CA2087504A1

CA2087504A1 - Administration of low dose hemoglobin to increase perfusion

Info

Publication number: CA2087504A1
Application number: CA002087504A
Authority: CA
Inventors: Robert J. Przybelski
Original assignee: Robert J. Przybelski; Baxter International Inc.
Current assignee: Baxter International Inc
Priority date: 1992-01-30
Filing date: 1993-01-18
Publication date: 1993-07-31
Also published as: US6022850A; US6090779A; US5334706A; US5614490A; JPH05255108A; US6046161A; JP2992855B2; US6117838A; US5510464A; JPH10306036A

Abstract

ADMINISTRATION OF HEMOGLOBIN TO INCREASE PERFUSION

Abstract of the Invention This invention provides a method to therapeutically increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from the least amount effective to increase perfusion, up to a dose of about 2500 mg per kilogram of body weight.

Description

~7~

ADMINISTRATION OF LOW DOSE HEMOGLOBIN TO
INCR~ASE PERFUSION

Back~round of the Invention sThe present invention relates to perfusion and specifically to the therapeutic use of hemoglobin in low doses to increase perfilsion.
Perfusion is supplying an organ or tissue with oxygen and nutrients via blood or a suitable fluid. Perfiusion is essentially the ~low of fluid to tissues and organs through a~eAes and capillaIies. Flow may be expressed as the ratio of 0pressure to resistance. If adequate oxygen and nutrients are not reaching tissues and organs, therapies to improve perfilsion may be employed.
Current management of hypotension, and its concurrent reduction in perfusion of tissues and organs, consists of the admir~istration of (i) vasopressors, (ii) positive ins~tropic agents, and/or (iii~ vascular volume expanders ~epending on 15the underlying etiology. Hypotension secondary to actual or relative hypovolemia, which also reduces perfusion, Lnitially is managed by administration of crystalloid or colloid solutions and/or blood products.
Therapeutics which increase blood pressure are employed in an attempt to increase perfusion. Vasopressor agents such as epinephrine, phenylephrine, 20metaraminol and methoxamine cause contraction of the muscles of capillaries and arteries. This increases resistance to the flow of blood and elevates blood pressure.
However, these drugs are not optimal ~or increas~g perfusion. They pose a risk of inducing excessive blood pressure; are known to cause arrhythmias; require int~arterial pressure monitoring; and tissue sloughing and necrosis may result if 25extravasation occurs. Moreover vasopressor agents may actually decrease the flow of oxygen and nutrients to tissues and organs. If the constriction of the capillaries and arteries increases resistance in proportion to the increase in blood pressure, the net flow, i.e. perfusion, will be unchanged. However, large increases ~n resistance result in decreased flow. At best a locali~ed increase in perfilsion in large vessels 30occurs while the flow in capillaries is reduced. Indeed, vasoperssor agents have bPen reported to result in decreased perfusion of vital organs. Moreover, because vasopressors increase venous pressure as well as ar~erial pressure, and, therefore can limit optimal fluid administration, such agents generally are given only after sufficient volume replacement with an appropriate fluid or blood. W. C.

2087-j~4 Shoemaker, A. W. Pleming, "Resuscitation of the Trauma Patient; Restoration of Hemodynamic Functions IJsing Clinical Algorithrns," Ann. ~3merg. Med., 15:1437 (1986). Dopamine hydrochloride is an inotropic agent used in the treatment of shock to increase blood pressure. It suffers from the drawbacks noted above for s vasoperssor agents. In addition, it has a ~ery smaU therapeutic window. Because the dose/response is extremely sensitive, dopamine rnust be carefully titrated, and invasive monitoring (arterial line) is required.
A class of therapeutics known as plasma expanders or volume replacements may be used ~o increase perfusion where significant blood loss has occurred. In 0 this therapy perfusion is increased by administenng volume replacement fluids such as albumin, Ringer's lactate, saline, or dextran solutions. A decrease in blood volume causes a decrease in pressure. By restoring the volume, some pressure, and thus flow can be restored. In addition9 these solutions do not carry oxygen or nutrients. So, while flow may be restored, oxygen delivery to the tissues is 5 reduced because oxygen and nutrient content of the blood is diluted. Hemodilut;on is beneficial in that it reduces viscosity of the blood, thus reducing resist~nce and increasing flow. But without the necessary oxygen and nutrients, this therapy isnot an optimal treatment for signific~nt blood loss.
Volume replacement with whole blood is currently the most e~lcacious 20 treatment where there has been significant bloodl loss. However, this cannot be used in a pre-hospital setting and its use requires a twenty minute wait for matching and typing, assuming a donor blood supply is available. Studies have also shown that the increased viscosity associated with infusion of blood may l~nit capillary blood flow. K. M. Jan, J. Heldman, and S. Chien, "Coronary Hemodynarnics and 25 Oxygen Utilization after Hematocrit Variations in Hemorrhage," ~n. J. Physiol., 239:H326(1980). There is also the risk of viral (hepatitis and/or H~V) or bacterial infection from transfused blood.
Hemoglobin solutions have been under investigation as oxygen carrying plasma expanders or blood substitutes ~or more than f~ty years. While no 30 hemoglobin solution is currently approved for use clinically, they are intended to be used to replace blood lost through hemorrhage. Their effectiveness as oxygen carriers has been demonstrated. However, their potential toxicity has been the focus of much research.

2~7~

Even srnall amounts of stroma (cell membrane) in hemoglobin solutions appear to be toxic. R. W. Winslow, "Hemoglobin-based Red Cell Substitutes,"
The Johns ~Ioplcins University Press, Baltirnore (1992). Such toxic effects include renal vasoconstriction and decreased renal flow as well as hypeFtension and bradycardia. In 1967 Rabiner utilized rigorous purification techniques to develop stroma-free hemoglobin which has prevented some of thc toxic effects encounte.red with prior hemoglobin solutions.
In connection with toxicity studies for hemoglobin solutions researchers noted an elevation in blood pressure as early as 1934. W. R. Arnberson, J.
Flexner, F. R. Steggerad~ et al., On Use of Ringer-Locke Solutions C:ontaining Hemoglobin as a Substitute for Normal Blood in Mammals," J. Cell. Comp.
Physiol, 5:359 (1934). Current toxicity studies of hemoglobin solutions continue to note a pressor effect. For exarnple, the replacement of blood with various bovine hemoglobin solutions in rabbits in a 1988 study was characterized by significants hemodynamic instability, with fluctuations of blood pressure and heart rate, and severe tachypinea. Of the various hemoglobin solutions tested in this study, thepurest (which comprised cross-linked hemoglobin) showed the least toxicity, but nevertheless "did produce a hypertensive reac~ion suggestive of a systemic vasoconstrictor effect". M. Feola, J. Simoni, P. (:h Canizaro, R. Tran, G.
Raschbaum, and F. J. Behal, "Toxicity of Polyme~zed Hemoglobin Solutions,"
Surg. Gynecol Obstet, 166:211 (1988). In 1975 Rabiner reported on the work of a Russian researcher who noted a beneficial effect following administration of 200-400 ml of a 3 % hemoglobin solution heavily contaminated with stroma lipid to each of 30 ~Iauma patients, in that there was a stabili~ation of blood pressure. S. F.
2s Rabiner, "Hemoglobin Solution as a Plasma lFxpander, " Fed. Proc., 34:1454 (1975). In 1949 Amberson et al. reported that the administration of 2300 ml of a10% to 14% hemoglobin solution (225 grams hemoglobin) restored blood pressure to norrnal in a patient who had suf~ered significant blood loss through hemorrhage.
W. R. Arnberson, J. J. Jennings, and C. M[. Rhode, "Clinical Experience with Hemoglobin-Saline Solutions," J. appl. Physioi., 1:469 (1949).
Although methods of measurement and reporting have been inconsistent, an increase in blood pressure and a fall in heart rate are frequently reported findings associated with administration of a vanety of hemoglobin solutions in an~mals and ~7~0l~

