Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (O2 ) carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration purification process to remove almost all undesirable Hb species (>0.2 µm and <500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Structure-activity relationship study on new hemorphin-4 analogues containing steric restricted amino acids moiety for evaluation of their anticonvulsant activity.

In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.

Potential anticonvulsant activity of novel VV-hemorphin-7 analogues containing unnatural amino acids: synthesis and characterization.

Herein, some new analogues of VV-hemorphin-7, modified at position 4 and 7 by the unnatural amino acids followed the structure Val-Val-Tyr-Xxx-Trp-Thr-Yyy-Arg-Phe-NH2, where Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) and Yyy is Dap (diaminopropanoic acid) or Dab (diaminobutanoic acid), were synthesized, characterized and investigated for anticonvulsant activity. The new synthetic peptide analogues were prepared by standard solid-phase peptide synthesis-Fmoc chemistry. A single intracerebroventricular (i.c.v.) injection at doses of 5, 10, and 20 µg/10 µl, respectively, was given before evaluation with timed intravenous pentylenetetrazole (ivPTZ) infusion test and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. To explain the structure-active properties of the modified peptides, some physicochemical characteristic was obtained. The FT-IR spectra and their second derivatives of the amide I, II, and III bands of the peptides show ß-sheet structure conformation. The calculation of isoelectric points, by potentiometric determination of dissociated constants, is in the range from 9.79 to 10.84. This study, for the first time, also reported on the reduction-oxidative potentials of the guanidine at Arg-moiety on such kind of peptides containing arginine and tyrosine residues in different medium and electrode surface. The VV-hemorphin-7 analogues 4 and 5 were the most active against the ivPTZ test, with the effect comparable to that of peptide 1 used as a positive control. Except compound 8, all other tested peptide analogues were ineffective to raise the threshold for the clonic seizures. The peptide analogue 5 showed 100% protection in the 6-Hz test, while the other seven VV-hemorphin-7 analogues have dose-dependent activity against psychomotor seizures comparable to 1. The novel peptides did not show neurotoxicity in the rotarod test.

Synthesis, characterization and anticonvulsant activity of new series of N-modified analogues of VV-hemorphin-5 with aminophosphonate moiety.

A new series of N-modified analogues of the VV-hemorphin-5 with aminophosphonate moiety have been synthesized, characterized and investigated for anticonvulsant activity. The novel peptide analogues were prepared by solid-phase peptide synthesis-Fmoc-strategy and were evaluated in the timed intravenous pentylenetetrazole infusion test (ivPTZ) and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. The redox potentials at glass carbonic electrode (GC) and the acid/base properties as pKa values of these peptide analogues were compared with the electrochemical behaviour of tyrosine- and tryptophan-containing peptides using different voltamperometric modes. Among the five tested peptide analogues, V3p was the most active against the ivPTZ test with effect comparable to that of the VV-hemorphin-5 (V1) used as a positive control. Dose-dependent elevation of the seizure threshold for myoclonic twitch and generalized clonic seizures was observed after i.c.v. administration of V2p, V4p and V5p as well as for forelimbs tonus in V4p peptides. The peptide analogues V2p-V5p were able to suppress dose-dependent psychomotor seizures in the 6-Hz test. In contrast, the V6p peptide showed either a pro-convulsant effect in the iv PTZ test or was inactive in the 6-Hz test. No changes in motor coordination were noted with the novel peptides. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of aminophosphonate moiety at position 1 of the VV-hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.

Anticonvulsant evaluation and docking analysis of VV-Hemorphin-5 analogues.

