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1.  PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues 
EJNMMI Research  2012;2:24.
Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN2/gastrin-releasing peptide receptors are overexpressed. We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart.
Derivatisation of a stabilised DOTA-BN(7–14)[Cha13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy.
The specificity and the binding affinity of the BN analogue for BN2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios (tumour-to-kidney, 0.6 vs. 0.4; tumour-to-liver, 8.8 vs. 5.9, 24 h p.i.). In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour growth. The therapeutic efficacy was highest for the PEGylated derivative of high specific activity administered in two fractions (2 × 20 MBq = 40 MBq) at day 0 and day 7 (73% tumour growth inhibition, 3 weeks after therapy).
PEGylation and increasing the specific activity enhance the pharmacokinetic properties of a 177Lu-labelled BN-based radiopharmaceutical and provide a protocol for targeted radionuclide therapy with a beneficial anti-tumour effectiveness and a favourable risk-profile at the same time.
PMCID: PMC3478187  PMID: 22681935
Gastrin-releasing peptide; Prostate cancer; 177Lu; Radionuclide therapy; PEGylation
2.  Hemoglobin Syracuse (alpha2beta2-143(H21)His leads to Pro), a new high-affinity variant detected by special electrophoretic methods. Observations on the auto-oxidation of normal and variant hemoglobins. 
Journal of Clinical Investigation  1975;55(3):469-477.
Family members from four generations were found to have polycythemia and increased whole blood O2 affinity (P50; 11 mm Hg; normal, 27 mm Hg). No abnormal hemoglobin bands were seen after electrophoresis on starch gel at pH 8.6 or agar gel at pH 6.0. Analysis of the oxygenated hemolysate by isoelectric focusing on polyacrylamide gel revealed two closely spaced bands. When deoxygenated hemolysate was analyzed in oxygen-free gels, the two components were more widely separated. About 40% of the patient's hemoglobin focused at a more acid pH than hemoglobin A, indicating a hemoglobin variant with impaired Bohr effect. Chromatography of globin in 8 M urea revealed two beta-chain peaks, the first of which was eluted at a lower buffer molarity than normal beta chain. Fingerprints of tryptic digests of the aminoethylated chains were done on silica gel thin-layer plates. Tp 14 from the abnormal beta chain had slower electrophoretic mobility and a greater Rf value. Amino acid analyses of this peptide gave values identical with those of betaTp 14, except that it contained one proline residue and no histidine. Since the one His in betaTp 14 is in position 143, hemoglobin Syracuse in alpha2beta2-143 His leads to Pro. Native Hb Syracuse could be separated from hemoglobin A on a carboxymethylcellulose column. The inclusion of 0.1 mM EDTA in the preparative buffers proved very useful in reducing the formation of methemoglobin. Oxygen equilibria of purified hemoglobin Syracuse showed high oxygen affinity (P50 value 12% that of hemoglobin A) and lack of cooperativity between subunits (Hill's n equals 1.1). The alkaline Bohr effect was about half that of hemoglobin A. The proline substitution at betaH21 disrupts the helical configuration and probably prevents the formation of salt bonds that are important in stabilizing the deoxy structure and contribute to the alkaline Bohr effect. Since beta143 His is a binding site for 2,3-diphosphoglycerate (2,3-DPG), it is not suprising that hemoglobin Syracuse had markedly impaired reactivity with 2,3-DPG. Hemoglobin Syracuse auto-oxidized more slowly than hemoglobin A, probably reflecting a slower rate of dissociation of oxygen from fully liganded hemoglobin.
PMCID: PMC301774  PMID: 234980
3.  Subunit dissociation of certain abnormal human hemoglobins 
Journal of Clinical Investigation  1969;48(1):126-138.
