Concordance of 3 alternative teratogenicity assays with results from corresponding in vivo embryo-fetal development studies: Final report from the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) DruSafe working group 2.

An IQ DruSafe working group evaluated the concordance of 3 alternative teratogenicity assays (rat whole embryo culture, rWEC; zebrafish embryo culture, ZEC; and murine embryonic stem cells, mESC) with findings from rat or rabbit embryo-fetal development (EFD) studies. Data for 90 individual compounds from 9 companies were entered into a database. In vivo findings were deemed positive if malformations or embryo-fetal lethality were reported in either species. Each company used their own criteria for deciding whether the alternative assay predicted the in vivo findings. Standard concordance parameters were calculated, positive and negative predictive values (PPV and NPV) were adjusted for the aggregate portfolio prevalence of positive compounds (established by a survey of participating companies), and positive and negative likelihood ratios (LR+ and iLR-) were calculated. Of the 3 assays, only rWEC data were robustly predictive, particularly for negative predictions (NPVadj = 92%). However, both LR+ (4.92) and iLR- (4.72) were statistically significant for the rWEC assay. When analyzed separately for rats, the NPVadj and iLR-values for the rWEC assay increased to 96% and 9.75, respectively. These data suggest that a negative rWEC outcome could defer or replace a rat EFD study in certain regulatory settings.

QT Interval Shortening With Isavuconazole: In Vitro and In Vivo Effects on Cardiac Repolarization.

The effects of isavuconazole (active moiety of isavuconazonium sulfate) on cardiac ion channels in vitro and cardiac repolarization clinically were assessed in a phase I, randomized, double-blind study in healthy individuals who received isavuconazole (after 2-day loading dose), at therapeutic or supratherapeutic doses daily for 11 days, moxifloxacin (400 mg q.d.), or placebo. A post-hoc analysis of the phase III SECURE trial assessed effects on cardiac safety. L-type Ca2+ channels were most sensitive to inhibition by isavuconazole. The 50% inhibitory concentrations for ion channels were higher than maximum serum concentrations of nonprotein-bound isavuconazole in vivo. In the phase I study (n = 161), isavuconazole shortened the QT interval in a dose- and plasma concentration-related manner. There were no serious treatment-emergent adverse events; palpitations and tachycardia were observed in placebo and supratherapeutic isavuconazole groups; no cardiac safety signals were detected in the SECURE study (n = 257). Isavuconazole was associated with a shortened cardiac QT interval.

Identification of a peptide directed against the anti-CD34 antibody, 9C5, by phage display and its use in hematopoietic stem cell selection.

A peptide sequence was identified by phage display technology that could be used as an alternative to chymopapain for the release of hematopoietic progenitor cells captured by anti-CD34 monoclonal antibodies. This was achieved by affinity selection screening (biopanning) of a random hexapeptide sequence phage display library. Four rounds of biopanning were performed to enrich for phage clones with specific affinity for anti-CD34 monoclonal antibody, 9C5. DNA sequence analyses of these phage clones revealed an enrichment of two predominant sequences, QQGWFP and TQGSFW. These two clones also shared a consensus sequence motif, QGxF, that exhibited 50% and 67% homology with a region spanning amino acids 14-19 of the mature CD34 antigen. Based on these data, synthetic peptides were generated and assessed for their ability to release 9C5 from CD34+ cells. Using a flow cytometric assay, it was found that the synthetic peptide, 9069N, effectively released 9C5 from the CD34-expressing cell line, KG1a, in a concentration-dependent manner (77% and 99% release of 9C5 at 0.14 and 0.70 mM peptide concentrations, respectively). In the Isolex 300i immunomagnetic selection system, this peptide was shown to be effective at releasing 9C5 sensitized CD34+ hematopoietic progenitors from sheep anti-mouse IgG Dynabeads. Thus, a synthetic peptide, which specifically and efficiently released immunomagnetically selected hematopoietic progenitor cells from paramagnetic beads, was identified. This reagent is a significant advance in the selection of hematopoietic progenitors in that it does not alter cell surface antigens. As such, further phenotypic characterization or immunoselection can be performed.

