Bovine hemoglobin anaerobically reacted with divinyl sulfone: a potential source for hypothermic oxygen carriers. The bifunctional reagent divinyl sulfone was anaerobically reacted with bovine hemoglobin to give a noncrosslinked intramolecularly-modified new derivative (HbBv-DVS). By employing a high molar ratio of divinyl sulfone to HbBv-DVS, it was possible to effect anaerobic intermolecular crosslinkage. The polymerized material (Poly HbBv-DVS) was shown to consist of a mixture of modified intermolecularly-crosslinked hemoglobins characterized by molecular masses ranging from 130 to -500 kDa. Some functional properties of HbBv-DVS and Poly HbBv-DVS have been evaluated in vitro and in vivo. Viscosities of HbBv-DVS solutions at temperatures as low as 15 degrees C and concentrations up to 14.0 g/dl were proved to be much lower than that of normal human blood at 37 degrees C (-4 cp). Poly HbBv-DVS (14.0 g/dl) was iso-oncotic (COP = 23 mm Hg) with plasma, and had viscosities of 3.37 and 4.57 cp at 37 and 15 degrees C, respectively. The clearance of Poly HbBv-DVS from the circulation was significantly delayed (T1/2 = 270 min), compared with those of HbBv and HbBv-DVS (T1/2 = 80 and 100 min, respectively. The P50 values were substantially increased (P50 = 52 and 61 mm Hg at 37 degrees C, 0.15 M Cl- and pH 7.4 for HbBv-DVS and Poly HbBv-DVS, respectively). Due to their right-shifted oxygen equilibrium curves, these derivatives still yielded P50 values of about 20 mm Hg at the low temperature of 15 degrees C, as compared with only 7 mm Hg for native bovine hemoglobin. These properties make HbBv-DVS and Poly HbBv-DVS potential new candidates for low-temperature organ perfusion.


Hemoglobin-based oxygen carriers (HBOCs): structural alterations that affect free radical generation. We examined how changes in oxygen affinity brought about by different chemical modifications of hemoglobins affect their oxidation-reduction reactions. The three modified hemoglobins studied were HbA-FMDA, HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin; and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHb to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide (Me2SO) to produce formaldehyde (HCHO). Relative to the reaction rate for HbAo (630 +/- 130 M/min) the rates of HCHO formation were roughly 70% for HbA-DBBF, 50% for HbA-FMDA and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rates of free radical formation, addition of mannitol or thiourea slowed both the rate of spectral changes and HCHO formation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation. HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays. The observed differences between HBOCs in these assays indicate differences in their ability to generate or interact with free radicals.


Stabilities and properties of multilinked hemoglobins. Crosslinking of both human and dog hemoglobin has been done with a variety of reagents to produce singly, doubly and multiply crosslinked hemoglobins. Succinate and glutarate diaspirins did not crosslink deoxy human hemoglobin in good yield, in contrast to the fumarate analog (DBSF). Deoxy dog Hb did not react well with DBSF, but oxy dog Hb did react, giving crosslinked tetramers as well as dimers on SDS electrophoresis. Crosslinking with a short, rigid reagent (difluorodinitrobenzene) resulted in a similar product for both oxy and deoxy hemoglobin that had high stability and oxygen affinity. The trilinker, tris-chloroethylamine, produced a more stable product than the corresponding crosslinker, bis-chloroethylamine. Double crosslinking oxy Hb with DBSF and dimethylpimelimidate or with DBSF followed by deoxygenation and recrosslinking with DBSF gave products with higher denaturation temperatures. The diaspirin double crosslinked product had high oxygen affinity.


Relationship between chemical properties and biological properties of pyridoxalated hemoglobin-polyoxyethylene. Pyridoxalated hemoglobin-polyoxyethylene (PHP) is a conjugate of human hemoglobin with alpha-carboxymethyl, omega-carboxymethoxypolyoxyethylene(POE). This conjugate is selected as an oxygen carrier for blood substitute because it can survive for a long time in the circulation and also it can transport the same amount of oxygen as red cell. Optimization of PHP has been done by changing the degree of the modification and reaction procedures in order to adjust viscosity and colloid osmotic pressure to physiological values. The oxygen carrying capacity was physically evaluated by oxygen equilibrium curves and biologically by an ATP content in perfused isolated liver. Structural relationship of PHP to the binding properties to haptoglobin was studied and the effect of the POE modification on the binding properties was observed when the number of POE per one hemoglobin molecule is over six. Based on the comparative study of solubility of met-PHP and met-SFH, the POE modification was suggested to reduce the toxicity of hemoglobin against organs. Finally physical properties of PHP at low temperature was discussed in relation to organ preservation.


Carbodiimide-mediated coupling of benzenepentacarboxylate to human hemoglobin: structural and functional consequences. We have examined the covalent modification of HbA with BPC (benzenepentacarboxylate) whose carboxyl groups were activated with EDC [1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide]. Reaction of deoxy-HbA at pH 8 with a 10-fold excess of BPC, preactivated with a 2-fold excess of EDC for 5 minutes, followed by anion-exchange chromatography, gives three components with p50 values of 1.15 (unreacted HbA), 11.7 and 7.6 mm of Hg at 20 degrees C (50 mM Bis-Tris pH 7.0). Component III does not dissociate into dimers upon dilution, but components I and II do. When deoxy-HbA is reacted at pH 6 with 10-fold BPC, preactivated with two-fold EDC for 5 minutes, the resultant HbA derivatives can be separated into three components, with p50 values at pH 7 of 14.2, 10.2 and 5.2 mm of Hg, respectively. All three components are stable tetramers. Oxygen binding by all of the covalent HbA-(BPC)x complexes is cooperative, pH sensitive, but IHP insensitive. The latter observation suggest that BPC is covalently bound to HbA's DPG/IHP binding site. This conclusion is corroborated by reversed phase HPLC analysis which shows that all five modified HbAs contain at least one modified beta chain. In addition, 4 of the 5 derivatives also contain modified alpha chains. No inter or intratetramer crosslinks are observed.


Stabilized hemoglobins as acellular resuscitative fluids. This study reports some recent work dealing with the stabilization of the tetramers of hemoglobin. It is shown that by using a variety of diacids, it is possible to increase the P50 above that of stroma free hemoglobin. In order to lengthen the retention times in the circulatory system, the stabilized hemoglobins were complexed with both hydroxyethyl starch polymers and polyol tetronic polymers. The resulting hemoglobin-polymer compounds were then freeze-dried. It was possible to reconstitute the powder by the addition of physiological saline when needed. The methods presented here appear to be appear to be as effective as using pyridoxal phosphate but at a fraction of the cost.


Synthesis and characterization of artificial red cell (ARC). An unsaturated lipid made from 2,4-octadecadienoic acid was used as a component of phospholipid vesicles. The vesicles (0.2 micron phi) was prepared by an extrusion method, and the unsaturated lipid was polymerized with gamma-ray. Vesicles constructed from the polylipid show enormous stability during long term storage in frozen state and can be handled easily. Negative effect could not be confirmed in vitro tests. This system satisfies all the physiological conditions, such as oxygen transport ability and solution properties as a blood substitute.


Liposome-encapsulated hemoglobin processing methods. An effective and safe red blood cell substitute is being developed based on double emulsion/evaporation techniques followed by high pressure homogenization to form liposome-encapsulated hemoglobin (LEH). Formulations are made up of hydrogenated phosphatidylcholine (PC, soy or egg), cholesterol, phosphatidylinositol (PI), and alpha-tocopherol in a molar ratio of 1:1:0.2:0.02, respectively. Resulting LEH-encapsulated hemoglobin (Hb) concentrations are greater than 80% of precursor Hb solutions. Met-Hb generation accompanying LEH processing appears to be small with only a 3% increase for encapsulated over precursor. These results correspond to an oxygen content for an LEH suspension sample (50% by volume LEH) of 15 volume% oxygen. Oxygen affinity and cooperativity values for LEH suspensions appear to be near the normal values expected for whole blood. The viscosity of LEH suspension samples (50% by volume LEH in phosphate-buffered saline containing 7.5 wt% albumin) were slightly higher than that of whole blood. The effect of shear rate on leakage of encapsulated Hb from LEH was small, i.e. 0.5% or less. Nearly total isovolemic exchange transfusion using a cannulated rat model demonstrates efficacy of LEH suspension samples. There appears to be no difference in rat internal organ weights between rats exchanged with control compared to rats exchanged with LEH. Circulation half-life following 50% isovolemic exchange-transfusion is about 15 to 18 hours.


Purification and quantitative determination of carboxymethylchitin incorporation into submicron bilayer-lipid membrane artificial cells (liposomes) encapsulating hemoglobin. The separation of carboxymethylchitin-coated hemoglobin-loaded liposomes (CMC-LEHb)s from the non-adsorbed CMC has been achieved by gel chromatography. This purification takes place at the physiological pH of 7.4 favoring HbO2 preservation. A comparative study between experimental techniques for the quantitative determination of the absorption of carboxymethylchitin (CMC) onto liposomes encapsulating hemoglobin (LEHb)s has been conducted. Results suggest that FT-IR spectroscopy gives a more accurate quantitative absorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. Quantitative bilayer and surface characterization show that the RBC membrane composition has been closely simulated by that of CMC-LEHbs in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively.


Large-scale blood substitute production using a microfluidizer. Microfluidization has been tested as a way to disperse phospholipids in aqueous hemoglobin solutions. Spherical and stable liposomes of 2 to 3 microns were obtained. Lipid incorporation (up to 85%) and hemoglobin encapsulation (up to 15%) in liposomes have been improved with respect to previous investigations. However, results show that a more efficient dispersion system using lower concentrations of lipid is required to obtain a high liposome hemoglobin concentration (limited actually to 150 g/l) and an economically and biologically suitable process for artificial blood production at large scale.


Oxygen-transport and solution properties of polylipid/Hb vesicles (ARC). Polymerized phospholipid vesicle encapsulating Hb (polylipid/Hb vesicle) was prepared from a mixture of unsaturated phospholipid, cholesterol and unsaturated fatty acid and polymerization by gamma-ray irradiation. The average radius of resulting vesicles was 203 +/- 39 nm and concentrated Hb (30 wt%) was efficiently encapsulated. gamma-Ray polymerization proceeds theoretically at low temperature (4 degrees C). P50 and oxygen transporting efficiency were adjusted to 40 mmHg and 40%, respectively. Oncotic pressure and solution viscosity can be controlled to the same values as blood.


