Clinical and anatomic pathology effects of serial blood sampling in rat toxicology studies, using conventional or microsampling methods.

UNLABELLED: As a general practice in rodent toxicology studies, satellite animals are used for toxicokinetic determinations, because of the potential impact of serial blood sampling on toxicological endpoints. Besides toxicological and toxicokinetic determinations, blood samples obtained longitudinally from a same animal may be used for the assessment of additional parameters (e.g., metabolism, pharmacodynamics, safety biomarkers) to maximize information that can be deduced from rodents. We investigated whether removal of up to 6 × 200 µL of blood over 24h can be applied in GLP rat toxicology studies without affecting the scientific outcome. METHODS: 8 week-old female rats (200-300 g) were dosed for up to 1 month with a standard vehicle and subjected or not (controls) to serial blood sampling for sham toxicokinetic/ancillary determinations, using miniaturized methods allowing collection of 6 × 50, 100 or 200 µL over 24h. In-life endpoints, clinical pathology parameters and histopathology of organs sensitive to blood volume reduction were evaluated at several time points after completion of sampling. RESULTS: In sampled rats, minimal and reversible changes in red blood cell mass (maximally 15%) and subtle variations in liver enzymes, fibrinogen and neutrophils were not associated with any organ/tissue macroscopic or microscopic correlate. CONCLUSION: Serial blood sampling (up to 6 × 200 µL over 24h) is compatible with the assessment of standard toxicity endpoints in adult rats.

Miniaturized blood sampling techniques to benefit reduction in mice and refinement in nonhuman primates: applications to bioanalysis in toxicity studies with antibody-drug conjugates.

Minimizing the number of animals in regulatory toxicity studies while achieving study objectives to support the development of future medicines contributes to good scientific and ethical practices. Recent advances in technology have enabled the development of miniaturized blood sampling methods (including microsampling and dried blood spots) applicable to toxicokinetic determinations of small-molecule drugs. Implementation of miniaturized blood sampling methods in the context of biotherapeutic drugs is desirable because a limitation to this type of medicine remains the total blood volume needed from a single animal to support toxicokinetic determinations of several analytes (parent drug, metabolites[s], antidrug antibodies, and so forth). We describe here the technical details, applicability, and relevance of new miniaturized blood sampling procedures in mice and nonhuman primates in the context of the toxicologic evaluation of biotherapeutic drugs consisting of antibody-drug conjugates developed for oncology indications. These examples illustrate how these techniques can benefit the reduction of animal usage in mouse toxicity studies by decreasing the number of animals dedicated to toxicokinetic determinations and the refinement of practices in nonhuman primate toxicity studies by decreasing the blood volume repeatedly drawn for toxicokinetic determinations.

Concise review: workshop review: understanding and assessing the risks of stem cell-based therapies.

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.

Hydroxyethylstarch 200 and 240 differently affect aortic distensibility but not viscosity and blood pressure upon acute isovolumic hemodilution.

OBJECTIVES: It has been shown that a hydroxyethylstarch solution significantly increases the aortic distensibility coefficient (ADC) as compared to other non-hydroxyethylstarch colloid solutions. In order to investigate whether the effect of hydroxyethylstarch on ADC is class-specific, we investigated the effect of two hydroxyethylstarch solutions (HES 200: Elohes and HES 240: Hesteril) on the ADC and compared them with two other colloid solutions: 5% albumin and fluid gelatin (Gelofusin) in a rabbit model of acute isovolumic hemodilution. METHODS: Twenty-eight male New Zealand white rabbits were anesthetized and randomly allocated to receive (n=7, each): albumin, hydroxyethylstarch-200, hydroxyethylstarch-240 and gelatin for acute isovolumic hemodilution by exchanging 13 ml.kg(-1) body weight of blood with an identical volume of the test solution. Blood viscosity, mean arterial pressure, aortic blood flow and heart rate were measured and ADC was calculated. RESULTS: All groups were comparable with respect to arterial pressure, heart rate and aortic blood flow velocity before and after isovolumic hemodilution. After hemodilution, ADC coefficient remained unchanged as compared with pre-hemodilution values with albumin, hydroxyethylstarch-240 and gelatin, whereas a sustained 3 fold increase was observed with hydroxyethylstarch-200. CONCLUSION: These results demonstrate that minor physicochemical differences between two hydroxyethylstarch solutions result in measurable differences in ADC and suggest that the clinical effects of colloids should probably be analyzed for each type of colloid and not for classes of colloids (e.g. hydroxyethylstarch or fluid gelatins).