WR2721 ameliorates the radiation-induced depression in reactivity of rat abdominal aorta to U46619.

Previous studies showed that 20 Gy whole-body gamma irradiation results in a decreased response of the abdominal aorta to the stable thromboxane A2 (TXA2) mimic, U46619. The present study evaluated the effect of WR2721 on this radiation-induced decrease in vascular responsiveness. Rats receiving WR2721 (200 mg/kg, i.p.) 20 min before irradiation showed no depression in vascular reactivity to U46619 compared to control. The abolition of the radiation-induced decrease in vascular responsiveness was not caused by a direct vasoconstrictor action of WR2721 or its metabolites. The vascular response of rat abdominal aortic rings to KCl was unchanged after in vivo exposure to ionizing radiation. WR2721 did not alter the vascular response to KCl. These studies confirm that exposure to whole-body ionizing radiation decreased abdominal aortic vascular responsiveness to U46619. This depressed vascular reactivity can be abolished by pretreatment with the radioprotectant, WR2721. These observations may provide a rapid initial screening method for evaluating the in vivo efficacy of radioprotectant drugs.

Reactivity of rat abdominal aorta to U46619 following whole-body gamma irradiation.

Rats exposed to 20 Gy whole-body irradiation demonstrated a depressed aortic responsiveness to the thromboxane mimic, U46619, 48 h postirradiation. The mechanism for this observed response was investigated. Shielding the abdominal aorta attenuated this altered vascular reactivity. Since this suggests that radiation exposure induces local changes in the aorta, vascular smooth muscle function was assessed with cumulative concentrations of KCl. Radiation-induced smooth muscle damage was insufficient to account for the decreased reactivity to U46619. Next, calcium availability for vascular smooth muscle function was evaluated and found not to be responsible for the radiation-induced depression in aortic responsiveness. Finally, the role that cyclooxygenase products play in the depressed contractile response was investigated. Indomethacin treatment prior to and for 48 h after irradiation attenuated the altered vascular reactivity to U46619. These data suggest that a radiation-induced increase in cyclooxygenase products may play a role in the decreased aortic reactivity to the thromboxane mimic.

Regional release of cyclooxygenase products after radiation exposure of the rat.

The present study evaluated the regional release of cyclooxygenase products 4 h following 20 Gy gamma irradiation. Thoracic shielding reduced the radiation-induced increase in immunoreactive thromboxane B2 (iTxB2) excretion to control levels while abdominal shielding partially attenuated the altered excretion of this cyclooxygenase product. To assess the role the kidneys play in the radiation-induced increase in iTxB2 excretion, an in situ isolated perfused rat kidney model was developed. The excretion rate of iTxB2 from irradiated isolated perfused kidneys was not significantly different from sham-irradiated perfused kidneys. Radiation exposure did alter renal cyclooxygenase product release in that the excretion of immunoreactive prostaglandin E2 (iPG2) and immunoreactive 6-keto-PGF1 alpha was significantly increased (P less than 0.05) in irradiated isolated perfused kidneys. These data show that radiation-induced increases in iTxB2 excretion are primarily due to altered extrarenal synthesis and/or metabolism of this arachidonate metabolite.

Thromboxane release from irradiated perfused rat lungs: role of oncotic agents.

