Chemistry for peptide and protein PEGylation.

Poly(ethylene glycol) (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules and surfaces for many pharmaceutical and biotechnical applications. In the modification of biological macromolecules, peptides and proteins are of extreme importance. Reasons for PEGylation (i.e. the covalent attachment of PEG) of peptides and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering biodistribution. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein. In this paper, we review PEG chemistry and methods of preparation with a particular focus on new (second-generation) PEG derivatives, reversible conjugation and PEG structures.

Pharmacodynamic and pharmacokinetic characterization of poly(ethylene glycol) conjugation to met-enkephalin analog [D-Pen2, D-Pen5]-enkephalin (DPDPE).

Poly(ethylene glycol), or PEG, conjugation to proteins and peptides is a growing technology used to enhance efficacy of therapeutics. This investigation assesses pharmacodynamic and pharmacokinetic characteristics of PEG-conjugated [D-Pen2,D-Pen5]-enkephalin (DPDPE), a met-enkephalin analog, in rodent (in vivo, in situ) and bovine (in vitro) systems. PEG-DPDPE showed increased analgesia (i.v.) compared with nonconjugated form (p < 0.01), despite a 172-fold lower binding affinity for the delta-opioid receptor. [125I]PEG-DPDPE had a 36-fold greater hydrophilicity (p < 0.01) and 12% increase in the unbound plasma protein fraction (p < 0.01), compared with [(125)I]DPDPE. [125I]PEG-DPDPE had a 2.5-fold increase in elimination half-life (p < 0.01), 2.7-fold decrease in volume of distribution (p < 0.01), and a 7-fold decrease in plasma clearance rate (p < 0.01) to [125I]DPDPE. Time course distribution showed significant concentration differences (p < 0.01) in plasma, whole blood, liver, gallbladder, gastrointestinal (GI) content, GI tract, kidneys, spleen, urine, and brain (brain, p < 0.05), between the conjugated and nonconjugated forms. Increased brain uptake of [(125)I]PEG-DPDPE corresponded to analgesia data. [125I]PEG-DPDPE in brain was shown to be 58.9% intact, with 41.1% existing as [125I]DPDPE (metabolite), whereas [125I]DPDPE was 25.7% intact in the brain (at 30 min). In vitro P-glycoprotein affinity was shown for [125I]DPDPE (p < 0.01) but not shown for [125I]PEG-DPDPE. In vitro saturable uptake, with 100 microM DPDPE, was shown for [125I]PEG-DPDPE (p < 0.05). In this study, PEG-conjugated DPDPE seems to act as a prodrug, enhancing peripheral pharmacokinetics, while undergoing hydrolysis in the brain and allowing nonconjugated DPDPE to act at the receptor.

Microcomputed tomography of kidneys following chronic bile duct ligation.

BACKGROUND: In hepatic cirrhosis, renal sodium and water retention can occur prior to decreases in renal blood flow (RBF). This may be explained in part by redistribution of the intrarenal microcirculation toward the juxtamedullary nephrons. To appreciate this three-dimensional spatial redistribution better, we examined the intrarenal microcirculatory changes using microcomputed tomography (micro-CT) in rats subjected to chronic bile duct ligation (CBDL). METHODS: Six kidneys from control rats and eight kidneys from rats that had undergone CBDL for 21 days were perfusion fixed in situ at physiological pressure, perfused with silicon-based Microfil containing lead chromate, embedded in plastic, and scanned by micro-CT. The microvasculature in the reconstructed three-dimensional renal images was studied using computerized image-analysis techniques. To determine the physiological condition of the rats, parallel experiments were conducted on six control and six CBDL rats to measure mean arterial pressure (MAP), RBF, glomerular filtration rate (GFR), urine flow (UF) rate, and sodium excretion by conventional methods. RESULTS: The percentage of vasculature in the renal cortex from CBDL rats was significantly decreased (10.8 +/- 0.4% vs. 16.8 +/- 2.7% control values). However, the vascular volume fractions of the medullary tissues were not significantly altered. There were no significant differences in the number of glomeruli between groups (36,430 +/- 1908 CBDLs, 36,609 +/- 3167 controls). The CBDL rats had a similar GFR than the controls but a reduced MAP, RBF, UF, and sodium excretion. CONCLUSIONS: The results indicate that after CBDL, there is a selective decrease in cortical vascular filling, which may contribute to the salt and water retention that accompanies cirrhosis.

Photoimmobilization of organophosphorus hydrolase within a PEG-based hydrogel.