Studies on the quality control of stroma-free hemoglobin. Analytical methods for phospholipids and blood group antigen were developed using HPLC-UV and EIA, respectively, for the quality control of SFH. These methods had sensitivities superior to those of conventional assays. In addition, the HPLC method could isolate PS, which might play an essential role in blood coagulation. The above analytical methods were applied to evaluate three representatives SFH preparation methods, 36,000xg centrifugation followed by the filtration with a 0.22 um filter, ultrafiltration with a 100 kd membrane, and filtration with the BMM-40 nm membrane. Both the BMM method and the ultrafiltration method were shown to remove more than 99.7% of phospholipids, and more than 99.99% of blood group antigen. On the other hand, the centrifugation preparation still contained much of both components. The BMM filter was originally developed as a virus removal filter. The virus removal efficiency of the BMM-40 nm was examined with a genetic technique, the PCR method. The BMM-40 nm was demonstrated to remove HB viruses with an efficiency of eight log or more. These results indicate that the BMM is a useful filter to prepare a stroma-free, virus-reduced hemoglobin solution.


Pilot scale production of pyrogen-free human hemoglobin for research. A pilot scale production facility for the preparation of 20 to 30 liters of stroma-free hemoglobin is described. The system is capable of producing pyrogen-free solutions for research purposes. It is not certified for production of parenteral solutions for human use, but the plan could be implemented to meet standards for such materials. Products of the facility should be of adequate quality to address most of the toxicity and efficacy issues facing further development of hemoglobin-based red cell substitutes.


Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies. During 1990 and 1991 the capability for repetitive, consecutive production of DCLHb solution to meet a rigorous and complete set of product criteria was demonstrated. In addition, through periodic monitoring of product stored under controlled conditions, the stability of all lots of DCLHb solution during frozen storage was demonstrated for more than a year. In this way, assurance was provided that the DCLHb solution used in preclinical testing met all product criteria throughout the biological testing period.


Large scale production and characterization of lyophilized pyridoxalated hemoglobin-polyoxyethylene (PHP). Lyophilized PHP as an oxygen carrier is prepared from outdated red cell and dicarboxymethylated polyoxyethylene. In order to apply PHP for a clinical use, a large scale production of high quality PHP has been studied. We have set up a 20 L scale production flow of PHP88. The product was tested to confirm the quality and lot-to-lot consistency. The blood group specific materials were weakly positive in stroma-free hemoglobin (SFH), however, were found negative in the PHP of this scale. The amount of phosphatidylethanolamine (PE) in purified SFH and PHP88 reconstituted solution was 0.19 +/- 0.04 and 0.03 +/- 0.01 ppm, respectively. Contamination of viruses such as HBV and Non A non B hepatitis virus could not be observed in the final product. Elimination and inactivation of HIV was validated through a spike test. The characterizations of the final products in 20 L scale were done through MW, P50, Hill coefficient, viscosity, and molecular weight distribution by SDS-PAGE and batch to batch consistency was also confirmed. The results show that production process is appropriate to eliminate the blood group materials, PE and virus, and produce PHP of high quality. Lyophilized PHP88 can be produced by addition of maltose and can be stored over 1 year.


Detection of membrane fragments in hemoglobin solutions. Two methods for the detection of membrane components in human stroma-free hemoglobin (SFHb) solutions are described. The first method is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml SFHb. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). The determination of both glycophorin alpha and phospholipid yields useful information on the purity of SFHb solutions, as was shown by determination of the purity of two SFHb solutions prepared in different ways.


Possible importance of chromatographic purification position in a blood substitute elaboration process. Purification of hemoglobin (Hb) solutions suggested as oxygen carriers is an imperative necessity. All processes currently used clear out the solutions of almost the totality of impurities that are able to negatively influence the transfusional efficiency of Hb. We have studied the stability (metHb measurement) of Hb purified by DEAE dextran chromatography (Spherodex, 10 mM phosphate buffer, pH 7.20) which eliminates lipopolysaccharides, enzymes and non heminic-proteins ... without denaturing Hb. In spite of the improvements due to this purification method on the transfusional efficiency of non modified Hb solutions, the lack of enzymes involved in the protection of Hb against autooxidation (superoxide dismutase, peroxidase, catalase ...) makes it much more vulnerable to the reagents used during the following chemical processes: pyridoxylation, polymerization or macromolecule binding, thereby leading to an important oxidation to metHb. These observations led us to question the optimal position of purification in a process of modified Hb preparation. We have shown the importance of this chromatographic position in working on pyridoxylated Hb bound to monomethoxypolyoxyethylene. Spherodex chromatography appears to be a very satisfactory purification method, provided it occurs only at the last step of the process. Many authors have not been attentive to this phenomenon which could be found with other Hb modifications. This therefore imposes to study the best place to incorporate a particular purification step into a hemoglobin preparatory procedure.


Development of analytical methods to evaluate SFH. For the determination of stroma content in SFH, two analytical methods were developed, the HPLC-UV for phospholipids and the enzyme immunoassay for blood group antigen. These analytical methods were applied to evaluate three representative SFH preparations. The 36,000 x g centrifugation method was shown to contain higher amount of stroma components. On the other hand, the BMM-40 nm and the ultrafiltration method with a 100 kd membrane was shown to remove more than 99.7% of phospholipids and almost all of blood group antigen from hemoglobin solutions. These results demonstrated that these analytical methods were very useful in evaluating any highly purified hemoglobin solutions.


Trace element analyses of diaspirin cross-linked hemoglobin solutions. A sensitive assay using inductively coupled plasma atomic emission spectroscopy (ICP-AES) has been applied to the measurement of trace elements in diaspirin cross-linked hemoglobin (DCLHb) solutions. Calcium, magnesium, zinc and iron were the only elements detected at greater than background levels. Ag, Al, As, B, Ba, Bi, Cd, Co, Cr, Cu, Mn, Mo, Pb, Sb, Se, Si, Sr, Ti, and V were present at concentrations either equal to the acid blanks or were not detected. Detection of non-heme iron (not incorporated into the hemoglobin porphyrin ring) in a 10 g/dL hemoglobin solution required the development of a special protocol. In this protocol a chelator, DTPA, was added to hemoglobin solutions to complex with both free and non-specifically bound non-heme iron. The resulting iron:DTPA complexes were separated from the hemoglobin molecules by ultrafiltration and the ultra-filtrate analyzed by ICP-AES. A modification of this assay in which the DTPA was omitted was used to measure the free non-heme iron in solution. Typical concentrations of chelatable (free and non-specifically bound) and solution (free) non-heme iron in DCLHb production lots at the completion of manufacture were 0.5-1.0 ppm and 0.1-0.3 ppm, respectively.


Endotoxin removed from hemoglobin solution using polymyxin-B immobilized fibre (PMX-F) followed by a new turbidometric endotoxin assay. Endotoxin contamination in modified hemoglobin can result in side effects. Accurate measurement and effective elimination of endotoxin are important in producing safe hemoglobin preparation. A new turbidometric endotoxin assay (Toxinometer) was studied, in which absorbance of wavelength was applied at 660 nm. This method is not affected by the presence of hemoglobin in solution. This way, toxinometer can accurately measure endotoxin concentration in hemoglobin solution. For the elimination of endotoxin, polymyxin-B immobilized fiber (PMX-F) was studied in-vitro and compared with commercial materials. The PMX-F was found to be a convenient and less expensive approach.


Conceptual design and cost estimate for a modified hemoglobin production plant. Polymerized, pyridoxalated stroma-free hemoglobin (Poly PSFH), made from outdated human blood, has excellent attributes for use as a blood substitute. A hypothetical larger scale process for the production of Poly PSFH is detailed, and facility design and cost estimates are presented. This paper gives the opportunity to discuss the engineering activities of process scope definition, utilities selection, and aseptic processing techniques, etc. Compliance with cGMP and other FDA regulation will also be discussed.


The purification and comparative analysis of hemoglobin from animal bloods. Various methods for the separation and purification of Hb from animal blood have been investigated to establish the optimal and highly reliable purification process. The Hb obtained from various sources, i.e., bovine, pig and human bloods were analyzed and compared using simple and fast analytical methods, including, electrophoresis, isoelectric focusing, and spectroscopy to study the characterization of protein structure. Further investigation along this line will enable us to realize the ultimate objectives of development for artificial RBC substitutes.


Diaspirin cross-linked hemoglobin solution as a resuscitative fluid following severe hemorrhage in the rat. The effect of diaspirin cross-linked hemoglobin (DCLHb) on mean arterial pressure (MAP) and heart rate (HR) was compared to Ringer's lactate (RL) and shed blood in a 70% lethal model of hemorrhage (35 cc/kg blood loss) in conscious rats. All animals resuscitated with DCLHb regardless of dose (17.5 and 35 cc/kg) and concentration (7% and 10% solution) exhibited complete restoration of MAP and HR which was maintained for at least 5 hrs. Hemodynamic responses in DCLHb-treated animals were not significantly different from 35 cc/kg blood-treated animals. In RL (105 cc/kg) treated animals the MAP was restored to 60-70% of baseline. 24 hr survival in animals resuscitated with fluids ranged between 88-100% and was not significantly different between treatment groups.


Dehydration and shock: an animal model of hemorrhage and resuscitation of battlefield injury. We have developed a porcine model of the anticipated military use of oxygen-carrying resuscitation solutions. The objective is to determine whether toxicity under adverse conditions will limit further development of hemoglobin-based products. Splenectomized immature female swine are used because of their extensive use in the evaluation of other resuscitation solutions. Five days prior to each experiment, central vascular catheters and a renal arterial flow probe are surgically placed in the animals. After recovery and weight gain has resumed, animals are placed in metabolic cages and deprived of water for 48 hours to produce hyperosmolar dehydration resulting in loss of approximately 7% of body weight. We remove 38% of estimated blood volume, 25 ml/kg, over one hour by a controlled logarithmic hemorrhage. Resuscitation is by administration of a fixed volume of test solution. Hemodynamic function is observed but not further therapy is given for three hours, a period corresponding to evacuation in the field. After this period, corresponding to arrival at a field hospital, the animals' blood is returned. Swine are then observed in metabolic cages for an additional 7 days while blood and urine are sampled daily. At the end of this period, animals are anesthetized, urinary catheters are implanted, and creatinine clearances are measured. Swine are than euthanized, and their tissues are examined. In a pilot study, resuscitation was performed with either Ringer's lactate, albumin, stroma-free hemoglobin, or cross-linked (alpha alpha Hb) hemoglobin. All animals survived.


Effect of a single replacement of one of Ringer lactate, hypertonic saline/dextran, 7g% albumin, stroma-free hemoglobin, o-raffinose polyhemoglobin or whole blood on the long term survival of unanesthetized rats with lethal hemorrhagic shock after 67% acute blood loss. This study is based on an unanesthetized lethal hemorrhagic shock rat model (67% blood volume bled in 2 stages). The 8 groups studied were as follows. 1. Control--no resuscitation fluid. 2. Reinfusion of rat's own shed whole blood. 3. Ringer lactate solution--3 times shed blood volume. The following 4 fluids were each given as equal to the shed blood volume. 4. 7.5g% NaCl hypertonic saline/6% Dextran 70, followed by 3 volumes Ringer lactate dextran. 5. Ringer lactate solution. 6. Human albumin 7g% in Ringer lactate. 7. Stroma-free hemoglobin in Ringer lactate 8. o-raffinose polyhemoglobin in Ringer Lactate. Blood pressure and other vital signs were recorded continuously during the control period, bleeding periods, infusion period and 60 minutes after infusion. After this all surviving animals were followed for 14 days with no other special treatments. Group 4 had transient (10 minutes) returned of blood pressure to control levels. The 3 groups with sustained blood pressure at control level when followed for 1 hour were groups 2, 6 and 7. The 2 groups with 100% survival on days 14 were group 2 and group 7.