The extent of dissociation of various hemoglobins into subunits was estimated from their elution volumes (Ve) on G-100 Sephadex. Under the same controlled conditions carboxyhemoglobins A, A3 (A1), F, S, and C all had the same elution volumes. The carboxy and cyanmet derivatives of hemoglobin Kansas (a variant with very low oxygen affinity) had a relatively high Ve, indicating a decreased mean molecular weight and therefore an increased tendency to form dimers and even monomers. Conversely, the liganded derivatives of hemoglobin Chesapeake (a variant with high oxygen affinity) had a relatively low Ve, suggestive of an impaired degree of subunit dissociation. Deoxyhemoglobin Chesapeake had a Ve identical with that of deoxyhemoglobin A. Cat hemoglobin, known to have an unusually low oxygen affinity, was found to have a higher Ve than human, dog, rabbit, rat, or guinea pig hemoglobins.
Haptoglobin is thought to bind αβ dimers in preference to the α2β2-tetramer. The comparative haptoglobin affinities of the human hemoglobins were measured by competition between the test hemoglobin and radioactive reference hemoglobin for haptoglobin binding sites. Hemoglobins A, F, S, and C all seemed to bind equally readily, but hemoglobin Kansas and cat hemoglobin showed a higher affinity, and hemoglobin Chesapeake a lower affinity.
These results are in accord with recently proposed models which predict that hemoglobins which have an increased degree of subunit dissociation will have a low oxygen affinity, and vice versa.
PMCID: PMC322198  PMID: 5765016
4.  Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC) 
Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.
The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo.
Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared.
Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.
PMCID: PMC2679726  PMID: 19374748
5.  A Long-Acting, MonoPEGylated Human Growth Hormone Analog is a Potent Stimulator of Weight Gain and Bone Growth in Hypophysectomized Rats. 
Endocrinology  2007;148(4):1590-1597.
Recombinant human Growth Hormone (GH) is used to treat growth hormone deficiency in children and adults, and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5 kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine-PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity and in vivopotency. In vitro bioactivity of GH was reduced 100- to 1,000-fold by extensive amine-PEGylation of the protein. Here we describe a homogeneously modified, monoPEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life and increased potency in vivo. The monoPEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20 kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH following sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable to that produced by daily administration of GH in hypophysectomized rats. Long-acting, monoPEGylated GH analogs such as PEG-T3C are promising candidate for future testing in humans.
PMCID: PMC1892190  PMID: 17234711
growth hormone; polyethylene glycol; rat; bone; growth
6.  Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol 
Video abstract
Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)2-Lys-NHS (20 kDa).
Methodology/principal findings
In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts.
α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.
PMCID: PMC3396394  PMID: 22802682
ribosome-inactivating proteins; alpha-momorcharin; momordica anti-HIV protein; antitumor; antivirus; (mPEG)2-Lys-NHS (20 kDa); immunogenicity
7.  PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity 
PLoS ONE  2011;6(12):e28611.
The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.
PMCID: PMC3237458  PMID: 22194865
8.  Design of Homogeneous, MonoPEGylated Erythropoietin Analogs With Preserved In Vitro Bioactivity 
Experimental hematology  2006;34(6):697-704.
Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the PEGylated Epo cysteine analogs have improved pharmacokinetic properties in vivo.
Materials and Methods
Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monoPEGylated derivatives of 12 cysteine analogs were prepared using 20 kDa-maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild type Epo (BV Epo) and a PEGylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats.
Biological activities of 17 purified Epo cysteine analogs and 10 purified PEGylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only PEGylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The PEGylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation.
Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monoPEGylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed EPO proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific PEGylation appears to improve the pharmacokinetic properties of Epo.
PMCID: PMC1634893  PMID: 16728273
Biochemistry  2007;46(15):4554-4564.
HexaPEGylated Hb, a non-hypertensive Hb, exhibits high O2-affinity which makes it difficult to deliver desired levels of oxygen to tissue. PEGylation of very low O2-affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2-affinity. Towards this goal, a doubly modified Hb with very low O2-affinity has been generated. The amino terminal of β-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans β-chain. To further modulate the oxygen affinity of Hb, the αα-fumaryl crossbridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (αα-fumaryl-HPPr-HbA) exhibits an O2-affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of αα-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(β). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2-affinity comparable to that of erythrocytes for developing blood substitutes.