man. Those resear~bers notmg the pressor effect of solutions include G. A. H.
Buttle, A. ~ekwick, and A. Schweitzer, "Blood Substitutes in Treatment of Acute Hernorrhage," I~ncet., 2:507 (1940). J. H. Miller and R. K. McDonald, "The Effect of Hemoglobin in Renal Function in the Human," J. Clin. Invest., 30:1033 s (1951). C. Elia, H. J. Sternberg, A. ~reenburg, and C;. W. Peskin, "Stroma-free Hemoglobin in the Resuscitation of H[emorrhagic Shock," Surg. ~7Orum, 25:201 (1974). G. S. Moss, R. DeWoskin, A. L. Rosen, H. I~vine, and C. K. Palani, "~ransport of Oxygen and Carbon Dioxide by Hemoglobin-saline Solution in the ~;Ced Cell Free Pr;mate," Surg. Gynecol Obstet., 142:3S7 (1976). J. P. Savitsky, J.
o Doczi, J. Black, and J. D. Arnold, "A Clinical Safety Trial of Stroma-free Hemoglobill," Clin. Phannacol Ther., 23:73 (1978). P. E. Keipert and T. M. S.
Chang, Pyridoxylated Polyhemoglobin as a Red Cell Substitute for Resuscitation of Le~hal Hemorrhagic Shock in Conscious Rats," Biomater. Med. Devices Artif.
Organs, 13:1 (1985). F. H. Jesch, W. Peters, J. Hobbhahn, M. Schoenberg, and 15 K. Messmer, "Oxygen-transporting Fluids and Oxygen Delivery with Hemodilution," Crit. Care Med., 10:270 (1982).
Two early anirnal studies in which hemoglobin solutions were administered following controlled hemorrhage and occlusion of the left coronaIy altery demonst~ated improved neyocardial blood flow compared to autologous blood or ~o dextr~m. G. P. Biro and D. Beresford-Kroeger, "The E~fect of Hemodilution with Stroma-free Hemogiobirl and Dextran on Collateral Perfusion of Ischemic Myocard;um in the Dog," Arn. Hrt. J., 99:64 (1980). M. 3~eola, D. Azar, and L.
Wiener, "Improved Oxygenation of Ischemic Myocardium by Hemodilution with Stroma-free Hemoglobin Solution," Chest., 75:369 (1979). Both of these studies 25 were exchange transfusions (1:1 or 2:1) of very large doses of hemoglobin.
Renal complications frequently have been have been associated with us~ of high doses OI hemoglobin solutions. Oliguria and decreased renal flow have been a common finding, although irnproved modifications of hemoglobin appear to have somewhat arneliorated this problem. N. I. Birndorf and H. Lopas, "E~fect of Red 30 Cell Stroma-free Hemoglobin Solution on Rellal Function in Morlkeys," J. Appl.
Physiol., 29:573 (1970). M. Reliharl, R. E Olsen, and M. S. Litwin, "Clearance Rate and Effect on Renal Function of Stroma-free Hemoglobin Following Renal 2 ~ 8 ~

Ischemia," Arm. Surg., 176:700 (1972). Other reactions such as fever, chills, flushing, nausea, and chest and abdominal pain are often experienced.
In sum, hemoglobin solutions at high doses in high volume administered as oxygen carrying blood substitutes haYe been reported to increase blood pressure,s and this effect has been characterized alterna~ely as toxic or potentially beneficia~.
Applicants have now discovered that, surprisingly, low doscs of hemoglobin in small volumes may be administered therapeutically to rapidly increase perfusion.

Summ~of the Invention 0 This invention provides a method to therapeutically increase per~usion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from the least amouont effective to increase perfusion, up to a dose of about 2500 mg per kilogram of body weight.

Detailed Description o the In_ention This invention is the use of low doses of hemoglobin to increase perfusion in any clinical setting where that positive change is desirable. This inchldes the administration of hemoglobin to increase blood pressure from abnormally low levels, as in shock of hemorrhagic, cardiogenic or septic origin or to increase blood pressure from normal levels to effect improved perfusion, as in stroke therapy.
The hemoglobin should be "stroma-free" as defined by Rabiner et al. in J.
Exp. Med. 126: 1127 to 1142 (1967), and is preferably alpha-alpha crosslinked, prepared by the method described by Przybelski et al. in J. Lab. and Clin. Med.
117: 143-151 (1991). It is preferably human derived, but may be of animal origin2s or recombinantly produced. It should be in a balanced electrolyte and buffer solution, and preferably is dissolved in one of the plasma expanders such as colloids (plasma, albumin) or crystalloids (saline, glucose, dextran, gelatins, Hemasol~ or Lactated Ringer's). l~e effect of hemoglobin is independent of the diluent used to make up the buLtc solution. The hemoglobin should be at a concentration of about 1 to about 207'o in solution, depending upon the application. The dose should be from about 30 mg per kg of body weight up to about 2500 mg per kg of body weight. The beneficial ef~ect will plateau after administration of about 125 mg per ~7~

kg of bs~dy weight. Doses greater than this but will not enhance the effect, although they will increase the duration of effest.
Conditions in which low dose hemoglobin would be used to rapidly increase perfusion would likely be emergent in nature. Such conditions include shock of 5 hemorrhagic, cardiogenic or septic origin. It would be particularly beneficial in septic shock in ~hich systemic vascular resistance is low causing low blood pressure despite high cardiac output. Under these conditions, hemoglobin would be administered as a bolus of approximately lOOmg (1.0 ml of 10% hemoglobin per kg) followed by volume expansioll with a commonly used crystalloid or colloid lo solution.
Use of hemoglobin to maintain adequate perfusion in a critical care setting would involve slow infusion of a crystalloid/hemoglobin solution 30 mg to deliver a minimum of hemoglobin per kg of body weight, while titrating to effect. The doseadministered should give a rise in mean arterial blood pressure equal to or above 5 normal physiologic levels. As used herein the term blood pressure shall mean the mean arterial blood pressure.
This invention has the following advantages over the prior art:
1. It can be administered rapidly (within ~1 minute) in small doses (30 mg/kg) as a bolus of approximately 75 + 25 ml or as a continuous infusion, 20 resulting in immediate restoration of blood pressure and perfusion in an adult. This markedly differs from current dose requirements of crystaLloid solutions of 3 times the volume lost, typically necessitating administration of many liters of fluid.Studies in our laboratory also indicate that successful resuscitation of hemorrhagic shock can be achieved with hemoglobin solution of one-sixth the dose of whole 2s blood.
2. The duration of effect on blood pressure of the lowest dose of hemoglobin is about 120 minutes, as compared to the transient (30 minute) rise in blood pressure achieved by administlation of Lactated Rhlgers, the most commonlyused initial resuscitation fluid. This should provide sufficient time to achieve30 definitive, corrective treatment.
3. It is preferably stroma-~ree and, therefore, does not require crossmatching or typing. This hastens the time in which de~mitive treatment enhancing oxygen delivery can be initiated.