Organophosphorous hydrolase (OPH) was physically and covalently immobilized within photosensitive polyethylene glycol (PEG)-based hydrogels. The hydroxyl ends of branched polyethylene glycol (b-PEG, four arms, MW = 20,000) were modified with cinnamylidene acetate groups to give water-soluble, photosensitive PEG macromers (b-PEG-CA). The b-PEG-CA macromers underwent photocrosslinking reaction and formed gels upon UV irradiation (>300 nm) in the presence of erythrosin B. Native OPH was pegylated with cinnamylidene-terminated PEG chains (MW = 3400) to be covalently linked with the b-PEG-CA macromers during photogelation. The effect of pegylation on the stability of the enzyme was determined. Furthermore, the effect of enzyme concentration, wavelength of irradiation, and photosensitizer on the stability of the entrapped enzyme was also investigated. The pegylated OPH was more stable than the native enzyme, and the OPH-containing gels exhibited superior stability than the soluble enzyme preparations.

Reductive amination using poly(ethylene glycol) acetaldehyde hydrate generated in situ: applications to chitosan and lysozyme.

Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities. For drugs bearing one or more amino groups, reductive amination is a potentially useful method for conjugation to PEG. PEG acetaldehyde has been used for this purpose, but its ease of polymerization under certain conditions and its susceptibility to air oxidation have caused some problems in its application. A simple and reliable method for preparation and use in reductive amination of PEG acetaldehyde hydrate generated in situ by hydrolysis of PEG acetaldehyde diethylacetal is demonstrated. PEG acetaldehyde diethylacetal is prepared in high yield and purity by reaction of PEG with chlorodiethylacetal in dioxane in the presence of finely powdered sodium hydroxide under heterogeneous conditions. PEG acetaldehyde hydrate is generated in solution by hydrolysis in aqueous acids. Solutions of the hydrate may be used directly, in conjunction with sodium cyanoborohydride, to effect reductive amination. We demonstrate application of these methods in PEGylation of lysozyme and chitosan to form water-soluble methoxy poly(ethylene glycol) (mPEG) derivatives and PEG-chitosan hydrogels.

Three-dimensional microcomputed tomography of renal vasculature in rats.

Current microscopic methods to view renal microvasculature reveal only a very limited portion of the total renal volume. Identification of connectivity for postglomerular vessels in the cortex and the medulla during functional states related to changes in sodium excretion will help better to understand the coupling of renal vasculature to tubular function. The purpose of this study was to investigate the possibility of visualizing the continuity of pre- and postglomerular vasculature using three-dimensional micro-computed tomography (micro-CT). Kidneys from normal rats were perfusion fixed in situ at physiological pressure, filled with latex microfil containing lead chromate, and embedded in plastic. The micro-CT scans of the intact kidneys were carried out on a rotating stage illuminated either by a synchrotron x-ray source or a conventional x-ray spectroscopy tube. Images were reconstructed by a filtered backprojection algorithm and volume-rendering techniques were utilized to display the vasculature. The reconstructed images clearly showed the large distribution vessels and the venous drainage of the kidneys, while pre- and postglomerular vessels and their vascular connections throughout the kidney were displayed in great detail. Efferent arterioles showed the characteristics of their peritubular capillary beds in the cortical and medullary regions. The vascular volume of the cortex was 27%, the outer stripe of the outer medulla 18%, the inner stripe of the outer medulla 30%, and the inner medulla 18%. In conclusion, micro-CT is a promising method to evaluate renal vascular architecture relative to physiological and pathological alterations.

Pressure dependency of canine intrarenal blood flow within the range of autoregulation.

The mechanism of pressure-induced natriuresis remains controversial. To assess whether intracortical or medullary renal blood flows (RBF) change with changes in renal perfusion pressure (RPP), global and regional RBFs were measured using the dynamic spatial reconstructor, a fast computed tomography scanner, in eight anesthetized dogs (group B) within the range of RBF autoregulation (RPP of 153.5 and 114.4 mmHg). Similar measurements were obtained in seven control dogs (group A) in which RPP was not manipulated. In group B, only inner medullary perfusion decreased (from 0.84 to 0.51 ml/min per cm3 of tissue, P = 0.03) with reduction of RPP, whereas global renal, intracortical, and outer medullary perfusions remained unaltered. In group A there was no change in global or regional renal perfusion. The change in inner medullary perfusion in group B (-34.7%) was significantly different (P = 0.021) from that in group A (+27.4%). Global, cortical, and total medullary RBFs (ml/min) and volumes did not change in either group. These results suggest that with changes in RPP, the only detectable change in intrarenal perfusion occurs in the inner medulla.

Measurement of renal perfusion and blood flow with fast computed tomography.

