Human serum albumin-porphyrin complexes with the ability to bind oxygen and therapeutic uses thereof

The invention is directed to human serum albumin-porphyrin (HSA-P) complexes which are capable of reversible oxygen binding and their uses. These complexes may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or in applications requiring plasma expanders. Methods for the production of these complexes are provided. In a specific example, HSA-P complexes are shown to exhibit reversible oxygen binding. In another example, the HSA-P complex does not exhibit appreciable vasoactivity.

TABLE OF CONTENTS 
1. INTRODUCTION 
2. BACKGROUND OF THE INVENTION 
2.1 BLOOD SUBSTITUTES 
2.2 HUMAN SERUM ALBUMIN 
2.3 HEME 
2.4 ALBUMIN/PORPHYRINS 
3. SUMMARY OF THE INVENTION 
4. BRIEF DESCRIPTION OF THE FIGURES 
5. DETAILED DESCRIPTION OF THE INVENTION 
5.1 Preparation of HSA-P 
5.2 Utilities of the Invention 
6. EXAMPLE PREATION OF HUMAN SERUM ALBUMIN-PICKET FENCE PORPHYRINS 
7. EXAMPLE: ABSORPTION SPECTRA OF OXYGEN BINDING BY THE HSA-PFP COMPLEX #1 
8. EXAMPLE: OXYGEN-BINDING OF HSA-PFP #1 
9. EXAMPLE: OXYGENATION CYCLES FOR HSA-PFP #2 
10. EXAMPLE: VASOACTIVITY EXPERIMENT FOR HSA-PFP #1 
INTRODUCTION 
The present invention is directed to human serum albumin (HSA)-porphyrin 
complexes, and their production and uses. Human serum albumin-porphyrins 
(HSA-P) produced by methods of the present invention may be used in 
applications requiring physiological oxygen carriers such as in blood 
substitute solutions, or in applications requiring plasma expanders. 
BACKGROUND OF THE INVENTION 
2.1 BLOOD SUBSTITUTES 
Treatment of many clinical conditions involving blood loss or blood 
deficiency requires supplementation with a source of donor blood or a 
blood substitute. A primary goal is to restore the circulation of oxygen 
through the body, a function that is physiologically mediated by the 
hemoglobin found in red blood cells. 
Transfusion of a patient with donated blood, while used widely, has a 
number of disadvantages. Firstly, there may be a shortage of a patient's 
blood type. Secondly, transfused blood may be contaminated with infectious 
agents such as hepatitis viruses, cytomegaloviruses, Epstein-Barr virus, 
serum parvoviruses, syphilis, malaria, filariasis, trypanosomiasis, 
babsiosis, pathogenic bacteria, and HIV (Bove, Progr. Hematol. 14: 
123-145, 1986). Thirdly, donated blood has a limited shelf life. 
An alternative to transfused blood involves the use of blood substitutes. A 
blood substitute is an oxygen carrying solution that also provides the 
oncotic pressure necessary to maintain blood volume. Two types of blood 
substitutes have recently been studied, fluorocarbon emulsions and 
hemoglobin solutions. 
Fluorocarbon emulsions, however, are not feasible blood substitutes, since 
they are known at times to block the immune system (Dellacherie, Crit. 
Rev. Ther. Drug Carriers 3:41-94, 1986). In addition, the use of 
fluorocarbons is limited to situations in which high partial pressures of 
oxygen can be administered. They do not have a sufficiently high oxygen 
binding capacity for use under normal physiological conditions. 
Native isolated hemoglobin, when used as a blood substitute, has a number 
of disadvantages. Firstly, large dosages are required (Walder, Biotech 
'88, San Francisco, Nov. 14-16, 1988). A single unit (450 ml) of a 10% 
hemoglobin solution contains 45 g of protein. Since it is estimated that 
ten million units of blood are used in the U.S. per year, the production 
of 450,000 kg of hemoglobin would be required. Secondly, as cited 
previously, the potential exists for contamination of the isolated 
hemoglobin by any number of infectious agents. Thirdly, although 
hemoglobin is normally a tetramer of 64,000 molecular weight, it can 
dissociate to form alpha-beta dimers which are rapidly cleared by the 
kidneys, therefore lowering the effective residence time of functional 
hemoglobin in the body. Fourthly, cell-free hemoglobin has too high an 
oxygen affinity to effectively release oxygen to the tissues due to the 
absence of 2,3 diphosphoglycerate (2,3 DPG). Efforts to restore 2,3 DPG 
have been unsuccessful since 2,3 DPG is rapidly cleared from the 
circulation (Snyder and Walder, "Chemically Modified and Recombinant 
Hemoglobin Blood Substitutes" in Biotechnology of Blood, 
Butterworth-Heinemann, pages 101-116, 1991). Finally, cell-free hemoglobin 
has been shown to act as a scavenger of nitric oxide in the body, a 
property which results in a vasoconstrictive effect on blood vessels 
(Kilbourn, R., et al., Biochem. Biophys. Res. Comm. 199:155-162, 1994). 
