Pharmacokinetic concepts — Drug binding,apparent volume of distribution and clearance

Summary Blood flow rate-limited physiological pharmacokinetic models have been used to examine the relationship between apparent volume of distribution and clearance or, more specifically between drug binding in blood, eliminating regions or noneliminating regions and clearance. The influence of binding on drug elimination depends on the driving force concentration in the eliminating region. In most instances this is likely to be free drug concentration in the region. Under these conditions, the results indicate that apparent volume of distribution and drug clearance from the blood should be treated as independent pharmacokinetic variables. Volume of distribution per se has no effect on clearance or on average steady-state blood levels. Drug binding in nonvascular regions (i. e. tissue binding) seems to be of limited importance except as a determinant of half-life. Although changes in tissue binding will affect partition coefficient and apparent volume of distribution, such changes will have no effect on average steady-state blood levels of either total or free drug.     

A five-drug remission induction regimen with intensive consolidation for adults with acute lymphoblastic leukemia: cancer and leukemia group B study 8811

The goal of this phase II multicenter clinical trial was to evaluate a new intensive chemotherapy program for adults with untreated acute lymphoblastic leukemia (ALL) and to examine prospectively the impact of clinical and biologic characteristics on the outcome. One hundred ninety-seven eligible and evaluable patients (16 to 80 years of age; median, 32 years of age) received cyclophosphamide, daunorubicin, vincristine, prednisone, and L-asparaginase; 167 patients (85%) achieved a complete remission (CR), 13 (7%) had refractory disease, and 17 (9%) died during induction. A higher CR rate was observed in younger patients (94% for those < 30 years old, 85% for those 30 to 59 years old, and 39% for those > or = 60 years old, P < .001) and in those who had a mediastinal mass (100%) or blasts with a T-cell immunophenotype. Eighty percent of B-lineage and 97% of T-cell ALL patients achieved a CR (P = .01). The coexpression of myeloid antigens did not affect the response rate or duration. Seventy percent of those with cytogenetic or molecular evidence of the Philadelphia (Ph) chromosome and 84% of those without such evidence achieved a CR (P = .11). Patients in remission received multiagent consolidation treatment, central nervous system prophylaxis, late intensification, and maintenance chemotherapy for a total of 24 months. After a median follow-up time of 43 months, the median survival for all 197 patients is 36 months; the median remission duration for the 167 CR patients is 29 months. Favorable pretreatment characteristics relative to remission duration or survival are younger age, the presence of a mediastinal mass or lymphadenopathy, a white blood cell count (WBC) less than 30,000/microL, L1 morphology, T or TMy immunophenotype, and the absence of the Ph chromosome. The estimates of the proportion surviving at 3 years are 69% for patients less than 30 years old, 39% for those 30 to 59 years old, 89% for those who had a mediastinal mass, 59% with WBC less than 30,000/microL, 63% with L1 morphology, 69% for T or TMy antigen expression, and 62% for those who lack the Ph chromosome. Fifteen patients (8%) had no unfavorable prognostic factors and have an estimated probability of survival at 5 years of 100% (95% confidence interval, 77% to 100%). This intensive chemotherapy regimen produces a high remission rate and a high proportion of durable remissions in adults with ALL.     

T-cell death by apoptosis in vertically human immunodeficiency virus- infected children coincides with expansion of CD8+/interleukin-2 receptor-/HLA-DR+ T cells: sign of a possible role for herpes viruses as cofactors?