Isolated lungs from 20 Gray (Gy) whole body irradiated rats were perfused with Krebs-Ringer bicarbonate plus 3% bovine serum albumin (KRB-BSA). The pulmonary effluent showed a 99% (p less than .05) increase in immunoassayable thromboxane B2 (iTXB2) release compared with non-irradiated lungs. Since both arachidonic acid and cyclooxygenase products bind to albumin, studies were performed to determine if omission or substitution of this protein oncotic agent would alter the radiation-induced increase in pulmonary iTXB2 release. Irradiated, isolated lungs perfused with media from which the BSA was omitted (KRB) did not demonstrate the radiation-induced increase in pulmonary iTXB2 release. Similarly, irradiated lungs perfused with media in which Dextran 70 (KRB plus 3% Dextran 70, KRB-Dextran 70) was substituted for BSA also did not show the radiation-induced increase in pulmonary effluent iTXB2 levels. These studies demonstrate the importance of including albumin as the oncotic agent in perfused organ systems when studying cyclooxygenase product release.

Calcium utilization associated with U-46619 and PGF2a vascular responses in rat lungs.

The pulmonary vasoconstrictor responses to U-46619 and PGF2a are calcium dependent. The purpose of this investigation was to determine to what extent extracellular and intracellular calcium pools are utilized during the dose-dependent pulmonary vasopressor responses induced by multiple doses of U-46619 and PGF2a. Increasing doses of these agonists were administered to isolated rat lungs perfused with Krebs-Ringer bicarbonate (KRB) or KRB not containing CaCL2. The data indicate that U-46619 uses predominantly extracellular calcium at low doses (0.1 microgram) and depends solely on intracellular calcium at the highest dose (0.4 microgram). In contrast PGF2a appears to use depletable intracellular calcium stores to achieve contraction.

Reticuloendothelial system blockade-induced humoral factor depletion and susceptibility to hemorrhagic shock.

This study was carried out to determine if reticuloendothelial system (RES) blockade-induced depletion of circulating alpha-2-glycoprotein opsonic activity resulted in increased susceptibility to hemorrhagic shock. RES blockade induced by the injection of gelatinized lipid emulsion was associated with a 45.9% decrease in phagocytic index and a 85.7% decrease in plasma alpha-2-glycoprotein opsonic activity. Animals subjected to RES blockade 30 min prior to hemorrhagic shock showed a decrease in time to decompensation and a decrease in maximum shed volume. These results are consitent with the concept that circulating levels of this opsonic protein are important in modulating RES phagocytic function and in host defense to shock.

Reticuloendothelial system depression with hemolyzed blood.

This study was carried out to evaluate the effects of hemolyzed blood and components of hemolyzed blood on RES function. Phagocytic index, determined using four different test particles in rats, was depressed 30 minutes following the injection of 0.3 ml/100 gm hemolyzed blood. Fractionation of hemolyzed blood into particulate and soluble fractions revealed that only the particulate material caused an RES depression. A decrease in phagocytic index was seen after the injection of hemoglobin-free erythrocyte ghosts, further indicating that the membrane components of hemolyzed blood are capable of depressing RES function. Injection of hemolyzed blood, as well as RES blockade, increased the mortality rate following endotoxin administration. Injection of hemoglobin had no effect on RES function or endotoxin shock susceptibility. It is concluded that hemolyzed blood causes a nonspecific depression in RES phagocytic function, as well as probably impairing endotoxin detoxification. These findings indicate that the hemolysis that accompanies burn injury may induce RES depression and increase susceptibility to shock states.

Cecal ligation and puncture is associated with pulmonary injury in the rat: role of cyclooxygenase pathway products.

The present studies evaluated the role cyclooxygenase products play in bacterial sepsis induced pulmonary injury in the rat. Lung injury was assessed by determining the pulmonary capillary filtration coefficient (Kf) and the lung lavage protein concentration four and 18 hours after cecal ligation and puncture. Four hours after cecal ligation, the Kf was unchanged from control, however, by 18 hours, the Kf was increased 171% (p < .05). Similarly, lung lavage protein levels were unchanged four hours after cecal ligation but were significantly (p < .05) elevated at 18 hours. On the other hand, pulmonary lavage immunoreactive thromboxane B2 (iTXB2) levels were increased both four and 18 hours after the initiation of sepsis. In order to determine if cyclooxygenase products played a role in the sepsis associated lung injury, ibuprofen was administered prior to cecal ligation. Ibuprofen pretreatment prevented the sepsis associated increase in both Kf and lung lavage protein concentration. These studies suggest that bacterial sepsis in the rat is associated with pulmonary injury and that early administration of ibuprofen ameliorates this damage.