2 ~

4. It can be purified by heat p~steurization and, therefore, free of infective vinuses including hepatitis and the human immunodeficiency virus. This is not tme of blood products.

5. It is hyperoncotic in nature and, thus, increases intravascular volume.
S Thi5 feature is of particular importance in resuscitation of patients in hemorrhagic shock. Recent reports of the improved results of resuscitation with hyperoncoticsaline solutions support the value of this addifional feature. P. A.. Mal~ingas and R.
F. Bellamy, "Hypertonic Sodium Chloride Solutions ~or the Prehospital Management of Traumat;c Hemorrhagic Shock:A Possible Improvement in the 0 Standard of Care?" Ann. Emerg. Med., lS-1411 (1986). S:. ~i. Wade, J. P.
Hannon, C. A. Bossone, and M. M. ~unt, "Superiority of Hypertonic Saline/Dextran over Hypertonic Saline During the First 30 Minutes of Resuscitation Fol1Owing Hemorrhagic Hypotension m Conscious Swine," Resuscitation, 20:49 ~1990). D. S. Prough, J. C. Johnson, D. A. Stump et al., "Effects of Hypertonic Saline Versus Lactated Ringer's Solution on Cerebral Oxygen Transport Dunng Resuscitation from hemorrhagic Shock," J. Neurosurg., 64:627 ~1986). J. D.
Schrnoker, J. Zhuang, and S. R. Shacl~ord, "Hypertonic ~Fluid Resuscitation Improves Cerebral Oxygen Delivery and Reduces Intracan~ial Pressure After Hemorrhagic Shock," J. Traurna, 31:1607 (19913.

6. The magnitude of its effect on blood pressure is non-dose dependent and self-limiting. Both low-doses and highdoses produce a 15-35% increase in mean arterial blood pressure. This important and surprising characteristic of the invention precludes the possibility of an overdose and the development of d~ngerous hypertension.
2s 7. It has some oxygen carrying ability thPreby increasing oxygen delivery to the tissues, albeit less than the oxygen carrying ability of high doses of hemoglobin solutions used as blood substitutes. However, even the small amount of oxygen carrying hemoglobin provide~ by this invention has a profound beneficial effect when perfusion is concomitantly increase~. Increased oxygen delivery is essential to organ viability and is a unique feature of hemoglobin.
8. It has a low viscosity which enhances flow through the microcirculation, thus preserving organ viability and function.

2 ~

Variations of hemoglobin, including modi~led hemoglobin, and intranaolecular or intennolecular cross-linked species, may be used în accordance with this invention to increase perfusion. The e~fect of hemoglobin is independent of the diluent used to make up the solution.
s Sarnple guidelines for the clinical administration of hemoglobin solutions in accorclance with this invention for three indications are set forth below:

I. To increase perfilsion in conditions of hypovolemic shock:
1. Inject 50 ml (5000 mg) or up to total estirnated volume lost of 10%
0 hemoglobin solution (or equivalent) as a bolus into a peripheral IV.
2. Inject hemoglobin solution within the first or "golden" hour of shock state to minimize duration of decreased perfusion.
3. Monitor cuff blood p~ssure every 15 minutes after administration until peak pressu~ obtained.
4. Monitor other cardiopulrnonary parameters according to standard practice.
5. Adminster other standard therapeutics, as desired or indicatecl.

II. To increase perfusion in conditions of cardiogenic or septic shock:
1. Inject 50 ml (5000 mg) IV bolus, or infuse up to 3000 ml (300 g) of 10% hemoglobin solution (or equivalent) at a rate of 1 ml/kg/min to achieve and maintain the desired effect.
2. Administer hemoglobin as early as possible after the development of a shock state to minimze the duration of decreasecl perfusion.
2s 3. Monitor blood pressure (directly or indirectly) every 15 minutes after administration until pPak pressure obtained.
4. Monitor other cardiopulmonary pa~meters according to standard practice.
5. Administer other standard therapeutics, as desired or indicated.
m. To increase perfusion in stroke conditions:
1. Infuse at least lO0 ml (10 g) of 10% hemoglobin (or equivalent) intravenously at a rate of 1 mVkg/min to achieve and ma~ntain the desired effect.

2~8730~

g 2. Administer hemoglobin solution as early as possible following the cerebrovascular accident to minimized the duration of decreased cerebral perfusion.
3. Monitor blood pressure (directly or indirectly) every 15 minutes after administration until peak pressure obtained.
s 4. Montior other cardiopulmonary parameters according to standard practice.
5. Administer other standard therapeutics as desired or indicated.

Numerous studies have been performed in our laboratory to determine safety 0 and efficacy, characterize the pressor response, define optirnal dosage, determine modifying factors, and d~f~ne agents to counteract the pressor e~fects of hemoglobin. These have been performed as top-load studies as well as hemorrhage and exchange-transfusion studies.

Example One Top-load Studies: Safety and Efficacy/O~timal Dose St~y Method: Conscious, unrestrained male Spraglle-Dawley rats (275-350G) with previously inserted indw~lling arterial and venous catheters were assigned to one OI the following treatment groups:
~o I. 10% hemoglobin solution at doses of 0.62S, 1.25, 2.5, 5.0, 10, 20, and 40 mVkg (equivalent to 62.5, 125, 250, 500, 1000, 2000, and 4000 mg/kg, respectively) (n=6-8) II. 8.3 % human serum album~n (40 mVkg) m. 10% hemoglobin solution at doses of 10 ml/kg (1000 mg/kg) and 20 ml/kg (2000 mg/kg) intra-arterial OR
10% hemoglobin solution at doses of 10mllkg (1000 mg/kg) and 20 mllkg (2000 mg/kg) intravenous Blood pressure and heart rate were continuously monitored for 6 hours after infiusion.
Results: 10% hemoglobin at doses from 1.25 to 5 mllkg (125 to 500 mg/kg) produced an ab~upt 25-30% increase in mean arterial pressure (MAP) that 2~7~