PEG-bovine hemoglobin: safety in a canine dehydrated hypovolemic-hemorrhagic shock model. An initial evaluation of PEG-bHb was performed using a modified hypovolemic shock model. PEG-bHb had a substantially longer intravascular half-life than native Hb and no measurable hemoglobinuria was observed in the canine. PEG-bHb allowed successful resuscitation with an oxygen carrying capacity of 14-22% over that of lactated Ringer's solution.


Resuscitation of bled dogs with pyridoxalated-polymerized hemoglobin solution. We bled 25% of estimated total blood volume, then infused pyridoxalated polymerized human stroma-free hemoglobin solution (PP-SFH) (10 g/dl) to dogs under anesthesia in a volume equal to the blood removed. Central hemodynamics, blood flow distribution to organs, and renal function were studied up to 2-3 hours following the infusion. Mean arterial pressure was reduced from 120 +/- 3 to 86 +/- 7 mmHg at the end of the 30-minute hypovolumic period and the cardiac output was reduced by 27%. Immediately following the PP-SFH infusion we observed a further fall in blood pressure (43%) caused by a fall in cardiac output which lasted for 10 minutes. Blood pressure was restored gradually with the continuation of the infusion and the cardiac output was restored and maintained well. During the hypovolumic period, blood flow to the heart, renal cortex, and liver were reduced, whereas normal flow to the renal medulla and brain were maintained. After the resuscitation, blood flow to the heart, brain, liver, and renal medulla significantly exceeded the normal range, but remained subnormal in the renal cortex. Glomerular filtration rate (GFR), urine flow, and electrolyte excretion were all reduced during the hypovolumic period and were not restored to the pre-bleed levels after the infusion.


Study of effect of the newly developed artificial blood "Neo Red Cells (NRC)" on hemodynamics and blood gas transport in canine hemorrhagic shock. The purpose of this study is to evaluate the newly developed liposome-encapsulated hemoglobin, named Neo Red Cells (NRC), in the treatment of hemorrhagic shock. The particle size of NRC is 180 +/- 88 nm, the hemoglobin concentration is 5.6 g/dl, the viscosity is 2 cp and P50 is 49.5 mmHg. The experiment was carried out on six mongrel dogs suffering hemorrhagic shock. Blood was extracted from the femoral artery and blood pressure became lower than 60 mmHg. NRC in amount equal to the amount of blood extracted was transfused immediately. Inhalating normal room air, the above manipulation was repeated 3-5 times. After 59% to 88% blood exchange using NRC, the total peripheral vascular resistance index (TPRI) was reduced and the cardiac index (CI) was increased, thereby alleviating the burden on the heart. The reduction of TPRI in the presence of hemorrhagic shock is presumed to be due to the small size of the NRC granules and their low viscosity. As the exchange rate increased, the oxygen consumption (VO2) increased remarkably, presumably due to the increase of CI and A-V difference of oxygen content. The conclusion of the study is that NRC is more suitable than natural blood for the treatment of hemorrhagic shock.


Modified hemoglobin solution as possible perfusate relevant to organ transplantation. A modified hemoglobin solution (<pyridoxylated hemoglobin>-<polyoxyethylene> conjugate solution, PHP solution) has very interesting characteristics such as oxygen-carrying property without corpuscular components. Experimental use of the PHP solution has shown promising possibilities as a perfusate relevant to organ transplantations. 1) Elongation of warm ischemic time in canine kidneys: Dogs survived even with the unilateral kidneys which had been exposed up to 4.5 hour warm ischemia and, thereafter, perfused with the PHP solution. 2) Elongation of perfusion preservation period of canine livers: Dogs survived with the transplanted livers which had been perfused for 48 hours with the PHP solution. 3) Successful perfusion of rat small intestine: Lewis rat intestines perfused and preserved for 12 hours with the PHP solution showed a higher survival rate compared with those with Collins or UW solution. 4) Removal of antibodies: By exchange transfusion with a total of 30-60 ml of the PHP solution, a Lewis rat hematocrit lowered to 5% while IgG went down to nil from 8970 mg/dl, IgA to 28 mg/dl from 118 mg/dl and IgM to 190 mg/dl from 897 mg/dl. This technique is expected to be applicable for removal of the naturally existing antibodies in xenotransplantation.


Does oxygen supply improve graft viability in liver preservation? While the clinical results of orthotopic liver transplantation have greatly improved, the viability of liver grafts and extension of the safe time for preservation are necessary factors in need of improvement. The liver is one of the organs most sensitive to anoxia. The addition of an oxygen carrying agent to the preservation solution was evaluated. Pyridoxalated hemoglobin-polyoxyethylene conjugate (PHP) is used as an oxygen carrier. Viaspan (UW) served as a control solution. Test solution (PHP+UW) composition was composed of a 1:1 mixture of PHP and UW solutions with hemoglobin 4.0g%, hydroxyethyl starch 2.5g%, osmolality 320 mOsm/kg H2O, and colloidal osmotic pressure 33 mmHg. The oxygen carrying capacity of PHP+UW solution is about 10 times higher than UW solution at 4 degrees C. Male Lewis rats (BW: 250-300 g) were divided into five groups. After flushing the solution via the portal vein, rat livers were harvested. Two preservation methods, simple storage and perfusion (0.1 ml/min/g liver), were studied at 4 degrees C for 24 or 48 hours. OxyHb, MetHb, pO2, pH, Na, K, GOT, and GPT of perfusate, hepatic mitochondrial functions after preservation, and tissue adenine nucleotides by HPLC were measured. Light microscopy on the tissue was also performed. No significant differences were noted in perfusate biochemical parameters. Oxygen consumption during the perfusion was significantly higher in the PHP+UW than in the UW group. Hepatic mitochondrial functions and tissue ATP levels were better preserved in perfusion than in simple storage, and in PHP+UW than in UW at 48 hours. The oxygen carrying agent, PHP, can provide significantly higher levels of oxygen to liver grafts and improve graft viability.


Machine perfusion of isolated kidney at 37 degrees C using pyridoxalated hemoglobin-polyoxyethylene (PHP) solution, UW solution and its combination. To preserve isolated kidney normothermically, PHP containing UW components were evaluated as perfusates. Kidneys were flushed out by Lactate Ringer solution immediately after isolation from mongrel dogs, and then connected to the perfusion circuit which consists of a preservation box, a reservoir of perfusate, a membrane oxygenator and a drive unit. PHP containing 140 mEq/l of Na+ and 4 mEq/l of K+ (PHP(E)), UW solution (UW) and UW components added PHP(E) were prepared and adjusted at pH 7.4 prior to use. Temperature and perfusion pressure were controlled at 37 degrees C and 100 mmHg, respectively. During 12 hour perfusion, remarkable changes in pH were seen in UW group and PHP group while higher oxygen consumption was noted in PHP(E)+UW group than that in PHP(E) group. The histological findings showed moderate damages of tubular epithelial cells and maintaining normal glomerular structure in PHP(E)+UW while severe damage of both tubulus in UW group were seen. There was no edematous degeneration in both UW and PHP(E)+UW groups, however, it was seen in PHP(E) alone. It was suggested that components of UW solution have positive effect on normothermic machine perfusion with PHP(E) solution.


Evaluation of a pyridoxylated hemoglobin polyoxyethylene conjugate solution as a perfusate for small intestine preservation. A new type of artificial blood, pyridoxylated hemoglobin-polyoxyethylene conjugate (PHP) solution, (developed by PHP research group of the department of health and welfare of Japan, and produced by Ajinomoto Co., Inc. Tokyo) as an oxygen-carrying component, has been recently devised using hemoglobin obtained from hemolyzed human erythrocytes. Recently, the studies using this solution as a preservation solution were performed in some instances. To examine the mechanism of improved viability using this solution as a preservation solution, we developed a model of orthotopic small intestine transplantation (OIT) in the rat. As a baseline study, we compared parameters of viability of the grafts preserved in Collins and UW solution to those preserved in PHP solution including a survival rate, a serum level total protein and albumin, and a change in body weight after transplantation. In our study, the simple hypothermia storage together with intestinal perfusion preservation with PHP solution was performed. Animals were divided into 6, 12, and 24 hr preservation groups. All of the rats survived after 6 hr preservation following transplantation. However, in 12 hr storage, five of six rats in PHP solution preservation survived and recovery in body weight after grafting was better than those with Collins and UW solution. We conclude that the PHP solution is, therefore, considered to possibly be a more suitable perfusate for small intestine preservation than Collins and UW solution.


A preclinical screening test for modified hemoglobin to bridge the gap between animal safety studies and use in human. The infusion of large amount of modified hemoglobin as blood substitute can potentially result in hypersensitivity and anaphylactic reactions, antibody-antigen reactions and others. Animal safety studies are important. However, response in animals may not be the same as in human. Before injecting into human, we may need to use an in-vitro screening procedure. One approach is based on testing the effects of modified Hb on complement activation (C3a) of human plasma. This paper describes this screening test. It also discusses how this may potentially be used. For instance using this to test for contamination from trace membrane fragments with blood group antigen or lipids, antibody-antigen complexes, endotoxin, trace fragments of microorganisms, residual amounts of some polymers, emulsifying agents, and organic solvents. There is also the possibility of obtaining plasma from a very large human population and analyse each of these to study the epidemiology of adverse reactions in different groups and types of patients.


Evaluating new red cell substitutes: a critical analysis of toxicity models. Because red cell substitutes (RCS) will improve oxygen delivery to tissues rendered hypoxic or anoxic they are presumed to be effective. Prior to clinical application the safety--toxicity of any proposed solution must be established. Models used to demonstrate efficacy do not necessarily evaluate toxicity. Infusion of human hemoglobin (Hb) solutions into animals raises one issue of immune response but obviates looking at the human response to similar materials. Differentiating the cause of an immunologic effect may be difficult. Effects seen in acute models may not have chronic implications; they may be transients of no consequence. Differentiating the effects of volume load intravascularly from the effects of the solution is also a problem. Identification of proper controls is essential for evaluation. However, the control solution may have problems of its own that must be defined. For any proposed RBS a menu of models exploring cellular, tissue, organ and organism responses to the solution, its residual modifiers and the products of metabolism are needed to define primary and secondary effects.


