Jonathan K. Armstrong

Antibody against poly(ethylene glycol) adversely affects PEG‐asparaginase therapy in acute lymphoblastic leukemia patients

Rapid clearance of poly(ethylene glycol)‐asparaginase (PEG‐ASNase) has been reported for up to one‐third of patients treated for acute lymphoblastic leukemia (ALL), potentially rendering their treatment ineffective. A 25% occurrence of an antibody against PEG (anti‐PEG) was previously reported in healthy blood donors. The objective of the study was to determine whether anti‐PEG was associated with rapid clearance PEG‐ASNase.

Covalent binding of poly(ethylene glycol) (PEG) to the surface of red blood cells inhibits aggregation and reduces low shear blood viscosity.

A simple method to coat human red blood cells (RBC) with PEG is described. Using a reactive derivative, monomethoxy‐PEG (mPEG) was covalently attached to the surface of RBC in aqueous media under mild conditions. The PEG coating dramatically reduced aggregation and low shear viscosity of RBC resuspended in autologous plasma, and inhibited RBC agglutination by blood group‐specific antibodies. Morphology and deformability of the PEG‐treated cells were unaltered. The PEG coating of the RBC surface may be of significant benefit in the treatment of a variety of diseases characterized by vaso‐occlusion or impaired blood flow, e.g., myocardial infarction, sickle cell disease. Am. J. Hematol. 56:26–28, 1997.

A local increase in red blood cell aggregation can trigger deep vein thrombosis: evidence based on quantitative cellular ultrasound imaging

Summary.  Background: Recurrent deep vein thrombosis (DVT) risk factors include a first idiopathic DVT, strongly suggesting the existence of undiagnosed and/or unidentified prothrombotic abnormalities. Objectives: To evaluate the impact of locally increased red blood cell (RBC) aggregation on DVT pathogenesis in a rabbit model. Methods: DVT presence, flow and aggregation were measured in situ with ultrasound. Greatly enhanced aggregation was achieved by covalent linkage of Pluronic F98 to the RBC surface; coating with Pluronic F68, which very mildly enhances aggregation, was used as a coating control. On day 1, endothelial damage and a partial stenosis were surgically created on the left femoral vein whereas the right femoral vein was not manipulated. Results: A thrombus was formed within 30 min in six out of seven left femoral veins of animals receiving a 30% volume blood exchange with F98‐coated RBC, whereas a thrombus occurred in only one out of seven veins in F68‐transfused controls. In vivo imaging using quantitative ultrasound confirmed increased aggregation in the thrombosed veins of the F98 group compared with the F68 group and the contralateral vessel. For each group, five animals were followed for 2 weeks before being killed. In F98‐transfused animals, lysis of clots occurred and the presence of chronic thrombi totally occluding the vein in three out of five animals was confirmed by histology. Conversely, in the F68 group, a single disorganized blood clot was observed in one out of five animals. Conclusions: A marked increase in RBC aggregation promotes thrombosis in rabbit femoral veins, confirming a pathophysiological role of locally altered hemorheology in the onset of DVT.

A new, pluronic-based, bone hemostatic agent that does not impair osteogenesis.

OBJECTIVEIntraoperative bone hemostasis can be accomplished using surgical beeswax (bone wax). However, bone wax locally interferes with osteogenesis, and its use is avoided when bone fusion is critical. We describe the use of a Pluronic copolymer blend as a biocompatible, absorbable, hemostatic agent. METHODSA rat femur defect model and a femur gap nonunion model were used. For each surgical model, 24 rats were divided into three treatment groups, i.e., those receiving bone wax implants, Pluronic (90% Pluronic P85/10% Pluronic F88) implants, or no implants (control group). After 10, 21, or 42 days, animals were killed and femora were removed for radiographic analysis and hematoxylin and eosin staining. RESULTSIn the femur defect model, no differences were observed between the Pluronic-treated and control groups; hematoxylin and eosin staining demonstrated bone formation and osteocytes within the defect. In the femur gap nonunion model, no fusions occurred in any group. Development of an osseous callus at the gap site was observed for the control and Pluronic-treated groups. In both models, rats that received bone wax implants exhibited no osseous growth. CONCLUSIONThe Pluronic blend exhibits handling properties similar to those of bone wax, readily achieves hemostasis, and does not inhibit bone regrowth. Pluronic compounds may serve as effective absorbable hemostatic agents for the treatment of bone bleeding in sites where fusion is critical. In addition, this copolymer blend may find use as a vehicle for the short-term release of pharmacological agents, which may further reduce the incidence of infections, reduce inflammation, and improve fusion rates.