persisted for 180 minutes. Hemoglobin at 10, 20, and 40 ml/kg (1000, 2000, and 4000 mg/kg), likewise, produced an immediate 25-30~; increase in MAP that was sustained for 240 to 300 minutes. Rlthough hemoglobin at 0.625 ml/kg (62.5 mg/kg) produced a 12% increase in MAP, it was not statistically significant 5 compared to baseline. Coincident with the increase in MAP, heart rate (HR) fell in all an~mals in~lsed with hemoglobm except at the dose of 0.625 ml/kg (62.5 mg/kg). Tlle duration of HR reduction corresponded to the duration of MAP rise.
Human serum albumin (HSA) and Ringers Lactate (RL) did not change MAP or HR significantly compared to baseline. Infusion of hemoglobin lOml/kg and 20 0 ml/kg intra-arterially and intravenously resulted in an abrupt increase in MAP and decline in HR that persistecl for 240-300 minutes. Statistically, there was no difference between the magTutude and duration of MAP and HR effect between the venous and arterial routes of administration. Cardiac output (CO) determinationswere performed in only 2-3 animals receiving hemoglobin at 20 mVkg or 8.3%
5 HSA (20 ml/kg). Although such few animals preclude statistical analysis, CO
values in animals receiving 8.3% HSA rose from a baseline of 30ml/min to 37 ml/min at the end of infusion. Calculated systemic vascular resistance (SVR) declined from 3200 units at baseline to 2300 units at the end of infusion. In contrast, CO values in animals receiving hemoglobin 20 mVkg (2000 mg/kg) 20 declined from baseline of 37 mUmin to 34 ml/lmin at ~he end of infiusion to 26 ml/min by 30 minute post-infusion. Calculated SVR almost doubled from a baseline of 2600 units to 4800 units at the end of infilsion, and 4300 units at 30 minutes post-infusion.
Conclusions: Intravenous topload infusion of up to 40 ml/kg (4000 mg/lcg) 2s of 10% hemoglobin solution ;s well tolerated in conscious rats. Doses of hemoglobin between 1.25 and 40 ml/kg (125 and 4000 mg/kg, respectively) elicit a30-35% increase in MAP ~hat persists between 180-300 minu~es depending on the volume infused. The lowest dose of hemoglobin (0.625 ml/kg or 62.5 mg/kg) produced a (12%) increase in MAP. Concomitant reductions in HR of 30-33~
30 from baseline that persist as long as MAP is elevated suggest a baroreceptor reflex response to the abrupt increase in MAP.

Example Two 2087~.J ~34 Chara~e ization of Pressure Response Study Methods: Conscious, unrestrained, male Sprague-Dawley rats with indwelling ar~erial and venous catheters were assigned randomly to one of four treatment groups:
s I. 37C 7% hemoglobin 4 ml/kg (280 mg/kg) infused at 0.34 ml/min II. 37C 7% hemoglobin 4 ml/kg (280 mg/kg) as a bolus m. 4C 7% hemoglobin 4 ml/l~g (280 mg/kg) in~used at 0.34 ml/min IV. 37C 7% hemoglobin 4 ml/kg (280 mg/kg) as a bolus MAP and HR were monitored continuously throughout the infusion and for 120 0 minutes posts-infusion.
Results: Admin~stration of 7% hemoglobin produced a rapid and sustained ~120 minutes) nse in MAP in all treatment groups. However, the maximum pressor response was greatest with wann (37) vs cold (4) bolus injections of hemoglobin (136 ~ 4 mmHg vs 119 + 6 mmHg). A similar, though less pronounced response occurred with wann (37) vs cold (4) infusions of hemoglobin (125 ~ 5 mmHg vs 118 + 5 mrnHg, respectively). VaIying the rate of administration of cold (4 ) solution did not alter the pressor response signiflcantly. However, the pressor response was attenuated with slow infusion (0.34 m~min) vs bolus administration of warrn (37) hemoglobin (124 ~ 5 mmHg vs 134 + 5 mmHg, respectively). In all cases, HR responded in a reciprocal manner to the changes in MAP. This reflex response was mora pronounced (302 11 bpm vs 351 + 8 bpm) with warm vs cold hemoglobin solution.
The magnitude and duration of the pressor response of 7% hemoglobin (4 mUkg or 280 mg/kg~ is affected by the temperature of the solution with a blunted2s response obselved with administration of cold (4) versus body tamperatura (37) solution. The rate of administration (bolus vs iT~sion at 0.34 ml/min) did not alter the pressor response significantly, regardless of the solution' s temperature.

EXalllpl8 Three echanism of Action Stu~
Methods: Conscious, male, Sprague-Dawley rats were instlumented with indwelling arterial and venous catheters for continuous monitoring of MAP and HR. The study was divided into two separate sets of experiments: endothelium andnitric oxide/L-NMMA experiments.

Endothelin S~dy Anirnals (1-4 per group) were assigned randomly to receive one s of four treatments:
I. Big ET (5 nM/kg), IV bolus II. Phosphoramidon (5 mg/lcg), IV bolus III. Phosphor~nidon (5 mg/kg) pretreatment (30 sec) plus Big ET (5 nM/kg) 0 IV. I:'hosphoramidon (5 mg/kg) pretreatment (30 sec) plus 7% hemoglobin (4ml/kg or 28Q mg/kg) IV
bolus Results: 7% hemoglobin (4 ml/kg or 280 mg/kg) elicited a rapid rise in MAP (105 + 2 mmHg at baseline vs 133 + 4 mmHg at 15 minutes post infusion) which peaked at 15 to 25 minutes and returned to baseline at 120 minutes.
Injection of B.g ET (5 nM/Kg elicited a similar, but more dr~natic MAP response ~98 + 4 mmHg at baseline vs 149 + 8 mmHg) which, l~ewise, peaked at 15 minutes and returned to baseline by 120 minutes. Phosphoramidon, an inhibitor ofpro-endothelin conversion to endothelin, given as a top-load injection (5 mg/kg)had no effect on MAP. However, when admmist~red as a 30-sec pre-treatment injection, phosphoramidon (~ mg/kg) attenuated the maximum MAP rise of both Big ET (5 nM/kg) and 7% hemoglobin ~4 mVkg or 280 mg/kg) by approximately 75% and 79%, respectively. lHR responded reciprocally with MAP with lowest HR' s occurring at maximum MAP. Phosphoramidon, as pre-treatment, also attenuated the magrutude of reduction in HR achie~/ed with both E~ and 7%
hemoglobin.