Effects of intravenous infusions of diaspirin cross-linked hemoglobin (DCLHb) on sheep. The purpose of this study was to compare the cardiopulmonary, hematologic, and immunologic responses of unanesthetized sheep to single, "topload", intravenous infusions of either 10 mL/Kg or 40 mL/Kg of Diaspirin Cross-Linked Hemoglobin, 10 mL/Kg or 40 mL/Kg of a Human Serum Albumin (HSA) solution oncotically adjusted with human serum albumin to approximately match the oncotic pressure of the DCLHb, or 10 mL/Kg of Erythrocyte Hemolysate solution prepared in a manner similar to that commonly described in the literature and referred to as "stroma free hemoglobin". Solutions were infused at a rate of 1 mL/Kg/minute and animals were monitored for 72 hours after infusion. These studies demonstrated that in sheep infusion of either DCLHb or HSA solutions was well tolerated and did not produce a significant increase in plasma C3a levels, an increase in the plasma concentration of thromboxane B2, or unexpected fluid shifts. In contrast, infusion of the Erythrocyte Hemolysate produced a greater than 10-fold increase in plasma C3a concentrations, a greater than 6000-fold increase in plasma TxB2 concentration, significant fluid shifts, and changes in a variety of other parameters consistent with induction of a dramatic inflammatory response. These results indicate that appropriately prepared and purified DCLHb solutions do not elicit an inflammatory reaction in sheep.


Pharmacokinetic studies in the rat on a o-raffinose polymerized human hemoglobin. We have studied the plasma half-life (T 1/2), oxygen-binding affinity (P50), organ distribution, and excretion of the individual molecular weight (MW) components of human hemoglobin polymerized with periodate-oxidized, ring-opened raffinose (oR poly-Hb), following transfusion in the rat. The model was an isovolemic 50% exchange transfusion in the conscious, chronically catheterized rat. Total plasma Hb levels yielded a (T 1/2) of 10 to 11 hr for oR poly-Hb. The T 1/2 values of individual MW components of the poly-Hb as determined by size-exclusion HPLC were approximately: 4 hr for the monomeric fraction (Hb)1, 9 hr for the dimer (Hb)2, and 15 hr for the fraction representing trimers to nanomers (Hb)3-9. The P50 values of plasma samples containing oR poly-Hb (collected from 0-24 hr after exchange) remained unchanged at 28 +/- 3 mmHg. oR stabilized and polymerized Hb were not excreted via the kidneys. Hepatic and renal distribution as well as plasma and renal clearance were determined by liquid scintillation counting using individual tritium [3H] labelled MW components purified from [3H]-oR poly-Hb: (Hb)1/2, (Hb)1, (Hb)2, (Hb)3&4, and (Hb) greater than 9. In kidney, uptake (determined by the relative concentration of radioactivity) decreased with increasing MW of the labelled component. Conversely, in liver, uptake increased with increasing MW. Plasma and renal clearance results were consistent with those obtained by HPLC analysis. Hematocrit levels returned from a 20% post-transfusion level to normal pre-transfusion levels (44%) within 10 days after the exchange.


Immunogenicity of diaspirin cross-linked human hemoglobin solutions. To assess the potential immunogenicity of human diaspirin cross-linked hemoglobin (DCLHb) solution, repetitive doses of this material were given intravenously to rhesus monkeys at monthly intervals and the immune response to this challenge was assessed. Serum samples collected at multiple intervals throughout the study showed no evidence of DCLHb specific IgG or IgM production. Intradermal skin tests performed one month after the final DCLHb infusion were also negative. These data demonstrate that DCLHb is not antigenic when administered intravenously to rhesus monkeys. In addition, screening of a panel of normal human sera for pre-existing anti-DCLHb IgG antibodies was negative, suggesting that this modified hemoglobin is unlikely to be antigenic in humans.


Immunological effects of hemoglobin, encapsulated hemoglobin, polyhemoglobin and conjugated hemoglobin using different immunization schedules. Repeated subcutaneous immunizing injections into rats of Freund's adjuvant containing rat hemoglobin or o-raffinose rat polyhemoglobin did not result in increase in IgG antibody titers. However, heterologous hemoglobin injected as above is antigenic (49.50 + 6.70 % CPM). Crosslinking heterologous hemoglobin into o-raffinose polyhemoglobin further increased its antigenicity (75.60 + 4.08). Thus, unlike homologous hemoglobin, cross-linking of heterologous hemoglobin increased its antigenicity. Liposome encapsulated homologous hemoglobin injected subcutaneously with or without Freund's adjuvant did not show antigenicity. Encapsulated heterologous hemoglobin resulted in a minimal increase in antibody titers (10.73 + 4.64) only with Freund's adjuvant, but no increase when injected without Freund's adjuvant. Conjugated heterologous hemoglobin (PEG-Hb) injected intravenously by itself at weekly intervals did not result in significant increase in antibody titers on the 5th week.


Liposome encapsulated hemoglobin: long-term storage stability and in vivo characterization. Liposome Encapsulated Hemoglobin (LEH) has been the focus of research and development at the Naval Research Laboratory in an effort to find a viable oxygen-carrying resuscitative fluid. Previous reports from our laboratory have shown that LEH binds and releases oxygen in a manner similar to red blood cells, and that it can sustain life when red cell hematocrits are decreased to critical levels. We have also reported on LEH with regards to preparative methods, scale-up feasibility, toxicity, hemodynamics, hemoglobin P50 modification by coencapsulation of organic phosphates, liposomal surface modification, and storage strategies. In this report, the issue of LEH efficacy following long-term storage in the dry state will be addressed. We have shown that hemoglobin, liposomes, and LEH may be successfully lyophilized and rehydrated to viable states. The modification of the LEH formulation by addition of the carbohydrate trehalose results in the successful lyophilization and storage of LEH. In vitro characterization of LEH stored in the dry state for up to six months includes measurement of oxygen-carrying capacity, liposome size retention, methemoglobin production, and the intraliposomal hemoglobin concentration. The in vivo studies report on physiological parameters such as circulation persistence, blood chemistry, and pathological examination in mice.


On the interaction of the liposomal membrane with blood components. Liposome-encapsulated hemoglobin (LEH) has been shown to be a viable candidate as a blood replacement. However, few data have been presented as to how LEH interacts with normal blood components. Liposomes were prepared from egg lecithin, cholesterol, and dicetyl phosphate or phosphatidic acid, and mixed with fresh blood plasma or whole blood. Erythrocyte osmotic fragility, prothrombin time (extrinsic coagulation efficiency), activated partial thromboplastin time (intrinsic coagulation efficiency), plasma clot stability in urea (fibrin stabilizing factor), and clot retraction (platelet activation) were measured. Although liposomes were found to bind extensively to erythrocytes, all tests indicated that the liposomes had no significant adverse effects, provided that normal levels of plasma Ca++ were maintained. The ability of liposomes to absorb Ca++ from the plasma was related directly to the amount of dicetyl phosphate or phosphatidic acid present and thus, presumably, to the presence of negatively charged species in the membrane. The mechanics of deformation of the LEH membrane were investigated by encapsulating Hemoglobin S in liposomes. Liposomes containing Hemoglobin S were found to sickle when deoxygenated, but not liposomes containing normal hemoglobin. Shape analysis of sickled liposomes yielded a deforming stress of 10(6) dynes/cm2, about 50 times greater than the reported limit for shear elasticity of the erythrocyte membrane.


Characteristics of polylipid/Hb vesicles (ARC) (in vitro and in vivo test). Polylipid/Hb vesicle, which is polymerized phospholipid vesicles (200 nm) encapsulating concentrated Hb solution, has been established by us as an artificial red cell (ARC). ARC can be easily adjusted to physiological conditions (viscosity, density and oncotic pressure etc) as the same of human blood. Our ARC has excellent blood compatibility (no effect on coagulation, no hemolysis and no red cell aggregation). Neither acute toxicity nor damage of internal organs was confirmed. Oxygen transporting capacity was almost the same as human blood, and the half life in blood stream was ca. 13 hrs.


Stability and blood compatibility of polylipid/Hb. The Polylipid/Hb vesicle is a new artificial red cell (ARC) based on liposome-encapsulated Hb. Advantages are derived from the stabilized liposomal bilayer membranes, obtained by polymerization of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phospho choline (DODPC). Furthermore, blood compatibility in vitro are good.


Intravascular reduction of methemoglobin in plasma of the rat in vivo. The formation and reduction of extracellular methemoglobin (metHb) in plasma was studied in vivo in conscious rats after isovolemic exchange transfusions with polymerized hemoglobin solutions. After exchange transfusions of 40 and 70% of the blood volume with hemoglobin solutions, containing less than 6% methemoglobin, the methemoglobin level remained below 15%, whereas exchange transfusions of greater than 90% resulted in an increase in the metHb level to about 30% after 24 hours. The reduction of metHb was studied after exchange transfusions with fully oxidized hemoglobin. A gradual decrease in the metHb level to 20% was observed after exchanges of 5 or 40%. A higher exchange transfusion (70%) resulted also in a decrease in the metHb level but only to approximately 40% in 24 hours. In another series of experiments the reduction of metHb was studied in vitro with isolated human erythrocytes. Incubation of the erythrocytes in the presence of oxidized polymerized hemoglobin (3 g%) resulted in a decrease in the percentage of metHb from 91% to 64%. In the presence of 0.2 mM ascorbic acid the metHb level declined to 22%, suggesting a synergistic effect. These results indicate (1) that there is a potent reducing mechanism present in blood that can reduce extracellular oxidized polymerized hemoglobin and (2) that isolated erythrocytes have a large capacity to reduce extracellular metHb, and may also play an important role in the reduction of extracellular metHb in vivo.


Effects of X-linked hemoglobin on in-vitro platelet function. The in-vitro function of platelets in 1:1 mixtures of fresh whole blood and 10% DBBF alpha-alpha cross-linked hemoglobin in Ringer's acetate buffer was assessed by quantitative aggregation after challenge with common agonists and compared to the function of platelets in similar dilutions of whole blood in saline. Whole blood aggregometry was performed after addition of ADP, collagen, and ristocetin. Aggregation was quantified by measuring the change in impedance as drawn on the chart recording. No difference in mean impedance change was noted between the groups of blood samples which were incubated and tested in the hemoglobin solution and those incubated and tested in saline. In addition, incubation and stirring of the above mixtures over a period of six minutes without the addition of agonists did not result in spontaneous platelet aggregation. We conclude that alpha-alpha cross-linked hemoglobin, in the concentration studied, does not affect in-vitro platelet function, as measured by whole blood aggregometry.


Effect of a bovine hemoglobin preparation (SBHS) on the response of two murine solid tumors to radiation therapy or chemotherapeutic alkylating agents. When tested in mice bearing the Lewis lung tumor with 2, 3, or 4 Gy daily for 5 days, SBHS produced a dose-modifying factor of 1.6 that was increased to 2.1 with carbogen. The addition of SBHS (1.32 gm protein/kg) to treatment with melphalan (MEL) resulted in a 2.2-fold increase in the tumor growth delay (TGD). The combination of SBHS with carbogen (6 h) produced a 3.6-fold increase in TGD compared with MEL alone. The addition of SBHS to treatment with cyclophosphamide (CTX) resulted in a 2-fold increase in the TGD. However, the combination of SBHS and carbogen was much more effective resulting in a 4.6-fold increase in TGD. There was a 1.3-fold increase in TGD with SBHS and CDDP compared with CDDP alone. The combination of SBHS and carbogen was a more effective addition to CDDP resulting in a 1.9-fold increase in TGD. The addition of SBHS to treatment with BCNU increased the TGD produced by BCNU by 1.5-fold. The combination of SBHS/BCNU and carbogen resulted in a 2.3-fold increase in TGD over that obtained with BCNU alone.