This vasoactivity may compromise the utility of cell-free hemoglobin in 
certain clinical conditions. 
2.2 HUMAN SERUM ALBUMIN 
Human serum albumin, a protein of 585 amino acids with a molecular weight 
of 66,500 daltons, is the most abundant protein in human plasma. It 
comprises 60% of the total protein, with a normal concentration of 42 
g/liter. It provides 80% of the osmotic pressure of blood, and is a very 
stable and soluble protein. It serves as a transport carrier for a variety 
of ligands, including fatty acids, amino acids, steroids, ions, and 
pharmaceuticals. It is able to shepherd hydrophobic ligands throughout the 
body. It was one of the first proteins to be crystallized, and the 
standard purification protocol was developed by Cohn in 1946 (Peters, 
"Serum Albumin", in Advances in Protein Chemistry, Academic Press, 1985). 
The three-dimensional structure of human serum albumin was determined by 
X-ray crystallography to a resolution of 2.8 .ANG.. Three homologous 
domains were identified and the principal ligand binding sites were 
localized (He and Carter, Nature 358:209-215, 1992; Carter and Ho, Adv. 
Protein Chem. 45:153-203, 1994). 
2.3 HEM 
Heme is a porphyrin in which a central iron atom is coordinately bound to 
the four pyrrole nitrogen atoms of the porphyrin ring. The physiological 
oxygen carriers, hemoglobin and myoglobin, contain a heme moiety that is 
the site of oxygen binding. Oxygen binds to the iron atom of free heme; 
however, only the ferrous (FeII) form of heme can bind oxygen. The binding 
of oxygen can rapidly lead to oxidation of the iron atom, creating ferric 
(FeIII) heme, which cannot bind oxygen. This oxidation reaction, 
therefore, has to be circumvented in order to optimize the capacity of the 
heme to reversibly bind oxygen for long periods. The three dimensional 
structure of the polypeptide moieties of myoglobin and hemoglobin result 
in a protective enclosure for the heme. This structure prevents the 
oxidation reaction which would occur upon the binding of oxygen by 
preventing the formation of an intermediate in this reaction, a sandwich 
dimer of two hemes with oxygen. Thus, the design of these molecules 
maximizes the oxygen binding capability of their heme moieties (Stryer, 
Biochemistry, Chapter 4, pp. 65-85, W. H. Freeman and Co., New York, 
1981). 
Efforts to modify hemes in order to design compounds which mimic the oxygen 
binding potential of the native heme in its hemoglobin or myoglobin pocket 
have resulted in the development of many porphyrin derivatives that have 
been tested for their ability to bind oxygen and exhibit resistance to 
oxidation (Traylor and Traylor, Ann. Rev. Biophys. Bioeng. 11:105-127, 
1982). Such porphyrin derivatives include capped porphyrins (Almog et al., 
J. Am. Chem. Soc. 97:226-227, 1975; Rose et al., Proc. Natl. Acad. Sci. 
79:5742-5745, 1982) and basket-handle porphyrins (Lexa et al., J. Am. 
Chem. Soc. 106:4755-4765, 1984) 
Picket fence porphyrin is such a modified heme. It was designed as a model 
compound to mimic the oxygen binding site of myoglobin and hemoglobin. 
Four axial bases are covalently attached to the porphyrin ring, 
effectively creating a protective enclosure for bound oxygen due to the 
great steric bulk provided (Collman et al., Proc. Natl. Acad. Sci. 