Nitric Oxicle and L-NMMA Study Animals (5-7 per group) were assigned randomly to receive one of four treatments:
I. L-NMMA (5 & 10 mg/kg) IV bolus II. L-arginine (200 mg/kg) IV bolus m. L-NMMA (5 mg/kg) plus L-arginine (50 & 100 mg/kg) IV
bolus 2~7~0~

IV. 7 % hemoglobin ~4 ml/kg or 280 mg/kg) plus L-arginine (200 mg/kg) IV bolus V. 7% hemoglvbin (4 ml/kg or 280 mg/kg) plus Nitroglycerin infusion (titrated at 10-lS0 mcg/min to effect) begun 15 minute post-hemoglobin s Results: L-N~A injections of 5 and 10 mg/kg increased MAP from 109 + 3 mmHg to 139 + 13 mmHg and from 106 + 2 mmHg to 146 + 6 mmHg, respectively. This resposlse peaked at 30 minute after injection, and lasted > 6hours. Administration of L-arginine (50 & 100 mg/kg) 30 minute after injection of L-NMMA reduced the L-NMMA's pressor e~fect signiflcantly (p<0.05). lBoth the 0 magnitude and durativn of this attenuation was greater at the higher dose of L-arginine. Injection of 200 mg/kg of L-arginine in no~not~nsive rais elicited an immediate drop in MAP that quickly rebounded to above baseline levels within 10 minutes. Injection of this same dose (200 mg/kg) of L-arginine 15 mins after a bolus injection of 7 % hemoglobin solution (4 ml/kg or 280 mg/kg) evoked a similar sudden and transient drop in MAP that was followed by an increase in MAP ~hat exceeded that which would be expected ~rom hemoglobin alone. Nitroglycerin ~NTG) infusion ~lO-150 mcg/min) begun at 15 min post 7% hemoglobin injection (4 ml/kg or 280 mg/kg) reduced the pressor effects of hemoglobin, decreasing MAP from a peak of 141 +7 mmHg to 113 +5 mmHg within minutes. Fifteen mimltes after discontinuation of NTG, the MAP remained significantly reduced from control values (115 ~4 mmHg vs 128 ~2 mmHg3, respectively.
Conclusions: Hemoglobin solution and Big ET (pro-endothelin) have similar pressor effects with respect to MAP peak e~fect time and duration.
However, absolute MAP increase is greater with :Big ~T ~han 7 % hemoglobin at the doses tested. Phospho~amidon, a metalloproteinase inhibitor, blunts the pressor effect oî both Big T and hemoglob~n solution, suggesting that the pressor effect is mediated, at least in part, by ET. Nitroglycelin, a prodrug of nitric oxide (NO3, reverses hemoglobin's p~essor effects, suggesting that exogenous NO may overridehemoglobin's binding of endogenous NO. However, L-arginine, at a dose exceeding that which reversed the pressor effect of L-NMMA, did not reverse the pressor effects of hemoglobin. This suggests that hemoglobin may also interfere with the synthesis of NO. Based on these F~dings, it is concluded that the pressor effects of hemoglobin are mediated, at least in part, by the release of endothelin - l4 -(E~, a potent vasoconstnctor, asld the inhibition of NO, an endothelin-derived relaxing factor. Ihus, hemoglobin's pressor effect is rnediated by an autoregulatory system which explains the wide margin of safety of this invention compared to other pressor agents.
s ~xample Four Use ~f Antihypertensive A~ents to Control PressQr Response Methods: Conscious, unrestrained, male, Sprague-Dawley rats (250-350g) with indwelling arterial and venous catheters were assigned to one of the following lO five treatment groups, with 6 to 8 animals in each group. MAP and HR were monitored continuously for 120 minutes following infusion.
I. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus II. 7 % hemoglobin (4 mVkg or 280 mg/kg) IV bolus plus P~azosin (2 mg/kg, I~ over 1 min) m. 7 % hemoglobin (4 mVkg or 280 mg/kg) IV bolus plus Propranolol (70 mcg/kg, I~ over 1 min) IV. 7 % hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus Verapamil ~0.25 mg/kg, IV over 1 min, repeated in 10 mins) ~J. 7 ~ hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus 20 Nitroglycenn (IV infusion titrated between 10-150 mcg/min to effect) Results: 7% hemoglobLhl infusion elicited an immediate increase in MAP
from 105 + 2 mmHg at baseline to 133 + 4 mmHg at 15 minutes which was sustained for 120 minutes. HR declined in a reciprocal manner. Injection of ~ osin (2 mg/kg) lS minutes after ;njection of hemoglobin, produced an 2s immediate, significar,t decrease in MAP from a maximum or 134 + 5 mrnHg to 102 + 11 mmHg with sustained rnaintenance of MAP near baseline levels for one hour. In response to the effect on MAP, HR was restored to ~aseline following P~zosin administration, and was sustained throughout the 120 minutes observationperiod.
Administration of Propranolol (70 mcg/kg~ 15 minutes after injection of hemoglobin did not significantly alter its pressor response. An obselved brief (3 to 4 minutes) reduction of MAP immediately ~ollowing Propranolol injection did not 2~7a ~

achieve statistical significance. Although HR returned near baseline levels, it,likewise did not achieve statistical significance.
Verapami] (0.25 mg/kg) transiently decreased MAP from a peak of 143 + 7 mmHg to 118 + 4 mmEIg within 2 minutes nf injection. However, MAP returned 5 to near baseline within 10 minutes. A second bolus injection of Verapamil produced a similar, transient effect. In response, HR transiently increased toward baseline; however, this did not reach statistical signiflcance.
Nitroglycerin (NTG) infusion over a dose range of 10 to 150 mcg/kg produced an immecliate and steady decrease in MAP from a peak of 141 + 7 0 mmHg to 113 ~ 5 mmHg. Fifteen minutes after discontinuation of NTG, MAP
still was signiflcantly reduced compared to baseline ~115 +4 mmHg vs 138 +
mmHg, respectively). HR returned to baseline by 15 mins of NTG infusion and remained at or above baseline for the remainder of the experimerlt.
Conclusions: The pressor effects of hemoglobin can be controlled readily 5 with clinically relevant doses of at least two commonly used anti-hypertensiveagents, Prazosin and Nitroglycerin. The transient effects of Verapamil on MAP
raise the question of whether a higher dose and/or continuous infusion might be more effective. Propranolol, at the dose tested, does not ef~ectively control hemoglobin's pressor effect.
Example Five Hemorrhag~Exchange Transfiusion Studies Resuscitation Sbudy Methods: Male Sprague-Dawley rats were anesthetized with an initial dose 25 of 1.2 ml/kg of a 3:7 mixture of xylazine (20 mg/ml) and ketamine (100 mg/ml)and thereafter given 0.6 rnl of the same anesthesia solution to maintain anesthesia.
Indwel~ing arterial and venous catheters and Cla~k-type heated electrodes were placed for eontinuous monitoring of MAP, HR and transcutaneous oxygen tension for 60 minutes post treatment. A sham group was not bled except for withdrawal 30 of two 1 ml blood samples, but was monitored the entire penod. All other animals were bled a total of 20 ml/kg (approximately one-third total blood volume) at a rate of 1 ml/min. Each rat was assigned randomly to one of six treatment groups:
(n=5-15 animals per group) 2~7~