Role of thromboxane in mediating the intrarenal vasoconstriction induced by unmodified stroma free hemoglobin in the isolated perfused rat kidney. Unmodified stroma free hemoglobin (SFH) increased renal vascular resistance (RVR) from 4.0 +/- 0.2 to 6.9 +/- 0.9 mmHg/ml/min in control isolated kidneys. In kidneys obtained from rats pretreated with the thromboxane synthetase inhibitor OKY-046 RVR increased from 3.9 +/- 0.3 to 4.8 +/- 0.4 mmHg/ml/min. The percent increase in RVR was greater in response to SFH in control kidneys (67 +/- 14%) than in OKY-046 treated kidneys (23 +/- 7%)(p less than 0.05). SFH resulted in a fall in glomerular filtration rate (GFR) from 0.75 +/- 0.09 to 0.21 +/- 0.04 ml/min in control kidneys while in kidneys from OKY-046 treated rats GFR fell from 0.65 +/- 0.04 to 0.54 +/- 0.06 ml/min. The percent fall in GFR was greater in vehicle treated kidneys (69 +/- 8%) as opposed to OKY-046 treated kidneys (19 +/- 7%)(p less than 0.05). The rate of urinary thromboxane excretion measured before the addition of SFH to the perfusate was 52 pg/min in the control group and 7 pg/min in the OKY-046 treated group(p less than 0.05).


Ultra-pure polymerized bovine hemoglobin blood substitute: effects on the coronary circulation. The effects of stroma-free hemoglobin (SFHgb) on the coronary circulation remain unclear. An intact canine model utilizing intracoronary adenosine to abolish the confounding effect of autoregulation was used to study maximal myocardial oxygen delivery during progressive hemodilution with polymerized bovine SFHgb. The circumflex coronary artery was instrumented with a flow probe, hydraulic constrictor, and proximal and distal catheters for adenosine infusion and distal pressure measurement, respectively. This preparation was used to generate diastolic coronary pressure-flow relations during maximal vasodilation. Maximal coronary conductance and maximal myocardial oxygen delivery were determined in two groups of 7 dogs each following hemodilution, first with 6% hetastarch (Control), followed by further hemodilution with ultra-pure, polymerized, bovine SFHgb. After hemodilution with SFHgb, maximal coronary flow increased slightly without evidence of coronary vasoconstriction. Since hemodilution with this material increases oxygen carrying capacity, maximal oxygen delivery is greater than Control, despite the very low canine hematocrit. These findings suggest: 1) SFHgb can provide adequate oxygen delivery to the myocardium despite extreme degrees of hemodilution, and 2) in this intact model, there is no evidence of adverse coronary vasomotion.


Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) on the endothelium-dependent relaxation in rat mesenteric arterioles. Effects of exchange-transfusion with a modified hemoglobin (pyridoxalated hemoglobin polyoxyethylene conjugate; PHP) on diameter of rat mesenteric arterioles responding to various vasoactive substances were examined using the image-splitter television microscopy method. The mesentery of the anesthetized rat was spread over the stage of a microscope and the microvasculature was visualized by transillumination. Exchange-transfusion with a 6%PHP solution (30 ml/kg) was performed and effects of topical application of norepinephrine (NE) and acetylcholine(ACh) on diameter of mesenteric arterioles were examined. By exchange-transfusion with PHP, NE induced a dose-dependent decrease in diameter, while ACh induced a dose-dependent increase in diameter of the arterioles preconstricted by NE. There was no difference in vascular responsiveness to ACh and NE before and after exchange-transfusion with PHP solution. Results suggest that circulating PHP moiety does not exert any action on function of endothelial cell to release endothelium-derived relaxing factor (EDRF) in rat mesenteric microvasculature in situ.


Effects of diasprin cross-linked hemoglobin (DCLHb) on cardiac function and ECG in the swine. The effects of DCLHb on cardiac function were measured in diazepam sedated (2-4 mg/min) swine. Mean arterial blood pressure (MAP), peak systolic left ventricular pressure (IVP), rate of left ventricular pressure development (dP/dt), pressure rate product (PRP), cardiac output (CO), and ECG were monitored continuously. Hemodynamic parameters were compared during control, DCLHb infusion (40 ml/min), one minute balloon occlusion (BO) of the proximal left anterior descending coronary artery (LAD) with a 2.5-3.5 F, 20 mm balloon angioplasty catheter, and four minute balloon occlusion with DCLHb (BO + DCLHb) perfusion (40 ml/min) of the LAD occluded region. Control hemodynamic values were as follows; MAP 91 +/- 6 mmHg, IVP 102 +/- 6 mmHg, dP/dt 2519 +/- 351 mmHg/min, PRP 15809 +/- 1515 mmHg.min, and CO 2.72 +/- 0.29 L/min. There was a significant reduction in cardiac function with BO as compared to control; MAP 16% decreases, IVP 14% decreases, dP/dt 34% decreases, and PRP 40% decreases. In contrast BO + DCLHb significantly increased all measurements of cardiac function, with the exception of CO as compared to control; MAP 32% increases, IVP 29% increases, dP/dt 20% increases, and PRP 19% increases. The S-T segment of the ECG was depressed by a significant 0.134 +/- 0.006 mv from control during BO. There was a significant decrease 0.093 +/- 0.045 mv in the BO + DCLHb group. These data demonstrate an increase in cardiac function and a decrease in S-T segment depression during balloon occlusion with DCLHb perfusion. Further studies are needed to define the mechanism of action of DCLHb mediated increases in cardiac function.


Lack of increased cardiac output during hemoglobin hemodilution can be reversed with sodium nitroprusside. To investigate a possible connection between EDRF or nitric oxide (NO) and the unchanged cardiac output (CO) during hemoglobin-hemodilution we infused nitroprusside (NP) in eight Hb-diluted dogs (Hct approximately 20%). Normal hypotensive doses of NP were not effective and supranormal doses (133.0 micrograms/kg/min) were needed to induce even a modest decrease in mean AoP (approximately 25 mmHg). With these NP doses, cardiac output increased 177%, diastolic AoP (afterload) decreased 30%, while systolic AoP and LVEDP (preload) were unchanged. Heart rate, LV contractility (pressure-volume function) and blood volume were not changed throughout the study. Normally, NP alone decreases both preload and afterload resulting in unchanged CO. In the Hb + NP dogs, CO increased because only afterload decreased suggesting a selective effect of Hb on venous and arterial smooth muscle relaxation. In hemodilution with nonhemoglobin colloids, CO increases primarily because the diluted blood offers less viscous resistance to ventricular ejection. It appears that in order for cardiac output to increases in the presence of Hb, some decrease in arteriolar resistance is needed, presumably to unmask the effects of reduced viscosity. These results suggest the unchanged CO during Hb-dilution is related to a selective effect of Hb on venous and arteriolar nitric oxide (EDRF) function.


Effects of modified hemoglobin solutions on the isolated rabbit heart. The effects of modified hemoglobin (Hb) solutions on the coronary vasculature were studied. Hearts were perfused according to Langendorff with constant flow of Tyrode solution. The solutions studied were stroma- free Hb, prepared by lysis of red blood cells in water (SFHb-lys), or prepared by swelling of red blood cells in hypotonic phosphate buffer (SFHb). The increase in coronary vascular resistance at a dose of 200 mg Hb/dl was 68% for SFHb-lys and 13% for SFHb, respectively. Addition of the modified Hb solutions HbNFPLP and polyHbNFPLP produced an increase in coronary resistance of 11% and 8%, respectively. The left ventricular developed pressure (LVDP) (control value 72 +/- 12 mm Hg) increased by 18 and 12 mm Hg, respectively, for a dose of 250 mg Hb/dl. When HbNFPLP was converted to its met-Hb form the increase in LVDP was reduced to 3 mmHg and the increase in perfusion pressure to 6 mm Hg. We conclude that elimination of stromal contamination from Hb solutions can diminish vasoconstrictor effects. The increase in cardiac pressure development and in coronary vascular resistance found for dilute modified Hb solutions is partly due to an improved oxygen transport to the heart.


Effect of stabilized hemoglobin as a component of cardioplegia on warm ischemic heart. Stabilized hemoglobin was utilized as a component of cardioplegia in isolated rat heart. While oxygen consumption and heart function was better preserved in long term after reperfusion, damage from reperfusion injury was suspected and reversed in part by diltiazem.


Heart protection by cardioplegic solutions containing oxyhemoglobin pretreated by carbontetrachloride and freeze-drying with sucrose. A technologically improved variant of native stroma-free oxyhemoglobin (SFH) pretreated by carbontetrachloride and freeze-drying with 240 mM sucrose were reconstituted in a properly diluted ionic solution to reach the final concentration of 66 g oxyhemoglobin/L, osmolality 280-320 m0sm and pH 7.4. Cardioplegia of isolated rat heart was induced and maintained by this solution without recirculation for 3 h at 20 degrees C prior to heterotopic allo-transplantation of the graft. Evaluation of the survival and performance of each graft after 24 h and extent of tissue necroses indicated that the given standardly produced SFH variant ensured reproducible heart preservation from ischemic and reperfusion injury similarly as did the renown crystalloid cardioplegic solution CUSTODIOL.


Effects of pyridoxalated hemoglobin polyoxyethylene conjugate(PHP) and stroma free hemoglobin(SFH) on pulmonary vascular responsiveness to various vasoactive substances in isolated perfused rat lungs. Pulmonary vascular responsiveness to norepinephrine(NE), angiotensin-II (ANG-II), acetylcholine(ACh) and nitroglycerin (NG) was examined in isolated perfused rat lungs. The lungs were perfused with pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a newly introduced modified hemoglobin, stroma free hemoglobin (SFH) or hydroxyethyl starch (HES) at a constant flow rate. Perfusion pressure (PP) and intra-tracheal pressure (ITP) were measured. Effects of intrapulmonary arterial injection of NE, ANG-II, ACh and NG on PP were examined. NE and ANG-II produced a dose dependent increase in PP in all groups. The response to NE in PHP or SFH perfused group was significantly greater than that in HES-perfused group. ACh induced a dose dependent decrease in PP in HES and PHP groups, while in the SFH group, ACh produced a dose dependent increase in PP. NG produced a dose dependent decrease in all groups. Results showed that stroma free hemoglobin (SFH) inhibits relaxation induced by endothelium-derived relaxation factor (EDRF), but PHP dose not exert inhibitory action on vascular relaxation induced by EDRF.