One mechanism proposed to play a role in T-cell depletion in human immunodeficiency virus (HIV) infection is apoptosis (activation-induced cell death). We assessed whether apoptosis is related to activation of T cells in vivo and its possible triggers. DNA was extracted from peripheral blood mononuclear cells (PBMC) taken from 16 vertically HIV- infected children and 9 HIV-negative children born to HIV-positive mothers (controls) and tested by agarose gel electrophoresis for the presence of DNA fragments specific for apoptosis. Signs of apoptosis were found on in vitro culture of PBMC from 12 of 16 HIV-infected children, but not in PBMC from the nine controls. Eleven of the 12 HIV- infected children with apoptosis showed an elevated (> 15%) proportion of CD3+/HLA-DR+ cells. This was due to an increased proportion of CD8+/HLA-DR+ cells, as shown in 7 of 7 further tested patients. In none of the probands an increased (> 5%) proportion of IL-2 receptor expressing CD3+ cells was found. T cells undergoing apoptosis were preferentially of the CD8+ phenotype. Expansion of circulating CD8+/interleukin-2 receptor (IL-2R)-/HLA-DR+ T cells is known to occur during active infection with herpes viruses. To investigate the possible role of herpes viral coinfections for apoptosis in HIV infection, we focused on Epstein-Barr virus (EBV) as an example for a herpes virus usually acquired during childhood. In 10 of 12 patients with apoptosis, we found increased levels of EBV genome in PBMC and/or tissues, indicating active EBV replication. By contrast, no increased burden of EBV was found in the four HIV-infected patients without apoptosis or in the controls. Our data indicate that in children the occurrence of apoptosis in HIV infection is closely related to activation of CD8+ T cells. Furthermore, primoinfection with or reactivation of herpes viruses, such as EBV, may substantially contribute to such T-cell activation and the ensuing apoptosis. Additional studies are warranted to evaluate the contribution of herpes virus-triggered apoptosis to the T-cell loss leading to the acquired immunodeficiency syndrome.     

The immunohistology of follicle lysis in lymph node biopsies from homosexual men

Follicle lysis is a characteristic alteration of B cell follicles described recently in lymph node biopsies from homosexual men. It consists of disruption of germinal centers by aggregates of small mature lymphocytes variably associated with erythrocyte extravasation. We studied the immunohistology of follicle lysis identified in lymph node biopsies from 11 homosexual men. The results indicate that follicle lysis has two principal immunohistologic features: (1) intrafollicular aggregates of small lymphocytes predominantly of polytypic mantle B cell phenotype (T015+/Leu-8+/mu+/delta+/k+ or lambda+), and (2) disruption of the normal, unified follicular meshwork of R4/23+ dendritic reticulum cells by these B cell aggregates. These structural alterations may affect the functional integrity of the germinal center as it pertains to the abnormal B cell effector function and the increased prevalence of B cell lymphoma recently documented in the acquired immunodeficiency syndrome and related disorders. Because dendritic reticulum cells weakly express the Leu-3 (T4) antigen, which is known to be an essential component of the receptor for human T- lymphotropic virus type III/lymphadenopathy-associated virus (HTLV- III/LAV) retrovirus infection, it is possible that retroviral infection of dendritic reticulum cells may play a role in the pathogenesis of follicle lysis.     

Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4-13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.     

Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide

A prospective randomized study was conducted comparing two conditioning regimens for the treatment of patients with chronic myeloid leukemia in chronic phase by marrow transplantation from HLA identical siblings. Sixty-nine patients received 60 mg/kg of cyclophosphamide on each of 2 successive days followed by 6 fractions of total body irradiation each of 2.0 Gy (CY-TBI), and 73 patients received 16 mg/kg of busulfan delivered over 4 days followed by 60 mg/kg CY on each of 2 successive days (BU-CY). There was no significant difference between the CY-TBI and the BU-CY groups in the 3-year probabilities of survival (0.80 for both), relapse (0.13 for both), or event-free survival (CY-TBI, 0.68; BU-CY, 0.71) or in speed of engraftment or incidence of venocclusive disease of the liver. The 4-year probabilities of survival and event- free survival for patients transplanted within 1 year of diagnosis were 0.86 and 0.72, respectively, for each group. Significantly more patients in the CY-TBI group experienced major creatinine elevations. There was significantly more acute graft-versus-host disease in the CY- TBI group. Fever days, positive blood cultures, hospitalizations, and inpatient hospital days were significantly more common in the CY-TBI group than in the BU-CY group. In conclusion, the BU-CY regimen was better tolerated than, and associated with survival and relapse probabilities that compare favorably with, the CY-TBI regimen.     

Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein A-II

Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate ("thromboplastin") or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible.     

A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.