Bacterial sepsis-induced decrease in lung vascular reactivity to 9,11-dideoxy-11a9a-epoxymethano-prostaglandin F2 alpha (U46619) in the rat.

This study determined whether a sepsis-associated increase in cyclooxygenase products altered the pulmonary vascular response to the thromboxane A2 mimic, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2 alpha (U46619). Rats were anesthetized (50 mg/kg of sodium pentobarbital i.p.), and sepsis was induced by cecal ligation and puncture. Four hours later, pulmonary effluent immunoreactive thromboxane (iTXB2) levels were significantly increased (156.8%) and pulmonary vascular reactivity to U46619 (50-200 ng) was significantly (P less than .05) decreased compared to lungs from nonseptic controls. This decreased vascular reactivity was not seen in lungs from cecally ligated rats challenged with angiotensin II (5-200 ng). Sham surgery did not alter pulmonary iTXB2 synthesis nor did it result in a depressed vascular response to U46619. Rats pretreated with ibuprofen (15 mg/kg i.v.) did not show the sepsis-associated increase in iTXB2 levels nor was a decrease in pulmonary vascular reactivity to U46619 observed. These data indicate that a sepsis-associated increase in TXA2 and/or other cyclooxygenase products can alter the pulmonary vascular response to the TXA2 mimic, U46619.

Effect of extravascular hemolysis on the RES depression following thermal injury.

This study was carried out to determine if the reticuloendothelial system (RES) uptake of damaged red blood cells (RBC) following thermal injury is sufficient to depress hepatic and splenic clearance function or to increase susceptibility to endotoxin shock. This was approached by determining the extent of the RES uptake of intact RBC (extravascular hemolysis) following thermal injury in rats, and by determining the effect on RES function of a similar degree of extravascular hemolysis induced in uninjured animals by the injection of heat damaged RBC. The RES uptake of three doses of heated RBC was characterized and the second largest dose caused a degree of extravascular hemolysis which was comparable to that associated with thermal injury. This latter dose of heated RBC depressed splenic clearance function but did not depress hepatic clearance function as determined using the smallest dose of heated RBC or formalinized sheep red blood cells at the test particles. Susceptibility to endotoxin shock was increased following the injection of the dose of heated RBC which stimulated the degree of extravascular hemolysis associated with thermal injury. Thus, the extravascular hemolysis associated with thermal injury is sufficient to depress splenic clearance function but not hepatic clearance function, and may contribute to the increased susceptibility to endotoxin shock following thermal injury. These findings further support the hypothesis that the hemolysis induced by thermal injury contributes to the depression of host defense associated with this form of injury.

CD34+ cell engraftment, ex vivo expansion, and malignant cell depletion following immunomagnetic selection.

This review describes the published preclinical and clinical data on the use of a manual or semiautomated immunomagnetic selection device, termed the Isolex system. Preclinical evaluation of hematopoietic progenitor cells (CD34+ cells) selected from bone marrow, peripheral blood leukapheresis products, and umbilical cord blood is reviewed with respect to differentiation (CFU-GM, BFU-E, and CFU-GEMM formation) and proliferation. The purities and yields of CD34+ cell products from clinical trials performed since 1994 are presented along with data on malignant cell depletion. On average, the Isolex system resulted in a final product median purity of 67% and a final product median yield of 64%. Positive selection of CD34+ cells with this device decreased residual tumor cell levels by 2-3 logs in autologous transplant products and reduced T cell levels by 3-4 logs in allogeneic grafts. To evaluate the clinical effect of these immunomagnetically selected cells, data on the rate of engraftment were reviewed. Autologous CD34+ cell transplantation resulted in recovery time from neutropenia (ANC > 500/microliter) of 9-14 days and recovery time from thrombocytopenia (platelet count > 20,000/microliter) of 10-20 days. These data showed that the Isolex system can positively select progenitor cells to reconstitute the hematopoietic system following myeloablative therapy.