VV-Hemorphin-5 is an endogenous opioid peptide of the Hemorphin family with affinity at opioid receptors. A series of C-amide analogues have been synthesized, based on the structure of VV-Hemorphin-5, modified at position 1 and 7 by the un/natural amino acids (Aa8-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Aa1-NH2 ) using SPPS, Fmoc-chemistry. The peptide derivatives were evaluated for their anticonvulsant activity in three acute seizure tests in male ICR mice, the maximal electroshock (MES), the 6 Hz psychomotor seizure test, and the timed intravenous pentylenetetrazole (ivPTZ) infusion test. Their neurotoxicity was assessed in the rotarod test. Among the tested peptide analogues, V4 showed anticonvulsant activity in the three seizure tests that was comparable to the VV-Hemorphin-5 (V1) used as a positive control. While V5, V6, and V7 peptide derivatives exhibited anticonvulsant activity in the MES and 6 Hz test, they were inactive (V7) or showed pro-convulsant effect (V5 and V6) in the i.v. PTZ test. At a dose of 10 µg/mouse the peptide V2 was effective against clonic seizures induced by PTZ. Motor coordination was not affected by newly developed analogues of VV-Hemorphin-5. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of nonproteinogenic and/or natural amino acids at position 1 and 7 of the VV-Hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.

Synthesis, characterization and nociceptive screening of new VV-hemorphin-5 analogues.

In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH2, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.

Mixtures of tense and relaxed state polymerized human hemoglobin regulate oxygen affinity and tissue construct oxygenation.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

HemoCD as a Totally Synthetic Artificial Oxygen Carrier: Improvements in the Synthesis and O2 /CO Discrimination.

HemoCD, which is composed of an iron(II)porphyrin such as 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) (Fe(II)TPPS) and a cyclodextrin (CD) dimer having a pyridine linker, represents a synthetic hemoglobin (Hb) model compound that exhibits reversible oxygen (O2 ) binding ability in aqueous solution at an ambient temperature. Therefore, hemoCD has the potential to be used as a totally synthetic artificial oxygen carrier. In this article, we describe the improvements of hemoCD related to its synthesis and O2 /CO selectivity. The synthesis procedure of the CD dimer of hemoCD was re-examined, and the CD dimer was successively synthesized from inexpensive ß-CD with a 38% yield (three-steps), which enabled us to obtain the CD dimer in gram-quantities. The O2 /CO selectivity of hemoCD was also markedly improved using an iron(II)porphyrin having a carboxylate group at the distal site of hemoCD.

"Inside-Out" PEGylation of Bovine ß-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to ß-cross-linked (ßXL-Hb) hemoglobin (Hb) tetramers through the Cys ß93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three ßXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50  = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50  = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Strain-promoted azide-alkyne cycloaddition for protein-protein coupling in the formation of a bis-hemoglobin as a copper-free oxygen carrier.

Conventional chemical approaches to protein-protein coupling present challenges due to the intrinsic competition between the desired interactions of reagents with groups of the protein as well as reactions with water. Biorthogonal Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC)-processes provide a basis to direct reactivity without functional group interference. However, the requirement for Cu(i) in CuAAC leads to complications that result from the metal ion's interactions with the protein. In principle, a similar but metal-free alternative approach to coupling could employ the reaction of an alkyne that is strained in combination with an azide (strain-promoted azide-alkyne cycloaddition, SPAAC). The method is exemplified by the combination of a cyclooctyne derivative of hemoglobin with an azide-modified hemoglobin. The bis-hemoglobin tetramer that is produced has properties consistent with those sought for use as a hemoglobin-based oxygen carrier (HBOC).

Synthesis of the Hemoglobin-Conjugated Polymer Micelles by Thiol Michael Addition Reactions.

Amphiphilic triblock copolymers mPEG-b-PMAC-b-PCL are synthesized using methoxyl poly(ethylene glycol), cyclic carbonic ester monomer including acryloyl group, and ε-caprolactone. Copolymers are self-assembled into core-shell micelles in aqueous solution. Thiolated hemoglobin (Hb) is conjugated with micelles sufficiently through thiol Michael addition reaction to form hemoglobin nanoparticles (HbNs) with 200 nm in diameter. The conjugation of Hb onto the micelle surface is further confirmed by X-ray photoelectron spectroscopy. Feeding ratio of copolymer micelles to Hb at 1:3 would lead to the highest hemoglobin loading efficiency 36.7 wt%. The UV results demonstrate that the gas transporting capacity of HbNs is well remained after Hb is conjugated with polymeric micelles. Furthermore, the obtained HbNs have no obvious detrimental effects on blood components in vitro. This system may thus have great potential as one of the candidates to be developed as oxygen carriers and provide a reference for the modification of protein drugs.