I. Sham ~[. No Resuscitation m. Autologous Shed Blood (20 mVkg) IV. Lactated Ringers 4û mUkg s V. 14% Hemoglobin 20 ml/kg (2800 mg/kg) VI. 14% Hemoglobin 10 mUkg (1400 mg/kg) All solutions were in~lsed at a rate of 1.7 ml/min Results: Following hemorrhage, the MAP fell to 40% of baseline (to approximately 40 mmHg) in all animals. Within 2 minutes of initiating 0 resuscitation infusion, whole-volume hemoglobin (20 ml/kg) raised MAP to abovebaseline levels (120 mmHg); half-volume hemoglobin (lOml/kg) raised MAP to baseline levels (100 mmHg); autologous shed blood raised MAP to approximately 75 mmHg; and Lactated Ringers raised MAP to 60 mmHg. By ~our minutes both hemoglobin groups had mean arterial pressures significantly higher than either the 15 Lactated Ringer's or blood glOUpS. By 6 minutes, there were no differences among the MAPs of the blood, full-volume, and half-volume hemoglobin groups, and all remained significantly higher than the no-resuscitaeion and I actated Ringers groups.
At 15 minutes post-resuscitation, the MAP of the Lactated Ringers group dropped to the level of the no-resuscitation group. A~ this same time, the MAP of both the 20 whole-volume and half-volume hemoglobin groups were significantly higher than those of the ~lood group.
HRs in all groups fell duling hemolThage. Within 2 minutes of resuscitation, HRs in both hemoglobin groups began to rise. By 4 minutes there were no significan~ differences in HR ~nong the resuscita~ed groups. However, by25 20 mins, the HRs of the Lactated Ringers group had fallen to the level of the no-resuscitation group, while that of the hemoglobin and blood groups remained nearbaseline levels.
All animals that were bled had a drop in transcutaneous oxygen tension (TCpO2) to approxirnately one-tenth their baseline level. Within S minutes of 30 resuscitation infusion, all groups, except the no-resuscitation group, had a rise in TCpO2 to at or near baseline levels. This trend continued in the blood and hemoglobin groups. In contrast, a large, persistent drop in TCpO2 occurred in the 17 ~ ;3~

Lactated Ringers group which, by 20 minutes, was not significantly dif~erent from the no-resuscitation group Measurement of serum lactate levels were not significantly different in all groups prior to hemorrhage. However, post-resuscit~tion serum lactate levels were 5 significantly increased in the Lactated Ringers and no-resuscitation groups, whereas the sham, blood, whole-volume, and half-volume hemoglobin groups had no significant change.
Hematocrit levels measured be~ore and 1 hour after hemorrhage shoed a significant drop in hematocrit all groups, except for the blood group.
Conclusions: 14% Hemoglobin solution promptly restores MAP, HR, TCpO2 after non-lethal hemorrhage. The restoration of TCpO2 with hemoglobin solution indicates blood flow peripherally and presumably to other organ systems is enhanced. A clinically significant fmding is that hal~-volume (10 ml/kg) hemoglobin solution is as efficacious in restoring MAP, HR, and TCpO2 as nearly 5 twice that volume of whole blood. The return of MAP to baseline before the hemogiobin solution was completely infused suggests that even a lower dose of the hemoglobin might be effective.

Example Six 20 Hemorrhage/Dose Optirnization Studv Methods: ~onscious, unrestrained male Sprague-Dawley rats (275-300g) with indwelling venous and a~erial catheters were bled 35 mVkg manually at 1 ml/min. Twenty mins after the bleed, the animals were assigned to one of the following treatment groups.
I. Non-resuscitated controlgroup II. Autologous shecl blood (35 ml/kg) m. Lactated Ringers (105 mVkg) at 3 ml/min IV. 7% Hemoglobin (17.5 mUkg=1225 mg/kg OR
35 ml/kg=2450 mg/kg) at 1 mVmin V. 10% Hemoglobin (17.5 ml/kg~1750 mg/kg OR
35 mVkg=3500 mg/kg) at 1 mVmin MAP, HR and pulse pressure were monitored for up to S hours.

2 0 ~ 7 ~ O ~

Results: MAP initially fell 31 + 3 mmHg following hemorrhage and returned to 57% of baseline within 20 minutes. T~is hypotension was associated with tachycardia. In the non-resuscitated group, MAP remained at 50 to 55 mmHg (from an average baseline of 99.9 + 4 mrnHg) throughout the observation period, s and plummeted just prior to death. At 24 hours, 11 out of 15 non-resuscitated animals were dead.
In anirnals resuscitated w;th Lactated Ringers (105 mVkg), MAP increased to 80% of baseline during the infusion, but fell to 60 to 70% of baseline at completion of infilsion and remained at this level throughout the observation period.
o All animals resuscitated with Lactated Ringers were alive at 24 hours, although all had significant tachycardia.
Animals resuscitated with Hemoglobin of either 7% or 10%, and at either doses (17.5 mVkg or 35 ml/kg) as well as animals resuscitated with shed blood had similar hemodynamic responses to resuscitation with an increase in MAP to near or 5 above baseline levels with a concomitant decrease in HR. A slightly 8reater increase in MAP (120 mmHg vs 110 mmHg) and a slightly lower HR (350 bpm vs 400 bpm) were noted in the anirnals resuscita~ed with 10% hemoglobin (both doses) at 60 min post ir~sion. However, at 120 to 300 minutes post-resuscitation, therewere no signiflcant differences between the hemoglobin and blood treated groups.At 24 hours post-resuscitation, 4 of 5 blood treated animals were alive; ~ of 9 anirnals resuscitated with 10% hemoglobin (17.5 ml/kg or 1750 mg/kg) were alive;7 of 8 of the 10% hemoglobin (35 mllkg or 3500 mg/kg) group were alive; 3 of 4 of the 7% hemoglobin (1?.5 ml/kg or 1225 mg/kg) group were alive; and 4 of 5 of the 7% hemoglob~n (35 ml/kg or ~450 mg/kg) treated a~unals were alive.
Conclusions: 7~ Hemoglobin solution is as ef~lcacious as a 10%
hemoglobin solution in restoring MAP and H[R following severe hemorrhage.
Furthennore, hemoglobin solutiQn at half the volume (17.5 ml/kg or 122S mg/kg) is as efficacious as blood in restoring ca~diovascular fimction and increasing survival ~ollowing hemorrhage.

Example Seven Hemorrhage Study ~37~

Methods: Conscious, York swhle (18 23 kg) with indwelling arterial and venous therrnodilution catheters were bled 30 ml/kg over a 20 min period and assigned to one of two treatment groups:
I. 7% hemoglobill (10 ml/kg; 700 mg/kg) n=6 s lI. Autologous shed blood (10 mVkg) n=6 Following a 2 hour stabilization period, an~mals received:
I. Lactated Ringers (20 ml/kg) lI. Autologous shed blood (20 mVkg) Blood samples ~or buffered base excess, hematocrit, and arterial blood gases as well 0 as hemodynamic measurements were obtained at basel.;ne, end of hemorrhage, end of first infi~sion, and end of second infiusion.
Results: Following hemorrhage, MAP fell 65% from baseline in Group I
ar~imals, and 62 5~ in Groups r[ anirnals. SVR ~ell in both groups. Post-hemorrhage HR in Group I decreased 37% from baseline in contrast to a 4%
increase in the shed blood group. This contrasting response is explained by the significant difference in baseline HR between group I (198 1~ 10 bpm) and group II
(153 _ 10 bpm).
Following administration of hemoglobin (10 ml/kg; 700 mg/kg), MAP rose by 18% ~from a baseline of 106 + 5 mmHg to 125 + 9 mmHg). This was accompanied by a 38 % decline in HR (from 198 + 10 mmHg at baseline to 124 +
~ mm~Ig), a 10% increase in stroke volume (SV) (from 31 ml/beat at baseline to 34 ml/beat), and an almost doubling of SVR from 18 +1.3 to 34 + 7.1 Ul~itS.
Following administration of autologous shed blood (10 ml/kg), MAP rose to 96 +6 mmHg, but remained 9% below the baseline of 104 + 8 mmHg. HR
2s remained at control levels and SVR increased from 23.3 + 2.8 at baseline to 31.4 + 5.2 units, w~lile SV remained below control values (31 ml/beat vs 25 ml/beat).At 2 hours post~ sion, animals in Group I ~hemoglobin) showed MAPs that remained above baseline, HRs above or close to baseline, continued elevation of SVR, and a deeline in SV to 21 ml/beat. At this same time, alLimals in Group II
(autologous blood) experienced further declines in MAP (86 + 6 mmHg) and HRs (137 ~ 7 bpm). S~ in this group declined, but remained above baseline levels at 27.8 + 4.1 units with essentially no change in SV.