Fast computed tomography (CT) is one of the few methods available to measure cortical and medullary renal blood flow (RBF) directly. Because these measurements are complicated by passage of the contrast medium into extravascular compartments, we used the residual opacity following the vascular blush as an index to account for extravascular iohexol. Kidneys of anesthetized dogs were examined in situ by fast CT following intra-aortic injections of iohexol. Perfusion was analyzed during a control period and three subsequent periods in which RBF was reduced by 10%, 30%, and 50%. Cortical microvascular distribution volume changed from 19.7 +/- 2.8% to 19.8 +/- 1.7%, 15.3 +/- 1.2%, and 9.9 +/- 1.7%, respectively, without significant alterations in cortical mean transit time. Microvascular distribution volume was divided by mean transit time to determine tissue perfusion. Cortical perfusion changed from 3.8 +/- 0.7 to 3.9 +/- 0.6, 3.1 +/- 0.5, and 2.2 +/- 0.5 mL.min-1.mL tissue-1. Total cortical blood flow (cortical perfusion multiplied by cortical volume) decreased from 164 +/- 32 to 159 +/- 31, 117 +/- 20, and 86 +/- 22 mL/min, respectively. Medullary microvascular distribution volume, mean transit time, perfusion, and total blood flow remained unchanged. Fast CT-determined total RBFs (cortex plus medulla) were similar to simultaneous electromagnetic flow measurements. These results indicate that renal regional perfusion is more dependent on the microvascular distribution volume than mean transit time and that variations in renal tissue perfusion with reduction of RBF are more apparent in the cortex than in the medulla.

The effect of a low-osmolar radiographic contrast medium on in vivo and postmortem renal size.

High osmolar radiographic contrast media (CM) are known to cause an increase in renal size. To examine the effect of low-osmolar CM on renal size, 14 anesthetized dogs received 12 intravenous bolus injections of 0.5 mL/kg iohexol (541 mOsm/L). The postmortem renal, cortical, and medullary volumes were determined by fluid displacement. Renal volumes of 18 control dogs were determined similarly. The mean (+/- SEM) postmortem renal volumes were 66.1 +/- 2.2 mL for the CM group and 52.3 +/- 3.3 mL for the control group (P = 0.003), whereas the cortical and medullary volumes were similar. Six dogs were also scanned by fast computerized tomography before and after iohexol administration. The in vivo whole renal and medullary volumes enlarged from 67.4 +/- 3.0 to 77.1 +/- 2.8 mL (P = 0.006), and from 28.5 +/- 2.0 to 35.1 +/- 1.1 mL (P = 0.026), respectively, while the cortical volume remained unaltered. These results suggest that even low osmolar CM may significantly increase renal volume, probably by causing tubular expansion.

Quantitation of the in vivo kidney volume with cine computed tomography.

The authors examined the utility of cine computerized tomography (CT) for noninvasive determination of whole kidney, cortical, and medullary volumes. The right kidneys of 14 anesthetized dogs were scanned after an intravenous bolus injection of iohexol, and their volumes determined after boundary identification. After the scans, the kidneys were excised at postmortem examination and their volumes determined by fluid displacement. The mean (+/- standard error of the mean [SEM]) postmortem and in vivo renal volumes were 66.1 +/- 2.2 cc and 78.2 +/- 2.4 cc, respectively (r = 0.86; P less than 0.001). The difference was consistent with the blood, filtrate, and urine contents of the in vivo kidney. The in vivo cortical and medullary volumes correlated poorly with their postmortem volumes because of difficulties in boundary definition. These results demonstrate the feasibility for fast and reliable in vivo whole kidney volume quantitation by cine CT.

Effect of captopril on renal function in hypertensive dogs with unilateral renal artery stenosis, studied with radionuclide dynamic scintigraphy.

The kidneys of five hypertensive dogs with experimental unilateral renal artery stenosis were examined by 99mTc-diethylenetriaminepentaacetic acid (DTPA) and 131I-hippuran radionuclide dynamic scintigraphy at 10 and 40 min (respectively) following the administration of intravenous bolus injections of captopril. Doses of 0.2, 0.5, and 1.0 mg/kg captopril reduced mean arterial pressure by 33 +/- 4, 31 +/- 7, and 51 +/- 4 mm Hg and increased plasma renin activity by 40.1 +/- 9.8, 57.6 +/- 3.2, and 34.4 +/- 15.2 ng A1/mL/h, respectively. The time-activity curves of both 99mTc-DTPA and 131I-hippuran indicated that renal excretory function in the stenotic kidney was compromised with all three doses of captopril. However, if nitroprusside was used to reduce the mean arterial pressure to a level comparable to that with captopril, there was no appreciable increase in plasma renin activity and renal excretory function was only partially affected in the stenotic kidney. One hour after the administration of 0.5 mg/kg captopril, the function of the stenotic kidneys was partially restored and, by two hours, the time-activity curves were comparable to control scans. These data demonstrate a reversible, time-limited suppression of stenotic kidney function by captopril in renovascular hypertension and provide support for the use of captopril in the diagnosis of renovascular hypertension by radionuclide dynamic scintigraphy.