Systemic hemodynamic and renal effects of unmodified human SFHS in dogs. Isovolumic exchange transfusion (25% of total estimated blood volume) was carried out in the anesthetized dogs using 9 g/dl of unmodified human stroma-free hemoglobin solution (SFHS). The objective was to determine the systemic hemodynamic, blood distribution and renal effects of SFHS over a 2-3 hour period post-exchange. At 30 minutes after the exchange, blood pressure rose from 114 +/- 117 to 133 +/- 22 mmHg, but this rise was not sustained thereafter. Mean pulmonary arterial blood pressure rose from 8 +/- 3 to 13 +/- 2 mmHg, and remained above the pre-exchange level up to 3 hours post-exchange. Cardiac output remained within normal limits. Significant flow-increments were seen at 30 minutes in heart, brain, liver, gut, and kidney, but these were also not sustained. A fall in glomerular filtration rate (GFR) occurred after the exchange and remained below the pre-exchange level. A reduction in urine flow at 150 minutes post-exchange was observed and was accompanied by a reduction in urinary electrolyte excretion. The findings suggest that the initial effects of the administration of unmodified stroma-free hemoglobin solution are those of peripheral vasoconstriction which does not appear to significantly restrict flow to the vital organs, such as heart and brain. Unmodified hemoglobin was found to cause a decrease in renal function.


Effects of pyridoxalated hemoglobin polyoxyethylene conjugate(PHP) on renal circulation in isolated perfused rat kidneys. We examined effects of perfusion of the kidney with pyridoxalated hemoglobin polyoxyethylene conjugate(PHP) solution on rat renal vascular responsiveness to norepinephrine(NE), angiotensin-II (AN-II), acethylcholine(ACh) and nitroglycerine(NG). The rat kidney was perfused with hydroxyethylstarch(HES) or PHP at a constant flow rate, using a pump. Perfusion pressure was monitored by a pressure transducer. Changes in PP induced by NE, AN-II, ACh and NG was examined. NE and AN-II applied intra-arterially induced a dose-related increase in PP in rat kidney perfused with both HES and PHP perfused groups. ACh and NG produced a dose dependent decrease in PP in both HES and PHP-perfused groups. There was no significant difference in response to ACh and NG between both groups. Results suggests that in rat vascular beds, PHP dose not interfere vascular relaxation caused by release of EDRF induced by ACh.


Artificial blood and extracorporeal circulation (ECC). To investigate a possible clinical use in ECC of artificial blood (AB) by means of a stroma free, polymerized and pyridoxylated hemoglobin a feasibility study was performed. Group I (5 calves) served as control. Group II (5 calves) received 2000 ml of a mixture of one part AB and one part Ringer's solution, group III (5 calves) 2000 ml of AB as priming solution. Blood samples were taken preoperatively, at 6 h, 24 h and then weekly up to 1 month. All animals survived and could be weaned from the ventilator immediately after the operation. Total hemoglobin was 10.6 before and 9.5 g/dl 12 h after the operation in mean in the group I and 11.5 and 11.9 for group II and III. Aortic mean pressure was increased in group II/III from preoperatively 105/95 mmHg to 124/135 mmHg after the end of cardio pulmonary bypass (CPB). The pulmonary vascular resistance increased in group II/III from 92/52 to 214/264 dyn*s*cm-5 in mean. This could not be observed in group I. Pulmonary resistance correlated with the concentration of free plasma hemoglobin.


The role of the kidneys in the excretion of chemically modified hemoglobins. Stroma-free hemoglobin (SFHb) can be chemically modified to prolong the intravascular retention (prevent renal filtration), and to improve oxygen delivering capability for use as a red cell substitute. Hb derivatives radioactively tagged with tritium [3H] or 14C were used to study their metabolic fate following clearance from the circulation. Fully conscious, chronically cannulated rats were treated by exchange transfusion (ET). Hb solutions tested were: PLPHb (Hb monovalently reacted with pyridoxal 5'-phosphate); HbXL (Hb crosslinked beta-beta with 2-nor-2-formylpyridoxal 5'-phosphate, or with bis-pyridoxal tetraphosphate); alpha alpha Hb (Hb cross-linked between the alpha-chains using bis-3,5-dibromosalicyl fumarate); and polyHb (polymerized with glutaraldehyde or o-raffinose). Plasma retention (T1/2) was significantly affected by dose and the degree of cross-linking. Urine flow rates all increased transiently above normal. In rats treated with any 64 kDa interdimerically cross-linked Hb, mild hemoglobinuria was evident and kidney tissue had the highest label concentration at all time points (1, 5, 10, 24, 48 hr, 7 d, and 14 days post-ET). For polymerized Hb derivatives, the amount of radioactivity in urine and kidneys was inversely related to the MW of the polyHb molecules. In all rats, regardless of the Hb derivative tested, the majority of radioactivity (dpm's) was excreted in urine. About 75% of all renal excretion of radioactivity occurred from 12-60 hours post-ET. This provided evidence that catabolism of cross-linked Hb's began early, and that the kidneys are primarily responsible for excreting smaller degradation fragments.


Effect of polymerization on clearance and degradation of free hemoglobin. In the present study we investigated the mechanism of prolongation of the plasma retention of free hemoglobin by polymerization. Polymerization of intramolecularly crosslinked hemoglobin with glutaraldehyde yields a mixture of large polymers, small polymers and monomers. In exchange transfusion experiments in rats we analyzed plasma samples by gel filtration to determine the clearance of polymers of different size. A positive correlation was found between polymer size and vascular retention. Furthermore, the clearance of large polymers appeared to be highly dose-dependent: after 20% and 70% exchange transfusions, we observed for large polymers a plasma half-life of 12 and 26 hours, respectively, whereas the half-life for 64 kD monomers was 4 hours in both cases. The degradation of hemoglobin was followed by measuring the bilirubin excretion. The infused heme was recovered as bilirubin within 72 hours. The delay between the disappearance of free hemoglobin from the plasma and the recovery as bilirubin was about six hours and was not affected by polymerization or dose. We conclude that polymerization prevents the operation of certain clearance mechanisms, while still allowing a route of clearance that is easily saturated. The intracellular degradation of heme into bilirubin is not affected by the modifications of hemoglobin and is not easily saturated.


Intravascular circulation and distribution of human 51Cr-DBBF stroma-free hemoglobin. Male B6C3HF1 mice were infused with 51Cr-labeled DBBF (bis 3,5-dibromosalicyl fumarate) crosslinked stroma-free hemoglobin (SFH). The intravascular halftime (T50) of the DBBF-SFH, determined from plasma hemoglobin levels, was 0.5 hours in the first 10 minutes and 4.3 hours during the next 50 minutes. At 24 hours, less than 5% of the DBBF-SFH remained. Elution of 51Cr was reflected in a lower T50 determined from the radioactivity levels: during the first 10 minutes the T50 was 0.3 hours; in the next 50 minutes it was 1 hour. The radioactivity sequestered in each organ in the first hour following DBBF-SFH infusion was as follows: 11.2% of the infused radioactivity was in the skin, 11.4% in muscle, 9.1% in the skeleton, and 5% in the liver. After 24 hours, the percentages in skin, muscle, skeleton and liver were 15.4, 10.3, 16.6 and 6.7% respectively. The percentage of infused radioactivity in the gastrointestinal tract and kidney at 1 hour and 24 hours ranged from 3.5 to 5.5%. Less than 0.4% was found in the spleen and lung. At 24 hours, 25% of the radioactivity was recovered in urine and 3% in feces.


Biodistribution studies of liposome encapsulated hemoglobin (LEH) studied with a newly developed 99m-technetium liposome label. A new method has been developed to label preformed liposomes with 99m-Technetium (99mTc) using hexamethylpropylenamine oxime (HMPAO). 99mTc is an ideal isotope for performing non-invasive dynamic biodistribution studies. This labeling method results in a high labeling efficiency (greater than 95%) and is stable as determined by both in vitro and in vivo studies. In vitro studies indicated that glutathione encapsulated in the LEH is important in the labeling process with 99mTc-HMPAO. In vivo studies with LEH were performed on 7 rabbits with dynamic scintigraphic 1 minute images performed from 1-120 minutes. Delayed images were performed at 20 hours followed by sacrifice and organ counting. Dynamic images reveal a gradual deposition of the LEH in the liver and spleen. Twenty hour biodistributions revealed 50% of the LEH remaining in the blood, 15% in the liver, 14% in the spleen, 3% in lungs, 3% in muscle with trace amounts in the brain, kidneys, and heart. Doses per gram were highest in the spleen with 12.5% of the injected dose per gram of spleen vs. 0.2% per gram of liver. This labeling technique is an effective method for non-invasively monitoring dynamic changes in liposome biodistribution and can be used to study the effects of various liposome modifications on biodistribution.


Effects of liposome encapsulated hemoglobin on the reticuloendothelial system. The effects of different doses (4, 10, and 25%) of liposome encapsulated hemoglobin (LEH) were measured on the Reticuloendothelial System (RES) and Kupffer cells (KC) by i) colloidal carbon clearance in the rat in vivo and in the isolated perfused liver, ii) magnetometry, and iii) histological analysis. At the highest dose, in vivo carbon clearance rates (k) were half the rate as in controls at 2 and 12 hours post-treatment. By 24 hours post-treatment clearance rates were at control levels. Empty liposomes (LIP) caused a 2-fold decrease in k at 2 hours only. With both LEH and LIP, the effects were less severe when rats were given the lower doses. Magnetometric studies showed a decrease in KC phagosomal motion in the LEH-treated (25%) rats at 2 and 24 hours that returned to control levels at 2 weeks. Perfused livers from rats treated with a low dose of LEH cleared carbon at the same rate as LIP and Krebs Ringer Bicarbonate (KRB) controls. Histological examination showed minimal tissue damage in all test groups. Thus, LEH and LIP have some short-term deleterious effects on KC and the RES probably due to RES blockade rather than cellular damage.


A double (exchange transfusion-carbon clearance) model for testing post-resuscitation reticuloendothelial function. A double exchange transfusion-double carbon clearance method was evaluated for assessing reticuloendothelial (RE) function following exchange transfusion with hemoglobin solutions. Fifty percent of estimated blood volume (3% body weight) was withdrawn from anesthetized Sprague-Dawley rats and isovolumically replaced with shed blood (SB, control), lysed shed blood (LB, pos. control), human stroma-free hemoglobin solution (SFH), or polyhemoglobin solution (PHS). Thirty minutes after the exchange transfusion, colloidal carbon was injected intravenously and its vascular clearance followed for 1 hour. Then, the 50% exchange transfusion was repeated and the second carbon clearance measured. The intravascular carbon clearance constants, K, and clearance half-times, T1/2, were calculated and compared. No apparent differences in RE function were seen among the groups after the initial exchange transfusion. However, following the second exchange transfusion significant (P less than 0.05) slowing of carbon clearance was observed in lysed blood treated animals. The RE function of SFH or PHS treated animals were not different (P less than 0.05) from that of SB animals. A double exchange transfusion-double carbon clearance method seems to reveal changes in RE function that are not apparent after a single exchange transfusion and clearance test.