Following the infusion of Lactated Ringers (20 ml/kg~, MAP remained elevated (120 ~ 3 mmHg), and HR declined to 160 ~ 17 bpm, and SY rose to within 10% of control levels.
Following in~usion of 20 ml/kg autologous blood, MAP rose to near, but s still below, baseline; HR and SVR declined, and SV increased above baseline levels.
Analysis of blood samples showed a decrease in venous pH in both groups following hemorrhage. This value rebounded slightly (~rom 7.28 to 7.33) following hemoglobin, but remained depressed at 7.28 following autologous blood.10 Venous pH rehlrned to nonnal in both groups following supplemental infusion of Lactated Ringers or autologous blood.
Buffer base excess (BE) dropped significantly in both groups following hemorrhage, and did not change significantly with in~sio~ of hemoglobin or autologous blood. By 2 hrs, Bl~ was retu~ing to baseline in both groups, and 5 increased in lboth groups following final treatment.
Conclusions: 7% Hemoglobin (10 mVkg or 700 mg/kg~ raises blood pressure more rapidly and to a greater extent than autologous blood, and replenishes buffer base excess equally well.

Example Eight Exchan~e Trans~usion Studv I
Methods: Conscious, unrestrained rats were bled a total of 60 ml/kg at a rate of 1 ml/min. Infusion of one of the following test solutions was begun after the initial 25 mVkg bleed while animals were bled an additional 35 ml/kg. (n=8 animals in each group).
I. 7% Hemoglobin (lO mVkg=700 mg/kg) follo~ed by L~ctated Ringers (50 ml/kg) to total volume lost (60 ml/kg) II. 7% Hemoglobin (20 ml/kg=1400 mg/kg) ~ollowed by Lactated Ringers (120 mVkg) m. Lactated Ringers (180 ml/kg) All infusions were given at a rate of l ml/min until completion of bleeding, andthen increased to 3 mVmin. MAP and HR were monitored continuously for 2 2~87~5~ll hours. Venolls blood samples were analyzed for blood gases, electrolytes, and hematocrit at baseline, end-of-resuscitation, and 1 hour post-resuscitation.
Results: Following the initial bleed of 25 mUkg, MAP fell to approxirnately 30 mmHg. By mid-transfusion (end-of-bleed), MAP was significantly higher and s near baseline level only in the group receiving the higher dose (20 ml/kg or 1400 mg/kg) of 7% hemoglob~n. This response was sustained for the entire observation period (120 rninutes). MAP in animals receiving the lower dose (10 mVkg or 700 mg/kg) of 7% hemoglobin rose to approximately 70 to 80 mmHg at the end of infusion, and was sustained for 120 minutes. In the Lactated Ringers group, MAP
0 was restored to only 60 mmHg (from a baseline of 98 mmHg) at the end of the infusion, and thereafter continued to decline with all animals dead within 60 to 90 minutes post-infusion. Animals in both hemoglobin groups survived longer: 90 +
9 minutes in the lower dose hemoglobin group and 277 + 50 minutes in the higher dose hemoglobin group.
Blood gas data showed lower HCO3, pC02, and pH levels (metabolic acidosis) in the Lactated Ringers group compared to the hemoglobin treated groups.
Serum K~ levels were significantly increased from baseline in the Lactated Ringers groups, but were only significantly increased at 1 hour post-resuscitation in both hemoglobin groups.
Conclusions: 7% Hemoglobin solution at a dose of 20 ml/kg (1400 mg/kg) followed by 3:1 Lactated Ringers is superior to lower dose 7% Hemoglobin (10 ml/kg or 700 mg/kg) followed by 1:1 L~ctated Ringers, and both hemoglobin doses were superior to 3:1 Lactated ~gers alone. This study demonstrates that 20 ml/kgor 140û mg/kg of 7 % Hemoglobin is sufficient for resuscitation following hemorrhage for up to 3 ~o 4 hours if adequate crystalloid is infused following hemoglobin administration.
This period of adequate tissue oxygenation may provide the necessary and critical time before defin~tive treatment is available.

Example Nine Exchange-Transfusion Studv I[

-22- ~87~

Methods: Conscious, unrestrained rats were bled a total of 70 ml/kg (approximately total blood volurne) at a rate of 1 ml/min. Infusion of one of the following test solutions was begun after an initial bleed of 35 ml/kg, while theanimals cont;nued to be bled an additional 3S ml/kg. (n=6-8 animals per group) s I. 7% Hemoglobin (20 ml/kg=1400 mg/kg) ~ollowed by Lactated Ringers (50 ml/kg) to total volume lost of 70 ml/kg II. Lactated Ringers (210 ml/kg) to total 3 tirnes the volume lost o m. 5 % lHuman Serum Albumin (HSA) 70 ml/kg All infusions were given at a rate of 1 ml/min until completion of the bleed, and then increased to 3 mVmin.
Venous blood sarnples were analyzed for blood gases, electrolytes and hematocrit at baseline, end-o~-resuscitation, and 1 hour post-resuscitation.
Results: MAP fell uniformly to approximately 35 to 40 mmHg at the end of the initial ~5 ml/kg bleed. Animals transfused with Lactated Ringers had a transient increase in MAP (to 50 mmHg) that fell precipitously even before the completion of infusion. HR also remained low during this treatment but increasedsignificantly by 20 minutes post resuscitation. By 60 minutes, the one remaininganirnal in tl~is group had tachycardia with a HR ~450 bpm in response to severe hypotension. Animals transfused with HSA had MAPs restored to approximately 60 mmHg. These animals had considerable tachycardia (450-500 bpm) throughout the observation period. By 60 minutes, one animal Ln the HSA group was alive.
Anirnals resuscitated with 7% hemoglobin had a restoration of MAP to baseline 2s during and up to 30 mins post-resuscitation. However, by 60 minutes, only 2 of the hemoglobin treated animals were alive, and their MAPs were decreasing. HR
was rapidly restored to or above baseline for at least 30 mins post-resuscitation. By 60 minutes, HR fell as circulatory f~nction collapsed. Survival time was not significantly different between the Lactated Ringers and HSA treated animals, but was significantly better in the hemoglobin-transfused animals.
All resuscitated animals were extremely acidotic by the end of resuscitation and at 1 hour post-resuscitation with significant drops in HCO3, pH and pC02.