75:1052-1055, 1978). When oxygen binds to the iron atom, five of the six 
coordination positions of this molecule are then occupied. As a result, 
formation of the intermediate in the oxidation reaction is prevented by 
the steric design of the molecule, and oxidation is prevented. Efforts to 
optimize the picket-fence structure currently continue through the use of 
molecular modeling (Wuenschel et al., J. Am. Chem. Soc. 114:3346-3355, 
1992). 
Liposome-bound picket fence porphyrins were shown to bind CO with highs 
affinity, but were subject to autoxidation, and no stable oxygenated forms 
were observed (Makino et al., Biochem, and Biophys. Res. Comm. 
108:1010-1015, 1982); these complexes were later shown to reversibly bind 
oxygen (Tsuchida et al., J. Chem. Soc. Dalton Trans., p. 1147-1151, 1984). 
2.4 ALBUMIN/PORPHYRINS 
Human serum albumin normally binds free heme in the body (heme-HSA); it 
acts as a scavenger for surplus heme released in hemorraghic conditions. 
The complex heme-HSA normally oxidizes to metheme-HSA. The resultant 
complex, methemalbumin, can be diagnostic for internal hemorrhage (Peters, 
supra). 
Albumin, with its known affinity for porphyrins, has been studied to 
determine the effectiveness of this protein carrier for the delivery of 
hydroxyethyl vinyl deuteroporphyrin and irreversible porphyrin aggregates 
in photodynamic therapy of tumors (Cohen and Margalit, J. Biochem. 
270:325-330, 1990). Certain albumin-porphyrin compounds have also been 
developed as anti-HIV agents, in which the porphyrin derivatives include 
hemin, proto-porphyrin, meso-porphyrin, iron meso-porphrin, 
hemato-porphyrin, iron hematoporphyrin, deutero-porphyrin copper 
chlorophyllin (International Publication No. WO 9303035 published Feb. 18, 
1993). 
Heme-HSA has never been shown to reversibly bind oxygen. The heme moiety is 
presumably not configured into the protein in such a way that it is 
shielded from the oxidation reaction that would occur if oxygen binds. 
However, optimization of human serum albumin as an oxygen carrier is 
provided by the HSA-porphyrin compounds of the present invention, in which 
formation of a complex between HSA and a suitable oxygen-binding moiety 
produces a mobile oxygen carrier which reversibly binds oxygen and thus 
can be used as a blood substitute. 
SUMMARY OF THE INVENTION 
The invention is directed to compositions comprising human serum 
albumin-porphyrin (HSA-P) complexes, methods for their production, and the 
use of these molecules as blood substitutes. The various porphyrins 
provided by the invention can bind oxygen reversibly, and they can be used 
to transport and deliver oxygen when bound to an HSA carrier. The 
invention further provides HSA-P complexes which do not exhibit 
vasoactivity. The invention also provides various modified porphyrins, 
including picket-fence porphyrin, as the oxygen-binding moiety in the 
HSA-P complex. 
The invention is illustrated by means of examples in which methods are 
given for the synthesis of HSA-P, and by examples in which the 
oxygen-carrying capacity of the HSA-P is demonstrated, and by an example 
in which the lack of vasoactivity of the HSA-P of the present invention is 
illustrated. 
BRIEF DESCRIPTION OF THE FIGURES 
FIG. 1. Structure of picket fence porphyrin #1: Fe meso-tetra (a, a, 
a,a-pivalamido-phenyl) porphine. 
FIG. 2. Structure of picket fence porphyrin #2: Fe meso-tetra 
(a,a,a,o-pivalamido-phenyl) porphine. 
FIG. 3. Absorption spectra of HSA-PFP #1 in the wavelength region from 450 
to 700 nm. Optical pathlength was 1 cm. The solvent was DMSO made 1 Mm in 
M-methylimidazole. The nominal concentration of #1 was 55 .mu.M. 
FIG. 4. Absorption spectra of the various forms of the HSA-PFP #1 with 
human serum albumin. Curve 1 corresponds to the carbon monoxide 
derivative; curve 2 to the oxygenated derivative and curve 3, the 
deoxygenated derivative. The pathlength was 1 cm. 
FIG. 5. Absorption spectra showing the raw data from an oxygen equilibrium 
experiment with 5 .mu.M HSA-PFP #1 complex. The complex was in 50 Mm 
Bis-Tris, pH 7.0 at 20.degree. C. The deoxygenated derivative has the 
highest molar absorptivity at 422 nm and the oxygenated derivative has the 
lowest absorptivity at 422 nm. Each intermediate spectrum corresponds to 
the partially oxygenated derivative at different oxygen concentrations. 