PMCID: PMC2572862  PMID: 17381072
10.  Effects of PEGylation on the physicochemical properties and in vivo distribution of organic nanotubes 
Application of organic nanotubes (ONTs) into drug nanocarriers ultimately requires validation in live animals. For improving the dispersibility in biological media and in vivo distribution, the outer surface of an ONT was functionalized with polyethylene glycol (PEG) via the coassembly of an ONT-forming lipid with 5–20 mol% of a PEG-tethered lipid analogue (PEG-lipid). Firstly, the effect of PEGylation on the psysicochemical properties of ONTs, such as morphology and dispersibility, was investigated. PEGylation of ONTs slightly reduced the average length and effectively prevented the aggregation in phosphate-buffered saline (PBS). The PEGylated ONTs even showed high thermal stability in aqueous dispersion at least up to 95°C. Secondly, differential scanning calorimetry and powder X-ray diffraction indicated that ~10 mol% of PEG-lipid was completely incorporated into the ONTs, while 20 mol% of PEG-lipid encountered a partial phase separation during coassembly. In the heating differential scanning calorimetry runs, the resultant PEGylated ONTs with 5 mol% PEG-lipid showed no sign of phase separation up to 180°C under lyophilized condition, while those with 10 mol% and 20 mol% PEG-lipid showed some phase separation of the PEG-lipid above 120°C. Finally, PEGylation significantly affected the tissue distribution and prolonged the persistence time in the blood in mice. Non-PEGylated ONTs was quickly cleared from the circulation after intravenous infusion and preferentially accumulated in the lung, while PEGylated ONTs was mainly trapped in the liver and could circulate in the blood up to 24 hours. This study provided valuable information of physicochemical properties and the in vivo distribution behavior of PEGylated ONTs for their potential application into drug nanocarriers.
PMCID: PMC4270402  PMID: 25540582
nanostructure; dispersibility; distribution
11.  Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands 
Bioconjugate chemistry  2011;22(3):445-454.
Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6–10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.
PMCID: PMC3063487  PMID: 21338093
polyethylene glycol; PEG; Toll like receptor 7
Journal of Clinical Investigation  1973;52(8):2060-2067.
Erythrocytosis associated with the presence of a hemoglobin with increased oxygen affinity has been reported for 10 hemoglobin variants, most of which demonstrate altered electrophoretic mobility. Several members of a family were found to have erythrocytosis, and both the whole blood and the hemoglobin exhibited increased oxygen affinity. Phosphate-free hemoglobin solutions had a normal Bohr effect and reactivity to 2,3-diphosphoglycerate. The electrophoretic properties of the hemoglobin were normal, but on peptide mapping of a tryptic digest of the isolated β-chains, a normal βT11 peptide and an abnormal βT11 with greater Rf were seen. Analysis of the abnormal peptide showed the substitution of leucine for the normal proline at β100 (helical residue G2).
The hemoglobin variant, designated Hb Brigham, serves to emphasize the necessity for detailed evaluation of the structure and function of hemoglobin in familial erythrocytosis even with electrophoretically “normal” hemoglobin.
PMCID: PMC302488  PMID: 4719677
13.  Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix 
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin has been solved.
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128.
PMCID: PMC3614163  PMID: 23545644
hemoglobin; deer mouse; oxygen affinity; Peromyscus maniculatus
14.  PEGylation of interleukin-10 for the mitigation of enhanced pain states 
The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of ~3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.
PMCID: PMC3604983  PMID: 19768789
glia; interleukin-10; intrathecal delivery; neuropathic pain; PEGylation
15.  Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations 
The journal of physical chemistry. B  2013;117(42):13058-13068.
Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation.
PMCID: PMC3973026  PMID: 23957820
Energy landscape; funnel; Gō model; molecular dynamics; machine-learning
16.  Effects of Shielding Adenoviral Vectors with Polyethylene Glycol on Vector-Specific and Vaccine-Mediated Immune Responses 
Human Gene Therapy  2008;19(12):1369-1382.