Rheologic behavior of sickle and normal red blood cell mixtures in sickle plasma: implications for transfusion therapy.

BACKGROUND:  Guidelines for transfusion in sickle cell disease usually define an upper hematocrit (Hct) limit of 0.30 to 0.35 to avoid blood hyperviscosity. In vitro viscosity studies of normal (AA) and sickle (SS) red blood cell (RBC) mixtures in buffer appear to confirm that this Hct limit is optimal for oxygen delivery to vascular beds as judged by the ratio of Hct to viscosity, with this ratio often termed “oxygen or RBC transport effectiveness.” In the absence of plasma, however, effects due to RBC‐RBC interactions mediated by plasma proteins cannot be assessed.

Ostene, a new water-soluble bone hemostasis agent.

Traditional formulations of bone wax are composed largely of beeswax and are well known to interfere with bone healing and cause inflammatory reactions. Ostene, a newly available bone hemostasis agent made of water-soluble alkylene oxide copolymers, was evaluated. The soft tissue response to Ostene was compared with bone wax and a polyethylene control after implantation into the paravertebral muscles of three rabbits. After 2 weeks, Ostene elicited no fibrous response, the polyethylene elicited a thin (less than 0.5 mm) fibrous response, and the bone wax was encased in a fibrous capsule 0.6 to 1.0 mm thick infiltrated with inflammatory cells. The effects of Ostene were compared with bone wax in a femur defect model in eight rabbits. Ostene showed no evidence of an adverse response in the cortical defect site, medullary cavity, or the surrounding tissue at 4 and 8 weeks. In contrast, bone wax at both time intervals elicited a foreign body response consisting of fibrous tissue infiltrated by macrophages, giant cells, and lymphocytes at the sites of the bone defects. Bone wax also displaced the bone marrow and interfered with bone ingrowth into the defects. Ostene provides the clinician a water-soluble bone hemostasis material that does not demonstrate the adverse tissue response or the interference with bone healing seen with the use of bone wax.

Inhibition of red blood cell-induced platelet aggregation in whole blood by a nonionic surfactant, poloxamer 188 (Rheothrx® Injection)

RheothRx Injection, an aqueous solution of a nonionic block copolymer (poloxamer 188) formulated for intravenous administration, was investigated as an inhibitor of red blood cell (RBC)-induced platelet aggregation at plasma concentrations of 0.05-5mgmL-1. Platelet aggregation was determined by measuring the fall in single platelet counts after mechanical agitation of 2mL aliquots of citrated whole blood in a 37 degrees C shaking waterbath. Inhibition of RBC-induced platelet aggregation of > 95% was observed for poloxamer 188 at a concentration of 1mgmL-1, and 41% inhibition was observed at 0.05mgmL-1. Poloxamer 188 was observed to be a more effective inhibitor of RBC-induced platelet aggregation than 2-chloradenosine (2-ClAd) or phosphoenolpyruvate/pyruvate kinase (PEP/PK). Studies using platelet rich plasma (PRP) showed that platelet aggregation could not be induced by shaking in the absence of RBC, though aggregation was induced by the addition of exogenous adenosine diphosphate (ADP). Poloxamer 188 did not inhibit ADP-induced platelet aggregation. We propose that poloxamer 188 protects RBC from mechanical trauma by non-specific adsorption of copolymer to the RBC surface (via the hydrophobic polyoxypropylene moiety), and that this effect prevents mechanical damage and hence leakage of ADP from RBC. RheothRx Injection has been shown to have value in the treatment of acute ischemic disorders such as myocardial infarction. The observation of significant inhibition of RBC-induced platelet aggregation at clinically relevant concentrations suggests that RheothRx Injection may have antithrombotic properties in vivo, and may therefore have potential not only in acute ischemia but also to prevent thrombosis within vascular prostheses or to prevent rethrombosis after angioplasty or endarterectomy.