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo.

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.

Inhibition of PDGFR phosphorylation and Src and Akt activity by GN963 leads to therapy of human pancreatic cancer growing orthotopically in nude mice.

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.

Human FGF-1 gene transfer promotes the formation of collateral vessels and arterioles in ischemic muscles of hypercholesterolemic hamsters.

BACKGROUND: Acidic fibroblast growth factor (FGF-1) has been identified as a potent mitogen for vascular cells, inducing formation of mature blood vessels in vitro and in vivo and represents one of the most promising approaches for the treatment of ischemic cardiovascular diseases by gene therapy. Nevertheless, and most probably due to the few experimental models able to address the issue, no study has described the therapeutic effects of FGF-1 gene transfer in subjects with peripheral arterial disease (PAD) exhibiting a clinically relevant cardiovascular pathology. METHODS: In order to assess the potency of FGF-1 gene transfer for therapeutic angiogenesis in ischemic skeletal muscles displaying decreased gene expression levels and sustained impaired formation of collateral vessels and arterioles, we developed a model of PAD in hamsters with a background of hypercholesterolemia. Hamsters fed a cholesterol-rich diet and subjected to hindlimb ischemia exhibit a sustained impaired angiogenic response, as evidenced by decreased angiographic score and histological quantification of arterioles in the ischemic muscles. RESULTS: In this model, we demonstrate that NV1FGF (a human FGF-1 expression plasmid), given intramuscularly 14 days after induction of hindlimb ischemia, promoted the formation of both collateral vessels and arterioles 14 days after treatment (i.e. 28 days post-ischemia). CONCLUSIONS: Our data provide evidence that NV1FGF can reverse the cholesterol-induced impairment of revascularization in a hamster model of hindlimb ischemia by promoting the growth of both collateral vessels and arterioles in ischemic muscles exhibiting significantly decreased levels of gene expression compared with control muscles. Therefore, this study underscores the relevance of NV1FGF gene therapy to overcome perfusion defects in patients with PAD.

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran.

Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P(50)=28-29 mm Hg). Under physiological conditions, the oxygen affinity (P(50)) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA(0) is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n(50)), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA(0). Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA(0) and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA(0). Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA(0), as shown by about 2-fold increase in autoxidation, and slightly higher H(2)O(2) reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T-->R transition may hinder its physiological role as an oxygen transporter.

Hemodilution with stroma-free [correction of stoma-free] hemoglobin at physiologically maintained viscosity delays the onset of vasoconstriction.

Solutions of modified cell-free hemoglobin, prepared from outdated red blood cells, have been developed during the past decade to circumvent the increasing need for allogeneic blood. Despite improvements in the safety and efficacy of these solutions, undesirable effects such as an increase in vascular tone leading to hypertension have not been fully resolved, which might hinder their clinical usefulness. To discriminate between the pharmacological and rheological effects of cell-free hemoglobin, we compared the effects of blood/cell-free hemoglobin mixtures of high versus low viscosity on hemodynamics and vascular hindrance, an index of vascular tone, which was normalized for blood viscosity. Anesthetized rats were subjected to 50% exchange transfusion with (1) high-viscosity solutions: whole blood (n=5) or red blood cells mixed with cell-free hemoglobin (Hb-Hv group, n=5); (2) low-viscosity solutions: cell-free hemoglobin (Hb-Lv group, n=5) or human albumin (n=5). Two hours after hemodilution, vascular hindrance remained unchanged in animals transfused with whole blood and albumin. Hb-Lv induced an immediate and sustained increase in vascular hindrance (208%). Conversely, in Hb-Hv animals, the vascular hindrance increase was delayed and smaller (27% to 147%), whereas peripheral resistance increased gradually (94% after 2 hours). Our results demonstrate the beneficial effects of cell-free hemoglobin in the presence of the animals' own red blood cells in maintaining physiological viscosity and limiting vasoconstriction because of the pharmacological properties of cell-free hemoglobin.