Clearance of differentially labelled infused hemoglobin and polymerized hemoglobin from dog plasma and accumulation in urine and selected tissues. Pyridoxalated hemoglobin polymerized with glutaraldehyde has been proposed as a hemoglobin based blood substitute. The preparations contain significant amounts of unpolymerized hemoglobin. We have prepared polymerized pyridoxalated hemoglobin labelled with 14C by reductive methylation free of unpolymerized hemoglobin and pyridoxalated hemoglobin labelled with 3H by reductive methylation to compare the handling of the two forms after infusion into dogs. Four dogs were examined sequentially. After three hours, 52.4 +/- 8.9% of the 3H label had disappeared from plasma whereas 21.7 +/- 5.8 of the 14C label had disappeared. The decrease of both labels occurred in a very close to linear fashion over the time period examined. From radioactivity in collected urine, it was calculated that 30.7 +/- 6.3% of the 3H and 9.0 +/- 2.7 of the 14C that had been cleared from plasma appeared in urine. The ratio of the specific radioactivity in tissue to the specific radioactivity of plasma indicated that extravascular accumulation of 3H label from unpolymerized hemoglobin occurred in kidney, heart and liver, with the kidney cortex exhibiting a very high concentration of the label. The specific radioactivity of both 3H and 14C label in liver suggested the substantial involvement of the reticuloendothelial system in the removal of both unpolymerized and polymerized hemoglobin from the circulation.


Hepatic reticuloendothelial function following resuscitation with hemoglobin solutions. Red cell substitutes could lead to depressed reticuloendothelial (RE) particulate clearance function. This hypothesis was tested using an animal model of hypovolemia-resuscitation. Anesthetized male Sprague-Dawley rats were subjected to 50% blood volume hemorrhage followed by isovolumic replacement with stroma-free hemoglobin (SFH, 7 gHb/dl), polyhemoglobin (PHS, 14 gHb/dl), or shed blood (SB). At 30 min post-transfusion, the liver was isolated and perfused with colloidal carbon. Hepatic RE function was assessed from the carbon clearance kinetics. In separate experiments, the hepatic Kupffer cells were isolated and cultured from rats that were previously hemorrhaged and transfused with normal saline solution. The cultured Kupffer cells were incubated with SFH or bovine albumin (ALB) and their phagocytic function assessed in-vitro. The hepatic carbon clearance following exchange transfusion with hemoglobin solutions was not significantly altered as compared to shed blood controls (P greater than 0.05). Similarly, phagocytic function of hemoglobin treated Kupffer cells was not significantly different (P greater than 0.05) from that of ALB treated cells.


Oxygen delivery from fluorocarbon emulsions--aspects of convective and diffusive transport. Perfluorocarbons (PFCs) deliver oxygen due to their physical characteristics and the convective delivery to tissues; this has been amply demonstrated in numerous animal models and in clinical trials. PFC emulsions have also been used for organ perfusion and augmentation of oxygenation to cell cultures. By reason of their small particle size, PFC emulsions penetrate collateral capillaries of an ischemic microcirculation, both supplying oxygenation and possibly restoring flexibility of acidotically stiffened erythrocytes by reinstituting aerobic metabolism. There is also evidence that PFCs can augment tissue oxygenation by increasing oxygen solubility in the plasma phase of blood; this improves diffusion gradients between plasma and tissues even at relatively low doses of PFC. This hypothesis is supported by the ability of PFCs to increase oxygen tensions in tissues such as blood, brain, liver, kidney, pancreas, skeletal muscle, retina etc. PFCs can also on occasions increase critical oxygen extraction coefficients and should therefore be effective in supporting oxidative metabolism in states of extreme cardio-respiratory insufficiency. This theory represents a new concept in oxygen delivery and may define a new class of therapeutic agents that can improve tissue oxygen supply in various pathological states involving low oxygen delivery and ischemia.


On the perfluorocarbon emulsions of second generation. Perfluorocarbon-emulsions of second generation were prepared by means of new perfluorocarbons (F-dimorpholines, F-dipiperidines and F-cyclohexylmorpholine), acting both as oxygen carriers and as interfacial active compounds (IFACs). The stabilizing effect of these IFACs is interpreted and a new theory is introduced. Also new classes of fluorosurfactants were synthesized and tested for biocompatibility. In PFC mixtures compounds of the type RFRH (RF = CmF2m + 1, RH = CnH2n + 1) are acting as IFACs but also as anchor-groups for lipophilic surfactants.


Stability and stabilization of fluorocarbon emulsions destined for injection. The factors that have an impact on the stability, and the mechanisms of degradation of fluorocarbon emulsions suited for intravascular use, are briefly reviewed. Various ways of prolonging shelf stability are discussed. The effectiveness of perfluoroalkylated surfactants and/or co-surfactants as stabilizers is demonstrated. New means of stabilizing fluorocarbon-in-water emulsions using molecular dowels are presented.


Phospholipid vesiculated fluorocarbons--promising trend in blood substitutes. Dispersions of fluorocarbons (PFC's) made with phospholipids have unusual properties, e.g. very high stability, due to the formation of thermodynamically stable vesicles. They can be used for high PFC concentrations as well as e.g. for ESR imaging.


Perfluoroalklylated phospholipids as surfactants and co-surfactants forinjectable fluorocarbon emulsions. Highly fluorinated phospholipids were investigated as sole surfactant, and as co-surfactant with egg yolk phospholipids (EYP), in the formulation of 50% and 100% w/v perfluorodecalin emulsions. The surfactant's capability to stabilize such emulsions improves with the length of the perfluoroalklylated tail and with the increase of its relative weight in the hydrophobic chain. As sole surfactant, 2, which has the longest fluorinated tail has the highest efficacy. As co-surfactant with EYP, a strong stabilizing effect is found when the total hydrophobic chain length is adjusted to the EYP membrane's thickness, which is the case of 1. Dispersions of the F-phospholipids do not modify cell growth and viability and show no hemolytic activity on human red blood cells at concentrations in the 60-100g/L range. Acute toxicity tests in mice indicate - i.v. DL50 greater than 2.75 g/Kg body wt.


Development of highly fluid, concentrated and stable fluorocarbon emulsions for diagnosis and therapy. A challenging aim in developing injectable fluorocarbon emulsions is to combine good flow characteristics (especially at low shear rates) with the high fluorocarbon concentration required for high oxygen delivery or effective contrast in imaging, long shelf life, and biological acceptability. A good balance of these sometimes conflicting objectives has been achieved with 90% w/v concentrated emulsions of various fluorocarbons, including the radiopaque oxygen carrier perfluorooctylbromide (PFOB, perflubron). The sterile emulsions have viscosities of about 20 cPs at a shear rate of 1 sec-1; the viscosity decreases rapidly with fluorocarbon concentration, and at 60% w/v the viscosity is less than that of human blood. The emulsions are suitable for injection as prepared, and are stable unfrozen for over a year.


Telomeric THAM-derived perfluoroalkylated surfactants for fluorocarbon emulsions. New fluorophilic and hydrophilic, cost efficient telomeric surfactants derived from tris(hydroxymethyl)acrylaminomethane were synthesized in 2 steps in 80% yield with respect to the perfluoroalkylated telogen. They demonstrate better ability to emulsify fluorocarbons than Pluronic f-68. The biological tolerance of these new surfactants is remarkable, the perfluorohexyl derivative was tolerated at doses of 4g/kg bw after i.v. injection in mice. None of the perfluoroalkylated THAM derivatives induces hemolysis of human red blood cells at concentrations up to 200g/l in physiological solutions.


Stabilization of perflubron emulsions with egg yolk phospholipid. Egg Yolk Phospholipid(EYP) has been used extensively as the primary surfactant in parenteral fat emulsions for many years. The simplicity, functionality and physiologic tolerance of EYP has contributed greatly to its success in the intravenous emulsion arena. The mechanism of stabilization in triglyceride emulsions is well understood; however, this is not the case with perfluorocarbon emulsions. Interfacial models, as well as emulsion stability studies, have been conducted utilizing EYP of varied composition in order to derive a structure/function relationship. Our studies indicate that minor components, total unsaturation, acyl chain length and presence of charged species have significant impact on the functional properties of EYP and the subsequent stability of the emulsion product. These findings contribute to our ability to design and manipulate natural surfactants with superior properties for use in medical applications of perfluorocarbon emulsions.


Application of computer-based experimental design to optimization of processing conditions for perfluorocarbon emulsion. Response surface methodology was applied during process optimization of a parenteral emulsion formulation containing perfluorocarbon (PFC). E-Chip, a computer software program, was used to design and analyze the optimization experiments. The levels of eight process variables of interest were varied: pre-emulsification temperature and mixing time, mixer speed, generator type, rate of PFC addition, homogenization temperature, pressure and number of passes. Dependent variables evaluated included emulsion median diameter, viscosity and percent unemulsified PFC. A predictive model describing the relationship between the dependent and process variables was developed. Median diameter of the emulsion was affected significantly by homogenization temperature and pressure, and marginally by the rate of addition of the PFC. Viscosity was not influenced by any of the process variables. Percent unemulsified PFC was slightly influenced by the number of homogenization passes. The model was then used to set optimum process conditions.


Particle size distribution of concentrated perfluorocarbon emulsions by sedimentation field flow fractionation. Alliance Pharmaceutical Corp's concentrated perflubron (perfluorooctylbromide; PFOB) emulsions are being developed for several medical applications. Sedimentation Field-Flow Fractionation (SdFFF) can provide accurate particle size distribution data when properly calibrated for those emulsions. Since no suitable particle standards are available with the proper density (approximately 1.9 g/cc) and size range (diameter = 0.1-1.0 microns), calibration of the mass distribution was done by comparing the SdFFF detector signal with the actual mass of perflubron extracted from fractions eluted at successive time points and measured by GC. The calibrated mass distribution can then be used to calibrate other instruments such as the photosedimentation instruments used for routine quality assurance measurements.


Drop size stability assessment of fluorocarbon emulsions. The aging of fluorocarbon emulsions prepared with natural egg yolk phospholipids (EYP) has been studied and a linear variation (r2 greater than 0.95) of the mean average volume of the droplets with time has been observed. The slope of the experimental lines, called "Stability Parameter, S" can thus be taken as a representation of the rate of aging of the emulsions. Examples are given of use of parameter S to assess the effect of formulation and processing parameters on the stability of diverse fluorocarbon emulsions. S is a useful tool to compare emulsions and ascertain any factors of stabilization/destabilization.