FIG. 6. The percent oxygen saturation of human hemoglobin A and HSA-PFP #1 
as a function of oxygen concentration (pO.sub.2 in mm Hg) is shown. 
--.quadrature.--HSA-PFP#1; --.diamond-solid.--: hemoglobin A. 
FIG. 7. Hill plots for 5 mM HSA-PFP #1 and human hemoglobin A in 50 mM 
Bis-Tris, pH 7.0 at 20.degree. C. using data derived from FIG. 6. 
--.quadrature.-- HSA-PFP#l; --.diamond-solid.--: hemoglobin A. 
FIGS. 8a-d Absorption spectra of HSA-PFP #2 cycling between the oxygenated 
(O.sub.2) (1) and deoxygenated (N.sub.2) (2) derivatives. 
FIG. 9. Effects of bovine cell-free hemoglobin (Hgb), picket-fence 
porphyrin human serum albumin (HSA-PFP) (#1) and heme-HSA in the 
concentration-contraction curves evoked by phenylephrine in rat aorta ring 
with endothelium, incubated for 4 hours in culture medium containing 
endotoxin (LPS), 200 ng/ml. Results are presented as mean +/- SEM of 4 
different experiments. --.quadrature.--: LPS treated; --.DELTA.--: 
LPS+10.mu.l/ml hemoglobin; --.smallcircle.-- LPS+20 .mu.l/ml HSA-PFP#2; 
--.gradient.--: LPS +20 .mu.l/ml HSA-heme.

5. DETAILED DESCRIPTION OF THE INVENTION 
The invention is directed to compositions comprising human serum 
albumin-porphyrin (HSA-P) complexes, methods for their production, and the 
use of these molecules as blood substitutes. The various porphyrins 
provided by the invention can bind oxygen reversibly, and they can be used 
to transport and deliver oxygen when bound to an HSA carrier. The 
invention further provides HSA-P complexes which do not exhibit 
vasoactivity. The invention also provides various modified porphyrins, 
including picket-fence porphyrin, as the oxygen-binding moiety in the 
HSA-P complex. 
The invention is illustrated by means of examples in which methods are 
given for the synthesis of HSA-P complexes, and by examples in which the 
oxygen-carrying capacity of the HSA-P complexes is demonstrated, and by an 
example in which the lack of vasoactivity of the HSA-P complexes of the 
present invention is illustrated. 
5.1 Preparation of MSA-P 
The invention provides human serum albumin-porphyrin (HSA-P) complexes. 
These are hybrid molecules that complex HSA with an oxygen-binding moiety. 
These HSA-P complexes provide several advantages over the prior art. 
First, the biocompatibility of human serum albumin (which is already used 
as a plasma expander and resuscitation fluid) allows the HSA-P complex to 
readily transport to and access numerous tissues and organs. Secondly, in 
a HSA-P complex preferred for use, such HSA-P complex does not have the 
vasoconstrictive potential of hemoglobin, i.e., the HSA-P complex does not 
promote high blood pressure. It is shown herein that an HSA-P complex does 
not have the vasoconstrictive potential of hemoglobin (Example 10, infra), 
a side effect which can limit its clinical effectiveness. 
Thus, in a preferred aspect, the invention provides for HSA-P complexes 
which are capable of reversible cycles of oxygenation and deoxygenation, 
and which does not exhibit significant vasoactivity. 
HSA-P complexes of the present invention may be screened for oxygen binding 
by any method known in the art, e.g., tonometry (Riggs and Wolbach, J. 
Gen. Physiol. 39:585-605, 1956) (see Example 7, infra) in order to 
determine the degree of saturation of the porphyrin moiety as a function 
of the oxygen partial pressure. This data can be extrapolated to a Hill 
Plot which allows for an assessment of the cooperativity of oxygen 
binding. 
HSA-P complexes of the present invention may be screened for reversibility 
of the oxygen binding by any method known in the art, e.g., 
spectrophotometrically by successive cycles of incubation with oxygen, 
followed by repeated vacuum evacuations with nitrogen purges (see Example 
8, infra). Characteristic changes in absorption that indicate oxy- and 
deoxy- complexes at a particular wavelength may be used to determine the 
potential of an HSA-P for use as an oxygen carrier. 