Many individuals have been previously exposed to human adenovirus serotype 5 (Ad5). This prior immunity has long been known to hinder its use for gene therapy and as a gene-based vaccine. Given these immunogenicity problems, we have tested whether polyethylene glycol (PEG) can blunt immune effects against Ad5 during systemic and mucosal vaccination. Ad5 vectors were covalently modified with 5-, 20-, and 35-kDa linear PEG polymers and evaluated for their ability to produce immune responses against transgene antigen products and the vector itself. We show that shielding Ad5 with different-sized PEGs generally reduces transduction and primary antibody responses by the intramuscular or intranasal route. In contrast, PEGylated vectors generally appear better at boosting antibody responses in Ad-immune animals. Displaying either glucose or galactose on PEG mediated increased transduction and antibody responses by the intranasal, but not the intramuscular, route. In naive animals, PEGylated vectors generated T cell responses that were equal to or better than those by unmodified Ad. Priming by PEGylated vectors generally enabled better subsequent T cell responses after boost. Priming and boosting by PEGylated vectors produced T cell responses after boost that were equal to or higher than those produced by unmodified vectors. These data indicate that PEGylation can enable more effective application of Ad5 and perhaps other Ad serotype vaccines during prime–boost vaccination.
PMCID: PMC2922072  PMID: 18778197
17.  A Better Anti-Diabetic Recombinant Human Fibroblast Growth Factor 21 (rhFGF21) Modified with Polyethylene Glycol 
PLoS ONE  2011;6(6):e20669.
As one of fibroblast growth factor (FGF) family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21) was modified with polyethylene glycol (PEGylation) in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients.
PMCID: PMC3108960  PMID: 21673953
18.  Preparation and Characterization of PEGylated Amylin 
AAPS PharmSciTech  2013;14(3):1083-1097.
Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys1. The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.
PMCID: PMC3755165  PMID: 23818080
amylin; diabetes; islet-associated polypeptide; PEGylation; receptor activity-modifying protein
19.  Structural and functional studies on hemoglobin Bethesda (α2β2145 His), a variant associated with compensatory erythrocytosis 
Journal of Clinical Investigation  1972;51(9):2299-2309.
Studies have been performed on a 12-yr-old Chinese girl with compensatory erythrocytosis due to the presence of hemoglobin Bethesda comprising about 45% of the red cell hemoglobin. Her parents and three siblings were normal. The oxygen affinity of her blood was markedly increased: under physiological conditions (pH 7.40, 37°C). P50 was 12.8 mm Hg (normal = 26.5 mm Hg). The red cell 2,3-diphosphoglycerate (2.3-DPG) level was normal. The abnormal hemoglobin could not be separated from hemoglobin A by zone electrophoresis at pH 8.6 or isoelectric focusing on polyacrylamide gel. However, after the hemoglobin was split into free α and β chains by treatment with p-hydroxymercuribenzoate (PMB) or 6 M urea, an abnormal β chain was readily demonstrated having a higher isoelectric point (more positive net charge) than normal βA. Structural analysis of the variant β chain demonstrated the substitution of histidine for tyrosine at position 145: hemoglobin Bethesda (α2β2145His). From earlier chemical and crystallographic studies, it has been postulated that this residue is a critical determinant of hemoglobin function. Hemoglobin Bethesda was separated from hemoglobin A by column chromatography. Oxygen equilibria of purified hemoglobin Bethesda revealed an extremely high oxygen affinity (exceeding that of isolated α and β chains), and markedly reduced cooperativity. The Bohr effect of hemoglobin Bethesda was 1/3 that of hemoglobin A. However, hemoglobin Bethesda showed a significant interaction with 2.3-DPG and inositol hexaphosphate.
PMCID: PMC292395  PMID: 4639015
20.  In Vitro Evaluation of the Efficacy of Liposomal and Pegylated Liposomal Hydroxyurea 
Breast cancer is one of the most frequent cancer types within women population. Hydroxyurea (HU) is a chemotherapy compound for treatment of patients with cancer diagnosis, including breast cancer associated with several adverse effects. In this study, we applied nanotechnology to decreased drug side effects along with improvement of therapeutic index. Liposomation is widely used in modern pharmacological developments in order to enhance the effects of the drugs. To achieve this, in this study a mixture of phosphatidylcholine and cholesterol was made up and HU was added to the resultant mixture, was then pegylated using Polyethylene Glycol 2000 to increase resistance, applicability and solubility. The mean diameters of nanoliposomal and pegylated nanoliposomal HU were measured by Zeta sizer device and obtained about 402.5 and 338.2 nm. The efficiency of non-pegylated and pegylated liposomal HU was 70.8 and 64.2, respectively. Releasing HU in both formulations was estimated about 25.8 and 21.7 %. Also, this study investigated the cytotoxicity effect of nanoliposomal and pegylated nanoliposomal HU using MTT assay. Results of this investigation showed that the cytotoxic properties of pegylated HU was 3.6 % more than those non-pegylated form, while was 38.93 % more than ordinary from of HU. This study showed that the stability, releasing pattern and cytotoxicity of the pegylated nanoliposomal HU is better than that of nanoliposomal HU.