Effect of red blood cells and red blood cell ghosts on reticuloendothelial system function.

Previous studies have shown that hepatic phagocytosis of red blood cell (RBC) stroma can depress reticuloendothelial system (RES) phagocytic function and increase susceptibility to shock. Since the RBC stroma used in these experiments contained substantial amounts of adherent hemoglobin, the present study was carried out to evaluate the role of the hepatic uptake of RBC membrane material on RES phagocytic function and susceptibility to endotoxin shock in rats. Neuraminidase-treated RBC which contained normal amounts of hemoglobin and RBC ghosts which were hemoglobin-free were used. Both preparations were removed from the circulation primarily by the liver. RES phagocytic function was depressed following the hepatic uptake of 29 X 10(8) neuraminidase-treated RBC and 26 X 10(8) RBC ghosts. RES uptake of neuraminidase-treated RBC was associated with an increase in susceptibility to endotoxin shock, but RBC ghosts did not affect shock susceptibility. Thus, RBC ghosts and intact RBC are equally effective in depressing RES phagocytic function, but RBC ghosts did not affect susceptibility to endotoxin shock.

The contribution of animal models to the development of treatments for hematologic recovery following myeloablative therapy: a review.

This review describes the role that animal models have played in the development of clinical procedures for growth factor and hematopoietic cell therapies following high-dose cancer chemotherapy, radiotherapy or both. Data are discussed describing animal models that add to the understanding of human hematopoiesis, including myeloid and lymphoid lineage localization and in vivo maturation. Finally, current animal models of cytokine and cell therapies are presented in the context of their contributions to early clinical trials and future therapies. These studies underscore the past and current contributions animal investigations have made to improving clinical therapies.

Thromboxane and prostacyclin synthesis following whole body irradiation in rats.

The effect of radiation on the mechanism and source of in vivo thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) synthesis was evaluated. Rats were irradiated with 2, 10, or 20 gray (Gy) whole-body gamma irradiation and showed an increase in urine TxB2 after either 10 or 20 Gy. Urine 6-keto-PGF1 alpha was elevated only after exposure to 20 Gy. Irradiation did not alter urine volume or osmolarity, nor was there a correlation between urine osmolarity and the urinary concentration of TxB2 r 6-keto-PGF1 alpha. Rats were pretreated with indomethacin to determine if radiation-induced alterations in urine TxB2 and 6-keto-PGF1 alpha could be suppressed. Pretreatment with indomethacin significantly decreased urine TxB2 and 6-keto-PGF1 alpha in both irradiated and nonirradiated animals. Finally, the sources of urinary cyclooxygenase products were investigated using an isogravitometric cross-perfusion system. These experiments demonstrated that urine TxB2 is derived from extrarenal sources, whereas 6-keto-PGF1 alpha is synthesized primarily by the kidney. It may be concluded that radiation exposure increases in vivo cyclooxygenase pathway activity by both renal and extrarenal tissues.

Inhibition of lipopolysaccharide-associated endotoxin activities in vitro and in vivo by the human anti-lipid A monoclonal antibody SdJ5-1.17.15.