In a preferred embodiment, the porphyrin in the HSA-P complex is picket 
fence porphyrin (PFP). As described in Section 2 above, PFP is a modified 
heme. By virtue of its structure, this molecule can be used to provide an 
oxygen-binding moiety to an exogenous protein. 
A HSA-P complex of the invention is that containing a PFP that is a 
compound of the following formula: 
##STR1## 
wherein 
M is Co, Fe, or Mn; 
R.sup.1 is C(CH.sub.3).sub.3 or (CH.sub.2).sub.n C.sub.6 H.sub.4 R.sup.2 ; 
R.sup.2 is H, CN, NO.sub.2, CO-phenyl, halogen, CF.sub.3, NHCOR.sup.3, 
CO.sub.2 R.sup.3, OR.sup.3, SO.sub.2 N(R.sup.3).sub.2, NR.sup.4 R.sup.5, 
or SO.sub.2 R.sup.6 ; 
R.sup.3 is H, C.sub.1 C.sub.6 alkyl or phenyl; 
R.sup.4 and R.sup.5, independently are H or C.sub.1 -C.sub.4 alkyl; 
R.sup.6 is C.sub.1 -C.sub.6 alkyl or phenyl; and 
n is 0-8. 
The invention provides various picket fence porphyrins that retain an 
oxygen binding capacity and are capable of binding the human serum albumin 
and serving as a mobile oxygen carrier. 
Human serum albumin may be prepared by any method known in the art such as 
purification from a natural source (including purification by 
polyacrylamide gel electrophoresis, immunoprecipitation or affinity 
chromatography), chemical synthesis, and recombinant DNA technology. The 
technique of Cohn involving successive cycles of precipitation from plasma 
to yield the 98% pure Fraction V albumin may be employed (Cohn et al., J. 
Am. Chem. Soc. 68:459-475, 1946). Recombinant DNA techniques may be used 
with the cloned gene (Hawkins and Dugaiczyk, Gene 19: 55-58, 1982) to 
express the recombinant protein in bacteria or any of other known 
expression systems in the art (see Current Protocols in Molecular Biology, 
Ausubel, F., et al., eds., Wiley and Sons, 1987). Alternatively, the 
protein may be purchased from known suppliers including Cutter 
Laboratories (Dallas, Texas), Abbott Laboratories (North Chicago, Ill.), 
and Sigma Chemical Company (St. Louis, Mo.). 
The porphyrins in the HSA-P complexes of the instant invention may be made 
by methods previously described (Collman et al., J. Am. Chem. Soc. 
97:1427-1439, 1975). Condensation of the appropriately substituted 
nitrobenzaldehyde with pyrrole yields a tetra-(nitrophenyl)porphyrin. 
Reduction of the nitro group using excess tin chloride in concentrated 
mineral acid yields the corresponding amine. Condensation of the 
tetra-amine with the appropriate acid chloride affords the substituted 
porphyrins of the instant invention. The requisite acid chlorides are 
either commercially available (Aldrich Chemical Co.) or can be made by 
standard methods from the corresponding carboxylic acids and thionyl 
chloride. Standard protecting groups may be necessary to prepare some of 
the requisite acid chlorides. Removal of the protecting groups can be 
affected under standard conditions following condensation of the amine 
with acid chloride. 
HSA-P complexes derivatives may be prepared by reacting a porphyrin, in a 
specific embodiment a picket-fence porphyrin, with carbon monoxide (CO) to 
form CO-PFP. This complex is then further reduced with dithionite. The 
CO-PFP is then mixed with HSA, and formation of a complex with HSA can be 
assessed by chomatography and ultrafiltration. Removal of the CO is 
accomplished by illumination of the sample with light in a tonometer with 
oxygen, yielding an 0.sub.2 -HSA-PFP complex. The oxygen can be removed 
with nitrogen, leaving the HSA-PFP complex. 
The invention also provides for HSA-P complexes in which the HSA is in the 
form of multimers, which in a preferred embodiment may be a dimer formed 
by the creation of a disulfide bond between HSA monomers, and which may 
prevent extravasation of the HSA-P from the circulation. Such dimers can 
be formed by the addition of mercuric chloride to a solution of HSA 
monomers, which causes the thiol-containing albumin to dimerize through a 
mercury bridge (Hughes and Dintzis, J. Biol. Chem. 239:845-849, 1964). 