Enhancing actions of peptides derived from the γ-chain of fetal human hemoglobin on the immunostimulant activities of monophosphoryl lipid A.

Hemoglobin and its structures have been described since the 1990s to enhance a variety of biological activities of endotoxins (LPS) in a dose-dependent manner. To investigate the interaction processes in more detail, the system was extended by studying the interactions of newly designed peptides from the γ-chain of human hemoglobin with the adjuvant monophosphoryl lipid A (MPLA), a partial structure of lipid A lacking its 1-phosphate. It was found that some selected Hbg peptides, in particular two synthetic substructures designated Hbg32 and Hbg35, considerably increased the bioactivity of MPLA, which alone was only a weak activator of immune cells. These findings hold true for human mononuclar cells, monocytes and T lymphocytes. To understand the mechanisms of action in more detail, biophysical techniques were applied. These showed a peptide-induced change of the MPLA aggregate structure from multilamellar into a non-lamellar, probably inverted, cubic structure. Concomitantly, the peptides incorporated into the tightly packed MPLA aggregates into smaller units down to monomers. The fragmentation of the aggregates was an endothermic process, differing from a complex formation but rather typical for a catalytic reaction.

Investigation of receptor binding and functional characteristics of hemopressin(1-7).

The orally active, α-hemoglobin derived hemopressin (PVNFKFLSH, Hp(1-9)) and its truncated (PVNFKFL, Hp(1-7) and PVNFKF, Hp(1-6)) and extended ((R)VDPVNFKFLSH, VD-Hp(1-9) and RVD-Hp(1-9)) derivatives have been postulated to be the endogenous peptide ligands of the cannabinoid receptor type 1 (CB1). In an attempt to create a versatile peptidic research tool for the direct study of the CB1 receptor-peptide ligand interactions, Hp(1-7) was radiolabeled and in vitro characterized in rat and CB1 knockout mouse brain membrane homogenates. In saturation and competition radioligand binding studies, [(3)H]Hp(1-7) labeled membrane receptors with high densities and displayed specific binding to a receptor protein, but seemingly not to the cannabinoid type 1, in comparison the results with the prototypic JWH-018, AM251, rimonabant, Hp(1-9) and RVD-Hp(1-9) (pepcan 12) ligands in both rat brain and CB1 knockout mouse brain homogenates. Furthermore, functional [(35)S]GTP γS binding studies revealed that Hp(1-7) and Hp(1-9) only weakly activated G-proteins in both brain membrane homogenates. Based on our findings and the latest literature data, we assume that the Hp(1-7) peptide fragment may be an allosteric ligand or indirect regulator of the endocannabinoid system rather than an endogenous ligand of the CB1 receptor.

Nanoscale Metal-Organic Framework-Hemoglobin Conjugates.

A metal-organic framework (MOF)-protein conjugate, NH2 -MIL-125(Ti)-hemoglobin [MIL-125(Ti)-Hb], was synthesized by a covalent postmodification strategy. The crystalline structure was maintained after chemical and protein modification. The content of grafted Hb was tuned by the stoichiometric ratio and reached 50 wt % if the mass ratio of MIL-125(Ti)/Hb was 1:1.25 in the feed. The oxygen-transporting capacity of grafted Hb was kept, and the P50 (the half O2 pressure saturated with O2 ) and Hill coefficients of the MIL-125(Ti)-Hb conjugate were found to be 22.9 mm Hg and 2.35, respectively, which are close to the respective values of free Hb. All the results indicate that the MIL-125(Ti)-Hb conjugate could be potentially used as an oxygen carrier.

Site-specific Substitutions Eliminate Aggregation Properties of Hemopressin.