~7~

Semm K~ levels were significantly elevated reflecting significant cellular damage from ischemia and hypoxia.
Conclusions: In this more severe transfusion-exchange model, 7 %
hemoglobin was superior to 3:1 Lactated Ringers, or isovolume 5 % HSA.
s However, in this model, hemoglobin solution was able to restore and maintain MAP for only 30 minutes. Although blood gases and chemistries were consider-ably better in the hemoglobin-treated an~mals, by 1 hour post-resuscitation animals were decompensat~ng and becarne as metabolically acidotic as anirnals in the other two groups. It is possible that increasing the volume of Lactated Ringers (3:1 vs 0 1:1) following hemoglobin in~usion may improve results.

Iixample Ten Tissue FI_w Study Methods: Conscious York swine with indwelling venous and right ventricular catheters were bled 30 ml/kg over 30 minutes. Following hemorrhage, venous blood samples were analyzed for base excess; when BE~ reached -5 to -10, infusion of 7% hemoglobin 5 mVkg (350 mg/kg) was ir~used at a rate of 1 ml/kg/min. Animals were monitored for 1 hour post~ sion at which time they were sacrificed for assessment of organ flow.
Results: MAP fell from a mean of 100 mmHg to 40 mmHg following hemorrhage and promptly returned to baseline following infusion of a very small volume of hemoglobin.
Flow to all organs except the adrenals and the liver, declined following hemorrhage. Following infusion of 7% hemoglobin, tissue flow increased to all 5 organ systems except the lung and the liver. Importantly, tissue flow had increased to above baseline levels to the heart and the brain. At 1 hour post-resuscitation, flow to all orgarls was increased except to parts of the splanchnic system.
Conclusions: Seven percent hemoglobin solution ef~ectively restored MAP
following acute hemorrhage. This is associated with an increase in perfusion to vital organ systems, and all other organs with the exception of the lung and theliver. This was achieved with doses as low as S mVkg (350 mg/kg) or one sixth ofthe blood volume lost.

- 24 - 2 Q 8 ~

E~xample Pleven Methods: Male, spontaneously hypertensive, anesthetized, and mechanically sventilated rats (350-400 g) with undwelling venous and arterial catheters were assigned randomly to one of the following treatment groups: (n= 9 animals per group).
I. Hematocrit 44%: blood volume increased with 8 ml donor blood lI. lHematocrit 37%: blood volume & hematocrit manipulated with 8 ml 0(560 mg) 7% hemoglobin III. Hematocrit 30%: blood volume and hematocrit manipulated by 5 ml (350 mg) exchange transfusion of 7 % hemoglobin plus an additional 8 ml (560 mg)7% hemoglobin IV. Hematocrit 23%: blood volume and hematocrit manipulated by 10 ml 15(700 mg) exchange transfusion of 7% hemoglobin plus an additional 8 ml (560 mg~

7% hemoglobin V. Hematocrit 16%: blood volume and hematocrit manipulated by lS ml (1050 mg) exchange transfilsion of 7% hemoglobin plus an additional 8 ml (560 mg) 7% hemoglobin 20VI. Hematocrit 9 %: blood volume and hematocrit manipulated by 20 ml (1400 mg) exchange transfusion plus and additional 8 ml (560 mg) 7% hemoglobin Maintenance fluids of 0.9 Na5Cl were nfused at 4 mVkg/hr and target hematocrits and blood volumes maintained for 30 minutes. Via a c~n~ectomy, the middle cerebral artery (~CA) was occluded. After 10 minutes of occlusion, 25lOOuCi-kg of C-iodoantipyrine was given. Brains were then removed, sec~ioned, and analyzed to define areas with cerebral blood flow (CBF) 0-10 ml/lOOg/minute and 11-20 ml/lOOg/min.
Results: There was no di~ference between the hematocrit 44% and hematocrit 37~ groups in areas of 0-10 and 11-20 ml/lOOg/minute CBF. In the 30other 4 groups, the areas of both of these low CBF's were less as hematocnt decreased, with the srnallest area of ischemia occurring in the hematocrit 9 % group (the group that received the largest dose of hemoglobin). Measurements of CB~ inthe hemisphere contralateral to the occluded MCA revealed a progress;ve increase 2o8r~3 ~

in CBF as herna~ocrit decreased (frorn 125.6 +18.8 ml with hernatocrit 44% to 180. 8 + 14.4ml w;th hematocrit 9 %).
Conclusions: H~pervolemic hemodilution with 7% hernoglobin effects a dose-related decrease in ischemia following 10 minutes of MCA occlusion in rats.s This occurs in association with increased perfi~sion (CB F) related to increased doses of hemoglobin.
While the foregoing embodiments are intended to illustrate a novel therapeutic method to increase perfilsion, they are not intended nor should they be construed as limitations on the inven~ion. As one skilled in the art would lo understand, many var~ations and modiflcations of these embodiments may be made which fall within the spirit and scope of this invention.

Claims

1. A method to increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from the least amount effective to increase perfusion, up to a dose of about 2500 mg per kg of body weight.

2. A method to increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from about 30 to about 2500 mg per kg of body weight.

3. The method of Claim 2 where the stroma-free hemoglobin is intramolecularly cross-linked hemoglobin, intermolecularly cross-linked hemoglobin or methemoglobin.

4. The method of Claim 2 where the stroma-free hemoglobin is administered in a physiologically compatible solution.

5. The method of Claim 2 where the stroma-free hemoglobin is dissolved in a colloid or crystalloid solution.

6. A method of treating hypotension secondary to hemorrhagic, cardiogenic, or septic shock in a mammal comprising administering stroma-free hemoglobin at a dose ranging from about 30 mg to about 2500 mg per kg of body weight.

7. A method of raising mean arterial blood pressure above normal levels in patients suffering a stroke comprising administering stroma-free hemoglobin at a dose ranging from about 30 to about 2500 mg per kg of body weight.

8. A method to increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from an amount effective to increase mean arterial blood pressure 50 that it is above normal physiologic levels, up to a dose of about 2500 mg per kg of body weight.

9. A method to increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from the least amount effective to increase mean arterial blood pressure so that it is above the pretreatment level, up to a dose of about 2500 mg per kg of body weight.

10. A method to increase perfusion in a mammal comprising administering stroma-free hemoglobin at a dose ranging from the least amount effective to give a rise in mean arterial pressure, up to a dose of about 2500 mg per kg of body weight.

11. A method to increase perfusion in a mammal following blood loss comprising the administration of stroma-free hemoglobin at a dose ranging from about 30 to about 2500 mg per kg of body weight and in a volume less than the volume of blood lost.

12. The method of Claim 11 where the stroma-free hemoglobin is administered in a volume of less than half the volume of blood lost.

13. The method of Claim 2 where the stroma-free hemoglobin is administered in a volume of less than 200 ml.

14. The method of Claim 2 where the stroma-free hemoglobin is administered in a volume of less than 50 ml.