Use of perfluorochemical emulsions in cancer therapy. Over the past 10 years, the use of perfluorochemical emulsions (PFCE) and carbogen or oxygen breathing has been explored as an adjuvant to radiation therapy and/or chemotherapy in the treatment of solid tumors. The rationale for the use of PFCE and oxygen breathing in this therapeutic setting is that solid tumor masses contain areas of hypoxia which are therapeutically resistant. Since x-rays and many chemotherapeutic agents require oxygen to be maximally cytotoxic and most normal tissues are well-oxygenated, the additional oxygen put in circulation by the PFCE should not increase the normal tissue toxicities produced by the various therapies. The largest body of preclinical work and all of the clinical studies in cancer conducted with PFCE, thus far, have been done with Fluosol-DA, 20%. Oxygen microelectrode studies have confirmed increased oxygenation in previously hypoxic tumor regions after the administration of Fluosol-DA and carbogen breathing. The preclinical studies have shown very positive effects with single dose and fractionated radiation in several rodent solid tumor models. Many widely used anticancer drugs including antitumor alkylating agents and adriamycin are enhanced by PFCE and carbogen breathing for longer time periods (6 h). More recently, several experimental concentrated PFCE preparations have become available and work with these is actively under way in several laboratories. Clinical studies with radiation and four or five chemotherapeutic drugs as single agents have indicated that Fluosol-DA followed by oxygen breathing can be administered safely in a variety of cancer therapeutic settings. Further clinical studies with Fluosol-DA are planned.


Preclinical evaluation of Oxygent as an adjunct to radiotherapy. These studies examine the potential value of a concentrated emulsion of perfluorooctylbromide (perflubron; Oxygent, Alliance Pharmaceutical Corp.) as an adjunct to radiotherapy. The effects of Oxygent on solid tumors were examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus O2, carbogen (95% O2/5% CO2), or hyperbaric oxygen (HBO) increased the effects of radiation on the tumors. Analyses of tumor cell survival curves and measurements of intratumor pO2 showed that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Neither treatment of the animals with carbogen, O2, or HBO alone nor treatment of air-breathing rodents with Oxygent produced changes of similar magnitude. Treatment with a vehicle emulsion containing all the components of Oxygent except the perflubron did not alter tumor radiosensitivity, showing that tumor radiosensitization required the oxygen-transporting perfluorocarbon, and did not result from any biologic or physiologic effects of other components of the emulsion. These studies also examined the effects of Oxygent on the radiation responses of mouse skin and bone marrow. Oxygent selectively increased the radiation sensitivity of tumors relative to these normal tissues, thereby increasing the therapeutic ratio and producing therapeutic gain. Oxygent appears to warrant further testing as an adjunct to cancer therapy.


Enhancement by perflusion emulsion (Oxygent) and carbogen breathing of the tumor growth delay of the FSaIIC fibrosarcoma after treatment with antitumor alkylating agents. Many anticancer drugs require oxygen to be cytotoxic, or are selectively cytotoxic toward cells under oxygenated conditions. The effects of the dilute perfluorochemical emulsion Fluosol-DA with a wide variety of chemotherapeutic agents have been explored; however, it has not been possible to determine the optimal level of circulating perfluorochemical emulsion with anticancer drugs because the volume of Fluosol that may be administered in limited. Using a concentrated 90% Perflubron emulsion, Oxygent, a wide range of perfluorochemical emulsion doses have been examined in combination with melphalan, cyclophosphamide and BCNU in a murine solid tumor model. When Oxygent was administered by injection i.v. just prior to the injection of melphalan (10 mg/kg), the greatest tumor growth delays were obtained with Oxygent levels between 4 and 12 g PFC/kg. With each of these drugs the greatest tumor growth delays were obtained when the drug was prepared in the emulsion and the combination injected i.v. In each case, each dose of drug was followed by 6 h. of breathing carbogen.


Enhancement of fractionated radiation therapy by an experimental concentrated perflubron emulsion (Oxygent) in the Lewis lung carcinoma. The concentrated Perflubron emulsion, Oxygent, in combination with breathing a 95% oxygen atmosphere, significantly enhanced the response of the murine Lewis lung carcinoma to fractionated radiation therapy. Administration of 4 to 12 g PFC/kg was most effective if administered prior to each x-ray fraction, however, alternated day and even once weekly administration of 8 or 12 PFC/kg and 95% oxygen breathing with each x-ray fraction resulted in significant improvements in tumor growth delay.


Effect of emulsion concentration on biodistribution of perflubron in tumor-bearing mice. Perflubron (perfluoroocytlbromide, PFOB) emulsion concentrations of 100%, 90%, or 60% w/v were administered to mice with and without 3 types of murine malignant tumor implants, and the distribution in blood, tumor, lung, liver and spleen were studied 48 hours after a dose of 10 or 3 g/Kg of PFOB. The most important changes were seen in the blood where the PFOB concentration [PFOB] was decreased in tumor bearing mice (TBM). Blood [PFOB] was also decreased in TBM and normal mice (NM) that received the 60% emulsion. Liver [PFOB] was increased in TBM. Lung [PFOB] was directly proportional to the emulsion concentration with the 10g/Kg dose. No major differences were seen in the biodistribution between the 100% and 90% emulsions using 10g/Kg, in spite of differences in composition and manufacturing history.


Cyclooxygenase inhibition with ibuprofen: effect on biodistribution of perflubron emulsions in mice with mammary tumor. The biodistribution of Perflubron (perfluorocytylbromide, PFOB) was studied by infusing 10 or 3 g/Kg doses of 90% and 100% w/v emulsions into Balb C mice with and without malignant mammary tumor implants. PFOB concentrations were measured in blood, liver, spleen, lung and tumor, and X-rays were taken 48 hours after infusion. Ibuprofen (IBU) was given intraperitoneally 10 mg/Kg dialy for 3 days to study the effects of cyclooxygenase blockade. The results showed that the blood [PFOB] in tumor-bearing mice (TBM) was only 3.6% of that measured in normal mice (NM), and the liver [PFOB] was increased by 59%. The lung [PFOB] increase with IBU therapy in NM was highly significant. IBU therapy resulted in a 98% and 468% increase in blood [PFOB] in normal and TBM, respectively. Smaller but statistically significant changes occurred in blood [PFOB] with saline and sham injections. Tumor [PFOB] was lower by 17% (P = .028) in mice treated with IBU. Smaller decreases were seen with saline and sham injections but did not achieve statistical significance. In conclusion, the presence of this malignant tumor resulted in changes that might influence the usefulness of PFOB as a contrast agent and as an adjuvant for radiation and chemotherapy. This study shows that cyclooxygenase inhibition significantly modified the pharmacodynamics of PFOB emulsions in blood, tumor and lung. The changes seen in the saline placebo and sham category were attributed to stress from procedures.


The use of Imagent BP in diagnostic imaging research and 19F magnetic resonance for PO2 measurements. Imagent BP (90% w/v perflubron emulsion) is radiopaque and serves as an X-ray contrast medium. Quantitative X-ray Computed Tomography, provides the means to non-invasively estimate tissue perflubron concentration providing three unique capabilities: 1) The use of the same animal for biodistribution and elimination analysis; 2) The precise geographic distribution of the agent to more accurately quantitate localized accumulations; and 3) The ability to gather physiologic data by monitoring the time dependent distribution of perflubron. It is known that the T1(-1) of 19F of perfluorochemicals is linearly related to the dissolved oxygen which allows the quantitation of PO2 in-vivo. We showed using perflubron in phanta that not only was T1(-1) linearly related to PO2 but also T2(-1) and both were insensitive to perflubron concentration. Since flow interferes with signal, the first in-vivo experiments have focused on stationary perflubron located within phagocytes. T1(-1) measured from this environment suggested a PO2 of 15-25 Torr. T1(-1) increased by nearly 50% when the FIO2 was increased from 20 to 100% reflecting an increase in intracellular PO2 on the order of 25 Torr.


Quantitation of perfluorocarbon blood substitutes in tissues using F-19 magnetic resonance spectroscopy. F-19 Magnetic Resonance Spectroscopy (MRS) has been used to quantitate the biodistribution of perfluorocarbon (PFC) blood substitutes in porcine tissues following intraperitoneal administration of a 20% w/v perfluorotributylamine (FC-43) based emulsion. PFC tissue concentrations were determined for spleen, liver and lung tissues from juvenile pigs ranging in weight from 12 to 15 kg. Typical average values (mmoles FC-43/gram tissue) ranged from 0.23-0.39 for spleen; 0.09-0.13 for liver; and 0.09-0.12 for lung. A description of this spectroscopy based quantitation technique will be presented. F-19 MRS is a specific, rapid, and accurate method for measurement of PFC tissue concentrations.


Protective effects of a novel perfluorochemical emulsion in photodynamic therapy. The effects of pre-injection of mice with a novel perfluorodecalin-based emulsion on the responses to photodynamic therapy (PDT) using the photosensitizer, metatetra (hydroxyphenyl) porphyrin (m-THPP), have been studied. Injection of emulsion after m-THPP and before illumination (activating wavelength 648 nm) protected skin against PDT-induced inflammatory effects, as reflected by decreases (P less than 0.05) in vascular permeability and oedema formation. However, there was no protection against epidermal cell loss. In contrast, injection of emulsion before sensitizer had no corresponding effect. A fall in mean dermal temperature of up to 6 degrees C occurred in mice injected with emulsion 1-2.5 h before illumination suggesting a decrease in skin blood flow which would reduce oedema formation. Possible mechanism(s) for this apparent protective effect are discussed.


Oxygent: a novel probe of tissue oxygen tension. We have examined the 19F NMR spectrum and relaxation behavior of Oxygent (an emulsion of perfluorooctylbromide). Each of the resonances exhibits a linear relationship between spin-lattice relaxation rate and oxygen tension at constant temperature. Oxygent provides enhanced sensitivity to changes in oxygen tension compared with other emulsions used previously. We have used Oxygent to determine the oxygen tension in the liver of a mouse.


A comparison study of rat pancreas preservation using perfluorochemical and fluorocarbon-emulsion as preservation medium. We reported previously the successful 72-hour cold rat pancreas preservation by using Perfluorochemical (PFC). The present study is to determine whether Fluorocarbon (FC) emulsion is as effective as PFC for long-term rat pancreas preservation. Lewis rat pancreases were stored in FC emulsion (4 degrees C) saturated by continuous supply of oxygen:carbon dioxide (95%:5%) (Group I) or by 100% pure nitrogen (Group II), or in PFC (4 degrees C) saturated by continuous supply of oxygen:carbon dioxide (95%:5%) (Group III) or nitrogen (Group IV) for 24 h and 48 h. Heterotopic pancreas transplantation into isogeneic diabetic rats were performed following preservation. Functional graft success rates following 24 h and 48 h cold storage were 71% (5/7) and 0% (0/5) in Group I, 71% (5/7) and 0% (0/5) in Group II, 100% (5/5) and 80% (4/5) in Group III, and 80% (4/5) and 0% (0/5) in Group IV, respectively. These results showed that, as an artificial blood substitute, the PFC with simple oxygen bubbling for 48-hour preservation of rat pancreas was much effective than FC emulsion, but not effective when saturated with nitrogen. We concluded that the PFC with saturated oxygen can obtain long-term successful preservation of rat pancreas. The direct oxygenation of the graft tissues is thought to play an important role in organ preservation.