Subsequent oxidation of this HSA dimer by treatment with iodine results in 
a formation of a disulfide bond between the cysteines to form a disulfide 
dimer (Straessle, J. Am. Chem. Soc. 76:3138-3142, 1954). HSA disulfide 
dimers may also be prepared by oxidation of HSA monomers with ferricyanide 
(Andersson, Biochem. et Biophys. Acta 117:115-133, 1966) or by oxidation 
at alkaline pH in the presence of oxygen. HSA multimers of the instant 
invention can also be prepared by crosslinking with any of known reagents 
in the art, including carbodiimide and glutaraldehyde. 
HSA-P complexes of the present invention may be screened for oxygen binding 
by any method known in the art, e.g., tonometry (Riggs and Wolbach, J. 
Gen. Physiol. 39:585-605, 1956) (see Example 7, infra) in order to 
determine the degree of saturation of the porphyrin moiety as a function 
of the oxygen partial pressure. This data can be extrapolated to a Hill 
Plot which allows for an assessment of the cooperativity of oxygen 
binding. 
HSA-porphyrins of the present invention may be screened for reversibility 
of the oxygen binding by any method known in the art, e.g., 
spectrophotometrically by successive-cycles of incubation with oxygen, 
followed by repeated vacuum evacuations with nitrogen purges (see Example 
8, infra). Characteristic changes in absorption that indicate oxy- and 
deoxy- complexes at a particular wavelength may be used to determine the 
potential of an HSA-P complex for use as an oxygen carrier. 
HSA-porphyrins of the present invention may be screened for vasoactivity by 
any method known to those skilled in the art, including but not limited to 
the use of in vitro models such as the phenylephrine-evoked contraction of 
endothelium (See Example 10, infra). 
5.2. Utilities of the Invention 
The HSA-P compositions of the present invention may be used as blood 
substitutes or as a blood plasma expander, in a pharmaceutical composition 
with an acceptable carrier, and with other plasma expanders, or in any 
application where a physiological oxygen carrier is needed. The 
pharmaceutical carriers may be such physiologically compatible buffers as 
Hank's or Ringer's solution, physiological saline, a mixture consisting of 
saline and glucose, and heparinized sodium-citrate-citrate acid-dextrose 
solution. The HSA-P complexes produced by the methods of the present 
invention can be mixed with colloidal-like plasma substitutes and plasma 
expanders such as linear polysaccharides (e.g., dextran), hydroxyethyl 
starch, balanced fluid gelatin, and other plasma proteins. Additionally, 
the HSA-PFP may be mixed with water soluble, physiologically acceptable, 
polymeric plasma substitutes, examples of which include polyvinyl alcohol, 
poly (ethylene oxide), polyvinylpyrrolidone, and ethylene 
oxide-polypropylene glycol condensates. Techniques and formulations for 
administering the compositions comprising the HSA-P complexes generally 
may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., 
Easton, Pa., latest edition. 
Pharmaceutical compositions for use in accordance with the present 
invention may be formulated in conventional manner using one or more 
physiologically acceptable carriers or excipients. 
The compounds may be formulated for administration by injection, e.g., by 
bolus injection or continuous infusion. Formulations for injection may be 
presented in unit dosage form, e.g., in ampoules or in multi-dose 
containers, with an added preservative. The compositions may take such 
forms as suspensions, solutions or emulsions in oily or aqueous vehicles, 
and may contain formulatory agents such as suspending, stabilizing and/or 
dispersing agents. Alternatively, the active ingredient may be in powder 
form for constitution with a suitable vehicle, e.g., sterile pyrogen-free 
water, before use. 
The compositions may, if desired, be presented in a pack or dispenser 
device which may contain one or more unit dosage forms containing the 
active ingredient. The pack may for example comprise metal or plastic 
foil, such as a blister pack. The pack or dispenser device may be 
accompanied by instructions for administration. 
Toxicity and therapeutic efficacy of such compounds can be determined by 
standard pharmaceutical procedures in cell cultures or experimental 
animals, e.g, for determining the LD.sub.50 (the dose lethal to 50% of the 
population) and the ED.sub.50 (the dose therapeutically effective in 50% 
of the population). The dose ratio between toxic and therapeutic effects 
is the therapeutic index and it can be expressed as the ratio LD.sub.50 
/ED.sub.50. Compounds which exhibit large therapeutic indices are 
preferred. 