The occurrence, induction, specificity and potential effect of antibodies against poly(ethylene glycol)

Specific antibodies against poly(ethylene glycol) (anti-PEG) were induced in animals following exposure to PEG-conjugated proteins and particles, resulting in rapid clearance of PEG-conjugated agents. In humans, induction of anti-PEG was observed following exposure to a PEG-conjugated drug, and pre-existing anti-PEG was identified in over 25% the healthy population. In clinical studies, the presence of anti-PEG was strongly associated with rapid clearance of PEG-asparaginase and PEG-uricase. PEGylation of therapeutic agents will continue to be of significant value in medicine to reduce immunogenicity, antigenicity and toxicity as well as markedly reducing renal clearance, while maintaining drug efficacy. It is important to recognize that PEG itself may possess antigenic and immunogenic properties. Further comprehensive studies are warranted to fully elucidate the effect of anti-PEG on PEG-conjugated agents and if confirmed in a prospective trial, patients should be screened and monitored for anti-PEG, and strategies developed to overcome the potential negative effect of anti-PEG on drug clearance to improve the effectiveness of therapy.

The Effects of a Soluble Polymer and Bone Wax on Sternal Healing in an Animal Model

PURPOSE This study compares the effects of a soluble polymer hemostatic material and bone wax on sternal bone healing. DESCRIPTION Median sternotomies were performed on 20 New Zealand White rabbits, and sufficient polymer (Ostene; Ceremed Inc, Los Angeles CA) or bone wax (Bone Wax; Ethicon Inc, Somerville, NJ) was applied to achieve bone hemostasis. After 6 weeks, sternal healing was assessed using roentgenograms, histology, and mechanical strength testing. EVALUATION Roentgenograms revealed normal bone healing in the polymer-treated group and nonunion in the bone wax group. Histology showed normal bone healing in the polymer group, with fibrotic scar tissue and the absence of new bone formation in the bone wax group. Mechanical strength testing showed that polymer-treated sternal segments were twice as strong as those treated with bone wax. They had a significantly higher flexural strength (2.53 +/- 0.43 vs. 1.29 +/- 0.37 megapascal [MPa]; p < 0.001) and Youngs modulus (0.315 +/- 0.056 vs 0.146 +/- 0.031 MPa; p < 0.001). CONCLUSIONS The application of the polymer hemostatic material to the sternum resulted in significantly stronger union compared with the use of bone wax.

The effect of pH and concentration upon aggregation transitions in aqueous solutions of poloxamine T701

Thermally induced aggregation transitions have been investigated for aqueous solutions of the poloxamine block copolymer T701-(OE(4)OP(13))(2)NCH(2)CH(2)N(OP(13)OE(4))(2)-using differential scanning calorimetry. The calorimetric signals obtained were fitted to a mass action model description of aggregation using a previously reported analytical procedure (Patterson et al., Langmuir 13 (1997) 2219). The presence of a central ethylene diamine moiety in the molecular structure renders the T701 molecule basic; this was confirmed and measured by acid/base titration. Basicity is shown to have an important impact upon aggregation. At low pH (2.5), the poloxamine exists in its protonated form and the bulk solution proton concentration is sufficient to suppress de-protonation, aggregation-as a consequence-is shifted to a higher temperature range. Any increase in pH reduces the temperature range over which aggregation occurs. The derived experimental calorimetric parameters, obtained from model fitting procedures, can be used to compute the fraction of poloxamine existing in an aggregated form, at any particular temperature. The data sets obtained were interpolated to show that at human body temperature (310.6 K) the fraction of poloxamine found in its aggregated form is zero at a pH of 2.5. However at a pH of 6.8, the percentage aggregation increases to about 85%. These aggregation characteristics of T701 have important implications for the design of drug delivery systems, which incorporate poloxamines.