Hemopressin is a naturally occurring and therapeutically relevant peptide with applications in hypertension, pain, addiction, and obesity. We had previously demonstrated that hemopressin converts into amyloid-like fibrils under aqueous conditions. However, the amino acid residues that modulate the aggregation propensity of hemopressin were not identified. In this study, we designed and synthesized 25 different analogs of hemopressin and analyzed their aggregation properties using the principle of dynamic light scattering. As a result, we were able to identify four conservative changes in the peptide sequence (Val(2) →DVal(2), Asn(3) →Gln(3) Leu(7) →Npg(7) and C-OH→C-NH2) that minimize aggregation propensity of hemopressin. The results indicate that hemopressin aggregation is cooperative in nature and involves contribution from multiple amino acids within the peptide chain. The analogs and the corresponding aggregation propensity data reported in this study would be useful for researchers investigating therapeutic properties of hemopressin, which have been hampered due to the tendency of hemopressin to aggregate in aqueous solutions.

Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase.

We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4, or 6 times that of RBC.

Haemoglobin wrapped covalently by human serum albumin mutants containing Mn(III) protoporphyrin IX: an O2 complex stable in H2O2 solution.

We describe the synthesis of a haemoglobin (Hb) wrapped covalently by recombinant human serum albumin mutants [HSA(Y161H)] containing Mn(III) protoporphyrin IX (MnPP), the Hb-[HSA(Y161H)-MnPP]3 cluster, highlighting the formation of its O2-complex stable even in H2O2 solution.

Swine hemoglobin as a potential source of artificial oxygen carriers, hemoglobin-vesicles.

Hemoglobin-based oxygen carriers (HBOCs) have been developed as a transfusion alternative and oxygen therapy. The Hb source is usually outdated donated human blood or cow blood obtained from cattle industries because of its abundance. This study examined the feasibility of using swine Hb ((S)Hb) for preparation of cellular-type HBOCs, hemoglobin-vesicles (HbV). Purification of (S)Hb from fresh swine whole blood was conducted with processes including carbonylation ((S)HbO(2) --> (S)HbCO), pasteurization (60 °C, 15 hours) and tangential flow ultrafiltration, with yield of 90%. Actually, differential scanning calorimetric analysis showed a denaturation temperature of (S)HbCO at 83 °C and assures its stability during pasteurization. Concentrated (S)HbCO together with pyridoxal 5'-phosphate (PLP) as an allosteric effector was encapsulated in phospholipid vesicles to prepare (S)HbV. After decarbonylation ((S)HbCO --> (S)HbO(2)), the oxygen affinity (P(50)) of (S)Hb changes mainly by PLP, and the influence of Cl(-) was small, in a manner similar to that of human Hb ((H)Hb). However, after encapsulation, vesicles of (S)HbV showed much lower oxygen affinity (higher P(50)) than (H)HbV did. Autoxidation of (S)HbV was slightly faster than (H)HbV. Although some differences are apparent in oxygen affinity and autoxidation rates, results clarified that (S)Hb is useful as a starting material for HbV production.

Resuscitation with polymerized human placenta hemoglobin attenuated hemorrhagic shock-induced lung injury.

This study was designed to investigate whether polymerized human placenta hemoglobin (PolyPHb) attenuated hemorrhagic shock-induced lung injury. A mean arterial pressure (MAP) of 30mmHg was maintained for 60 min. Then, all the rats were randomly resuscitated with hetastarch, whole blood, or PolyPHb. The result indicated that PolyPHb greatly improved the MAP and pulmonary function, and significantly reduced the release of inflammatory cytokines, histopathological changes, and pulmonary edema. Therefore, our findings suggest that PolyPHb could reduce pulmonary injury after hemorrhagic shock, and this effect was probably associated with the depressed inflammatory response.

Glutathionylation induced structural changes in oxy human hemoglobin analyzed by backbone amide hydrogen/deuterium exchange and MALDI-mass spectrometry.

Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and ß globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: ß86-102, ß1-14, α34-46, ß32-41, ß130-146, ß115-129, ß73-81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.