The data obtained from animal studies can be used in formulating a range of 
dosage for use in humans. The dosage of such compounds lies preferably 
within a range of circulating concentrations that include the ED50 with 
little or no toxicity. The dosage may vary within this range depending 
upon the dosage form employed and the route of administration utilized. 
For any compound used in the method of the invention, the therapeutically 
effective dose can be estimated initially from cell culture assays. A dose 
may be formulated in animal models to achieve a circulating plasma 
concentration range that includes the IC50 (i.e., the concentration of the 
test compound which achieves a half-maximal inhibition of symptoms). Such 
information can be used to more accurately determine useful doses in 
humans. 
Subjects for treatment with the compounds of the present invention include 
humans and other mammals such as monkeys, chimpanzees, rodents, pigs, 
cows, horses, dogs, cats, and particularly primates. 
6. EXAMPLE: PREATION OF HUMAN-SERUM ALBUMIN-PICKET FENCE PORPHYRINS 
A 500 .mu.M stock solution of Fe meso-tetra (a,a,a,a-pivalamido-phenyl) 
porphine (PFP #1) (FIG. 1) or Fe meso-tetra (a,a,a,o-pivalamido-phenyl) 
porphine (PFP#2) (FIG. 2) (both purchased from Porphyrin Products, Provo, 
Utah) was prepared in 100% dimethyl sulfoxide (DMSO) containing 1 mM 
N-methylimidazole. 
The spectrum of this material (60 .mu.M) (PFP#2) after degassing, is shown 
in FIG. 3, spectrum 1. Addition of carbon monoxide (CO) results in 
spectrum 2, indicating that the PFP is partially reduced (Fe.sup.2+). 
Addition of dithionite further reduces the PFP (spectrum 3). The CO-PFP 
was mixed with HSA (Albumin-USP 25%; Cutter Pharmaceuticals, Dallas, Tex.) 
in 50 mM Bis-Tris pH 7 (3 ml PFP (60 .mu.M) with 7.5 ml HSA (14.5 .mu.M); 
measured PFP/HSA ratio (Fe/protein) was 0.9. The spectrum of the mixture 
is still indicative of CO-PFP. The mixture was concentrated on an Amicon, 
PM-30 membrane (no color in filtrate) and then passed through a Sephadex 
G-25 column. The spectrum of the fractions that contain HSA is shown in 
FIG. 4, spectrum 1. It is evident that a CO-PFP-HSA complex has formed and 
that the complex is stable (i.e., remains in the reduced state in aqueous 
solution). Removal of CO through illumination of the sample with bright 
light, in a tonometer filled with oxygen, results in a O.sub.2 -PFP-HSA 
complex (spectrum 2). The oxygen can be removed by nitrogen (spectrum 3). 
The S PFP/HSA ratio after Sephadex was 0.65. 
7. EXAMPLE: ABSORPTION SPECTRA OF OXYGEN BINDING BY THE HSA-PFP COMPLEX #1 
A 5 .mu.M PFP-CO/HSA complex was prepared in 50 mM Bis-Tris pH 7.0. This 
solution was then pipetted into a tonometer, treated with N.sub.2, and 
rotated in a water bath at 25.degree. C. for 10 minutes under a lamp to 
allow for equilibration. This was followed by the addition of 1 ml of 
O.sub.2 (5.75 .mu.M) and subsequent equilibration for 5 minutes as above 
except in the absence of the lamp. The oxygen titrations were monitored 
spectrophotometrically and resulted in FIG. 5. 
The family of curves shown in FIG. 5 show varying degrees of saturation of 
HSA-PFP #1 as a function of oxygen concentration. Although the shape and 
position of the absorption peaks differ from those of hemoglobin, the 
family of spectra seen in FIG. 5 are qualitatively the same as one would 
see in an oxygen equilibrium experiment done with hemoglobin. 
8. EXAMPLE: OXYGEN BINDING OF HSA-PFP #1 
The P.sub.50 for HSA-PFP #1 and human Hemoglobin A (HbA) were determined by 
tonometry (Riggs and Wolbach, 1956, J. Gen. Physiol. 39:585-60.5). 