PMCID: PMC3903928  PMID: 24478555
Breast cancer; Hydroxyurea; Liposome; Pegylated liposome
21.  In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection 
DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream.
A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution.
We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing.
The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.
PMCID: PMC2950395  PMID: 20957159
gene delivery; DOTAP; polyethylene glycol (PEG); biodistribution; lung cancer; xenograft tumor model
22.  Site-Selective Glycosylation of Hemoglobin with Variable Molecular Weight Oligosaccharides: A Potential Alternative to PEGylation 
Poly(ethylene glycol) (PEG) conjugation (i.e. PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids, however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with various sized carbohydrates, up to a molecular weight (MW) of 10,000 Da, thus, serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with various sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of a similar MW to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (504 to ~10,000 Da). Hb was selected based on the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb’s biophysical properties was investigated in vitro.
PMCID: PMC3353419  PMID: 22489605
Glycosylation; glycoprotein synthesis; hemoglobin cysteine 93; PEGylation; carbohydrate; saccharide
23.  PEGylated Cationic Liposome – DNA Complexation in Brine is Pathway-Dependent 
Biochimica et biophysica acta  2013;1838(1):398-412.
Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) – which modulates the electrostatic interaction between oppositely charged vesicles and DNA – on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150 mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5 mol% to 10 mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10 mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150 mM.
PMCID: PMC3891919  PMID: 24060564
CL-DNA; Lipoplexes; DNA; cationic lipid; PEG; PEGylated; salt; steric stabilization; SAXS; gene therapy
24.  Effects of T-state and R-state stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide signaling 
Recent data suggest that transitions between the relaxed (R) and tense (T) state of hemoglobin control the reduction of nitrite to nitric oxide (NO) by deoxyhemoglobin. This reaction may play a role in physiologic NO homeostasis and be a novel consideration for the development of the next generation of hemoglobin-based blood oxygen carriers (HBOCs, i.e. artificial blood substitutes). Herein we tested the effects of chemical stabilization of bovine hemoglobin in either the T- (THb) or R-state (RHb) on nitrite reduction kinetics, NO-gas formation and ability to stimulate NO-dependent signaling. These studies were performed over a range of fractional saturations that is expected to mimic biological conditions. The initial rate for nitrite-reduction decreased in the following order RHb > bHb > THb, consistent with the hypothesis that the rate constant for nitrite reduction is faster with R-state Hb and slower with T-state Hb. Moreover, RHb produced more NO-gas and inhibited mitochondrial respiration more potently than both bHb and THb. Interestingly, at low oxygen fractional saturations, THb produced more NO and stimulated nitrite-dependent vasodilation more potently than bHb despite both derivatives having similar initial rates for nitrite reduction and a more negative reduction potential in THb versus bHb. These data suggest that cross-linking of bovine hemoglobin in the T-state conformation leads to a more effective coupling of nitrite reduction to NO-formation. Our results support the model of allosteric regulation of nitrite reduction by deoxyhemoglobin and show that cross-linking hemoglobins in distinct quaternary states can generate products with increased NO yields from nitrite reduction that could be harnessed to promote NO-signaling in vivo.
PMCID: PMC3115472  PMID: 21277987
Hypoxia; blood flow; oxygen sensing; blood substitute; nitrite reduction
25.  The acetylation of hemoglobin by aspirin. In vitro and in vivo. 
Journal of Clinical Investigation  1975;56(1):201-207.
The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.
PMCID: PMC436570  PMID: 237937

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