Volume expansion with modified hemoglobin solution, colloids, or crystalloid after hemorrhagic shock in rabbits: effects in skeletal muscle oxygen pressure and use versus arterial blood velocity and resistance.

Therapeutic goals for hemorrhagic shock resuscitation are the increase of cardiac output and oxygen delivery. The possibility exists that because of microcirculatory effects, different volume expanders result in different tissue oxygen delivery and oxygen use. In a rabbit model of resuscitation from hemorrhagic shock (50% blood loss), we compared the effects of an hemoglobin-based O2-carrying solution (HbOC) with those elicited by albumin, hydroxyethyl starch (HES), or saline on systemic hemodynamics, skeletal muscle O2 pressure (PtiO2), and interstitial concentration of lactate (LACi) through the combined implantation of a microdialysis probe and a sensitive O2 electrode into the hind limb. Hemorrhagic shock induced a 50% decrease in mean arterial pressure (MAP), femoral artery blood flow (BF), and PtiO2. After resuscitation, there were statistically significant differences among the volume expanders. The increase in MAP was faster with HbOC and colloids, and slower with saline, mainly obtained by vasoconstriction for HbOC and by increased BF with albumin and HES. The maximum MAP values were significantly higher for HbOC compared with the other volume expanders. HbOC and colloids induced a faster increase in PtiO2 as compared with saline, but maximum PtiO2 values were not different among the volume expanders. Tissue oxygen use as estimated by LACi increased transiently at the beginning of volume expansion with similar maximum values. Animals resuscitated with saline had significantly higher LACi concentrations after the onset of volume expansion as compared with HbOC but not with colloids. Our results demonstrate that there are measurable differences in MAP and BF upon resuscitation with the four different solutions and there is a slower increase in tissue PtiO2 with saline than with colloids associated with significantly increased LACi consistent with delayed reoxygenation upon resuscitation with saline.

Hemoglobin-based oxygen carriers do not alter platelet functions: study of three chemically modified hemoglobin solutions.

OBJECTIVE: Chemically modified hemoglobins are being developed as potential oxygen-carrying blood substitutes (HBOCs). Clinical and preclinical data demonstrate the vasoactive properties of HBOCs by trapping of nitric oxide, which is also known to have platelet inhibitory activities properties. This study evaluated the effects of three structurally different HBOCs (Hb-Dex-BTC, alphaalpha-Hb, and o-raffinose-poly-Hb) on platelet functions in vitro to compare to those elicited by plasma substitutes, such as hydroxyethylstarch. DESIGN: Platelet activation state was assessed using platelet-rich plasma diluted to 20% (v/v) with the different solutions, by main measuring glycoproteins (GPIb, GPIIb/IIIa, and P-selectin) using flow cytometry. Aggregation was assessed by impedance aggregometry on whole blood hemodiluted to 20% (v/v) with the solutions. SETTING: Biological hematology department of the university hospital of Nancy-Brabois. PATIENTS AND PARTICIPANTS: Ten healthy volunteers consent and informed of the study who denied taking any drugs at the time of the experiment. MEASUREMENTS AND RESULTS: None of these solutions induced activation nor modified reactivity of platelets as measured by the surface expression of glycoproteins GPIb, GPIIb/IIIa, and P-selectin. Moreover, none of these solutions induced platelet aggregation when added alone, nor modified the aggregation patterns of platelets induced by collagen (0.5 microg/ml) and thrombin receptor agonist peptide (12.5 microM). CONCLUSIONS: The three tested structurally different HBOCs, as with hydroxyethylstarch, did not alter platelet functions in vitro.