unbound prednisolone levels were assessed by high performance liquid chromatography and equilibrium dialysis (i). The immunosuppressive activity of each sample was determined as the inhibition of the mixed lymphocyte reaction (MLR). To quantify immunosuppressive activity we defined the parameter "area under the inhibition time curve" (AUIC) which is calculated using the trapezoidal rule similar to the pharmacokinetic measure of area under plasma concentration time curve (AUC). The MLR was modified for the kinetic studies: (a) adding 50% of plasma instead of the usual 10%, a situation closer to that in vivo, which increases the sensitivity of the system; (b) harvesting on day 3 instead of the usual day 5 which reduces the variability in response (2). With these conditions we found a total prednisolone clearance rate of 2.17 ± 0.56 ml/min kg (X±SD) and an unbound prednisolone clearance rate of 9.6 ± 2.42 ml/min kg; oral bioavailability, the ratio of AUCoral/AUCiv, measuring total prednisolone was 0.74 ± 0.09 vs. 0.59 ± 0.08 when free concentrations are measured. The biological bioavailability, i.e. AUIC following the oral dose divided by the AUIC following the iv bolus dose, was 0.85 ± 0.25. A correlation was found between AUIC and both total and unbound AUC (P<0.05). Conclusion: The clinically relevant MLR inhibition can be related to interpatient variable prednisolone kinetics. i) 2)
Frey FJ, Frey BM, Benet LZ, Clin. Chem. 25:1944-1947 (1979) Frey BM, Frey FJ, Benet LZ, Cochrum KC, Int. J. Immunopharmacol.
2: in press
Supported by NIH Center Grant GM 26691 and the Swiss National Foundation.
(1980)
16
CHARACTERIZATION OF ADENYLATE CYCLASE-LINKED CELL SURFACE RECEPTORS PRESENT ON SPECIFIC POPULATIONS OF NORMAL AND NEOPLASTIC LYMPHOID CELLS S.W. Burchiell, 2 and N.L. Warner 2 The University of New Mexico College of Pharmacy I and School of Medicine 2 Albuquerque, NM 87131, USA. In previous studies, we have shown that various murine tumors within lymphoid lineages can be characterized on the basis of cell surface antigens thought to be indicative of specific subsets of normal lymphoid cells. Because such tumors seem to represent discrete stages of differentiation of normal lymphoid cells, they appear to be in a state of differentiation arrest. Therefore, we have utilized such tumor cells as a model system for studying lymphocyte differentiation. In this particular study, we have characterized drug/hormone receptors present on the surface of normal and neoplastic lymphoid cells by examining pharmacologic agents that can stimulate adenylate cyclase-linked receptors. By monitoring intracellular levels of cyclic AMP via radioimmunoassay, we can identify functional receptors that are present on the surface of cells. The lack of cyclic AMP elevation, however, does not preclude the possibility that hormone receptors are present on the surface of cells in an uncoupled fashion. Therefore, ligand binding assays were employed to examine those murine tumors that were incapable of responding to pharmacologic agents, as measured by cyclic AMP alterations. In agreement with past investigators, we have found that normal B cells and T cells can respond to beta-adrenergic agonists (isoproterenol), PGE2, cholera toxin, and histamine, as determined by cyclic AMP elevation. Our work with murine tumors shows that T lymphomas are, in general, far more responsive to cyclic AMP-induced alterations produced by isoproterenol than are B lymphomas or macrophage-monocytic tumors. Interestingly, T lymphomas differentially respond to histamine, yet have good responses to isoproterenol, PGE2, and cholera toxin. This result suggests that there may be distinct populations of T cells that can be identified on the basis of whether they possess histamine receptors. All of the B lymphomas tested were very responsive to PGE2 and cholera toxin, but these cells showed less sensitivity to histamine and isoproterenol. As a group, macrophage tumors were far less sensitive to alterations in cyclic AMP levels produced via these agents, a result that is analagous to that observed with highly purified normal macrophage populations.
17
AREA UNDER B.M. Frey~ Schools of CA 94143,
THE INHIBITION TIME CURVE OF MIXED LYMPHOCYTE CULTURES F.J. Frey~ F.C. Losada~ K.C. Cochrum and L.Z. Benet Medicine, Dentistry, and Pharmacy, University of California, USA
San Francisco,
With the ultimate goal of developing rational individualized dosage regimens for prednisolone, we examined the relation between the pharmacokinetics of prednisolone and an in vitro measure of immunosuppressive activity. Eight to 15 plasma samples were obtained from 9 patients with oral autoimmune mucocutaneous diseases on chronic corticosteroid treatment following both iv and oral dosing (30-50 mg/day). Total and unbound prednisolone levels were assessed by high performance liquid chromatography and equilibrium dialysis (i). The immunosuppressive activity of each sample was determined as the inhibition of the mixed lymphocyte reaction (MLR). To quantify immunosuppressive activity we defined the parameter "area under the inhibition time curve" (AUIC) which is calculated using the trapezoidal rule similar to the pharmacokinetic measure of area under plasma concentration time curve (AUC). The MLR was modified for the kinetic studies: (a) adding 50% of plasma instead of the usual 10%, a situation closer to that in vivo, which increases the sensitivity of the system; (b) harvesting on day 3 instead of the usual day 5 which reduces the variability in response (2). With these conditions we found a total prednisolone clearance rate of 2.17 ± 0.56 ml/min kg (X±SD) and an unbound prednisolone clearance rate of 9.6 ± 2.42 ml/min kg; oral bioavailability, the ratio of AUCoral/AUCiv, measuring total prednisolone was 0.74 ± 0.09 vs. 0.59 ± 0.08 when free concentrations are measured. The biological bioavailability, i.e. AUIC following the oral dose divided by the AUIC following the iv bolus dose, was 0.85 ± 0.25. A correlation was found between AUIC and both total and unbound AUC (P<0.05). Conclusion: The clinically relevant MLR inhibition can be related to interpatient variable prednisolone kinetics. i) 2)
Frey FJ, Frey BM, Benet LZ, Clin. Chem. 25:1944-1947 (1979) Frey BM, Frey FJ, Benet LZ, Cochrum KC, Int. J. Immunopharmacol.
2: in press
Supported by NIH Center Grant GM 26691 and the Swiss National Foundation.
(1980)