The present study evaluated the effect of a novel anti-lipid A monoclonal antibody, termed SdJ5, on the in vitro production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta by endotoxin- or lipopolysaccharide (LPS)-challenged human peripheral blood mononuclear cells (hPBMC). In addition, the present study determined whether SdJ5 could neutralize the in vivo toxicity of LPS. SdJ5, at a concentration equal to or greater than 3 micrograms/ml, specifically inhibited TNF-alpha and interleukin-1 beta production by hPBMC stimulated with every type of LPS and lipid A assessed. SdJ5 also showed a significantly greater inhibition of cytokine production than a nonrelevant human immunoglobulin M myeloma control. The SdJ5-mediated inhibition of TNF-alpha production was rapid, as the simultaneous addition of the SdJ5 and LPS still resulted in a marked decrease in hPBMC cytokine synthesis. The ability of SdJ5 to neutralize in vivo toxicity was also determined by using LPS from four different strains of gram-negative bacteria. LPS, when preincubated with SdJ5, resulted in a significant decrease in the 24-h mortality rate compared with that for the control. These studies show that the anti-lipid A monoclonal antibody SdJ5 can modulate LPS-induced cytokine production in vitro and increase the survival rate of rats challenged with lethal doses of LPS.

Role of calcium in U 46619 and PGF2 alpha pulmonary vasoconstriction in rat lungs.

The role of calcium and calmodulin during U 46619 and PGF2 alpha-induced pulmonary vasoconstriction was studied in isolated rat lungs perfused with Krebs-Ringer bicarbonate (KRB) or calcium-free KRB. In lungs perfused with KRB, bolus injections of U 46619 (0.2 microgram) and PGF2 alpha (40.0 micrograms) resulted in a 48.0 +/- 4.0 and 23.9 +/- 2.5% increase in mean pulmonary artery pressure, respectively. During lung perfusion with KRB without calcium, the U 46619 response decreased to 31.1 +/- 7.5% whereas the PGF2 alpha response increased to 34.6 +/- 4.1%. Repeated challenges with PGF2 alpha in the KRB without calcium resulted in reduction of the response to 11.8 +/- 1.2%; the U 46619 response was unaltered. The intracellular calcium blocker, 8-(N,N-diethylamino)-octyl-3,4,5, trimethoxybenzoate HCL (TMB-8) significantly attenuated the pressor response to U 46619 at low doses and PGF2 alpha at high doses. The calmodulin inhibitor trifluoperazine (TFP 100 microM) attenuated the vasoconstrictor response to U 46619 by 54%, whereas the PGF2 alpha was unchanged. However, in the calcium-free KRB, TFP attenuated the pressor response to both U 46619 and PGF2 alpha. The U 46619 pressor response depends on intracellular and extracellular calcium to achieve calmodulin-dependent vasoconstriction. PGF2 alpha requires extracellular calcium to replenish depletable intracellular calcium pools and is independent of calmodulin activation.

Comparison of binding specificity and the function of two human IgM anti-lipid A monoclonal antibodies.

The interactions of two anti-lipid A monoclonal antibodies (mAb)--HA-1A and SdJ5-1.17.15--with their antigenic sites on lipid A, were compared using a dot-blot assay and lipid A structural analogues, as well as lipid A-high-density lipoprotein (HDL) complexes. The reactivities of both mAb were affected by the type of fatty acid side chains and by the phosphate group on the glucosamine residue II; however, the interaction of SdJ5-1.17.15 appeared to be more markedly affected by the fatty acid side chains. A determination of the biological significance of these antigenic differences was made. Human peripheral blood mononuclear cells (hPBMC) challenged with Escherichia coli 055:B5 lipopolysaccharide (LPS) pre-incubated with SdJ5-1.17.15 released significantly less tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), compared to hPBMC exposed to vehicle preincubated LPS. HA-1A did not attenuate the in vitro release of either cytokine. The ability of both mAb to neutralize the in vivo toxicity of LPS was also evaluated. Rats administered E. coli 055:B5 pre-incubated with SdJ5-1.17.15 had a significantly reduced 24-hr mortality rate compared to vehicle controls. HA-1A did not attenuate the in vivo mortality rate. Therefore, the reactivity of anti-lipid A mAb with the antigen is preferentially affected by different residues on the lipid A moiety. Thus, the differences in biological activity seen with SdJ5-1.17.15 and HA-1A may be due in part to differences in their recognition sites on lipid A.