Specifically, a HSA-PFP solution was placed in a gas-tight vessel which 
has an attached spectrophotometer cell. The solution was deoxygenated by a 
series of repeated vacuum evacuations followed by nitrogen purges. After 
the deoxygenated state was obtained, a "deoxy" spectrum was obtained. 
Next, a series of metered oxygen additions were made with a spectrum taken 
after each addition yielding a set of curves from which can be calculated 
(using established extinction coefficients) the degree of saturation of 
the heme sites with oxygen as a function of the oxygen partial pressure 
(FIG. 6). The percent oxygen saturation of human HbA and HSA-PFP #11 as a 
function of oxygen concentration is shown. The oxygen affinity (P.sub.50) 
of HbA is high (2 mm Hg) relative to that of HSA-PFP #1 (10 mm Hg). This 
data is transformed into the Hill plot shown in FIG. 7. The degree of 
cooperativity of HbA is high (n50=2.7) relative to that of HSA-PFP #1 
n50=l.6). 
9. EXAMPLE: OXYGENATION CYCLES FOR HSA-PFP #2 
HSA-PFP #2 was deoxygenated in a tonometer as described in Example 7. A 
deoxy spectrum was obtained. The oxygenated form of the molecule was 
obtained by adding pure O.sub.2 to the tonometer. The oxygen spectrum was 
obtained. 
FIG. 8 (a-d) show four successive cycles of deoxygenation/oxygenation. The 
decrease in the difference spectra for 8(a-d) can be attributed to 
autoxidation of the Fe.sup.2+ to Fe.sup.3+ in the HSA-PFP #2 complex. The 
Fe.sup.3+ derivative cannot bind oxygen. 
10. EXAMPLE: VASOACTIVITY EXPERIMENT FOR HSA-PFP #1 
Male Wistar rats (300-400 g) were euthanized by intraperitoneal injection 
of sodium pentobarbitol (50 mg/kg). The thoracic aortas were excised and 
stored in cold modified Krebs-Ringer solution containing NaCl 118.3 mM, 
KCl 4.7 mM, MgSO.sub.4 1.2 mM, KH.sub.2 PO.sub.4 1.2 mM, CaCl.sub.2 2.5 
mM, NaHCO.sub.3 25 mM, Ca EDTA 16 .mu.M, and glucose 11.1 mM (control 
solution). Arteries were cleared of fat and connective tissue and cut into 
rings. For some experiments the endothelium was removed mechanically by 
placing rings on filter paper wetted with the control solution, inserting 
the tip of a forceps into the lumen, and rolling the ring back and forth 
on the filter paper. The presence of the endothelium was confirmed by 
determining the relaxation to acetylcholine (10.sup.-6 M) in arteries 
contracted with phenylephrine (10.sup.-6 M). The rings were placed in 
24-well multiwell plates with Dulbecco's Modified Eagle's Medium and Ham's 
F-12 Medium (DMEM/F12) (1 ml), in the presence or absence of endotoxin 
(LPS) 200 ng/ml, for 4 hours. After incubation, the rings were suspended 
in organ chambers containing 10 ml of control solution (37.degree. C., pH 
7.4) and aerated with 95% O.sub.2 and 5% CO.sub.2. Rings were stretched 
progressively to 2.5 to 3 g of tension. Changes in isometric tension were 
recorded with a force transducer connected to an analog-to-digital input 
board (Scientific Solutions, Inc., Solon, Ohio) in an IBM 386/30 mHz 
personal computer. The aortic rings were rinsed three times with warm 
control solution, rested for 30 minutes, and the incubated with bovine 
cell-free Hgb, Heme-HSA, or HSA-PFP for 5 minutes before a 
concentration-contraction curve to phenylephrine (10.sup.-9 to 10.sup.-5 
M) was obtained. 
FIG. 9 shows that the HSA-PFP #1 at 5 mg/ml had no significant effect on 
phenylephrine-evoked contraction of rat aortic rings treated with 
endotoxin. 
The present invention is not to be limited in scope by the specific 
embodiments described herein. Indeed, various modifications of the 
invention in addition to those described herein will become apparent to 
those skilled in the art from the foregoing description and accompanying 
figures. Such modifications are intended to fall within the scope of the 
appended claims. Various publications are cited herein, the disclosures of 
which are incorporated by reference in their entireties.