MR imaging-based volume measurements of the hippocampal formation and anterior temporal lobe: validation studies.

An experiment was designed to compare the accuracy and reproducibility of three different techniques for in vivo magnetic resonance (MR) imaging-based volume measurements of brain structures. These techniques were tracing, thresholding, and random marking. Anterior temporal lobe (ATL) and hippocampal formation (HF) volumes in 10 volunteers were measured from MR images, as were four cylinders of known volume. The upper limit of accuracy of in vivo volume measurements is estimated to be within 0.1 cm3 of true volume for the HF and 0.9 cm3 for the ATL with a combined tracing-thresholding technique. Intra- and interobserver variations were estimated from the pooled standard deviations of HF and ATL measurements. With the combined tracing-thresholding technique, the coefficient of variation for HF measurement was 1.9%; for the ATL measurement, it was 0.7%. The results indicate that MR-based volume measurements of these brain structures can be made with high precision and reproducibility.

Distribution of prostaglandins E2 and 6-keto-F1 alpha production in dog kidneys.

Little is known about the distribution of prostaglandin E2 (PGE2) and prostacyclin (PGI2) production in the canine kidney. To determine the basal and stimulated profiles of PGE2 and PGI2 production along the corticomedullary axis of the dog kidney, a slice (0.5 mm thick, 10-50 mg) was obtained from six equally spaced zones along the axis (zone 1, medullary crest; zones 2 and 3, inner medulla; zone 4, outer medulla; and zones 5 and 6, cortex) and was divided into equal halves. One half of the slice was incubated with Krebs-Ringer buffer containing arachidonic acid (6.6 x 10(-4) M), bradykinin (9.4 x 10(-6) M), or indomethacin (10(-5) M), whereas the remaining half of each slice was similarly incubated in Krebs-Ringer buffer alone. The production of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) was determined by radioimmunoassay. Under basal conditions, both PGE2 and 6-keto-PGF1 alpha were highest in the innermost zones of the inner medulla (PGE2, 3,328 +/- 549 pg/mg; 6-keto-PGF 1 alpha, 1,611 +/- 129 pg/mg) and decreased exponentially to low levels in the cortex (PGE2, undetectable; 6-keto-PGF1 alpha, 13 +/- 2 pg/mg); this production was inhibited by indomethacin. Arachidonic acid significantly increased the production of PGE2 in all zones of the kidney and the production of 6-keto-PGF1 alpha only in zones 3-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrarenal mechanisms that regulate sodium excretion in relationship to changes in blood pressure.

Because pressure-related natriuresis may be central to the regulatory role of the kidney on blood pressure, it is important to understand the relationship of humoral systems involved in the control of renal hemodynamics and tubular function. The preglomerular endothelial synthesis of prostaglandin I2 and endothelium-derived relaxing factor seem to modulate autoregulatory control by the afferent arterioles and the release of renin by the juxtaglomerular apparatus. The release of renin is followed by an increase in angiotensin II in the renal interstitium, which is responsible for adjusting the vascular tone of the efferent arterioles and vasa recta and for stimulating proximal tubular reabsorption of sodium. Variations in medullary circulation induced by angiotensin II could alter medullary interstitial pressure and the medullary production of prostaglandins E2 and I2 and, ultimately, could modulate sodium reabsorption in the medullary thick ascending limbs and the collecting ducts.

Alterations in blood pressure by derangement of the mechanisms that regulate sodium excretion.

Understanding the sequence of events responsible for pressure-related natriuresis and their pathophysiologic alterations may be useful in distinguishing various types of essential hypertension of renal origin. The perturbation of a distal step in the sequence is likely to be reflected in a simple physiologic defect. For instance, pathophysiologic alterations in the medullary production of prostaglandin E2 might directly influence natriuresis and diuresis because of its modulatory effect on tubular reabsorption of sodium and water. Perturbation of more proximal steps in the sequence could influence all the distal events as well. For instance, prostaglandin I2 and endothelium-derived relaxing factor may be produced by the preglomerular vasculature in response to alterations in renal perfusion pressure and may modulate the release of renin from the juxtaglomerular cells. Thus, variations in the production of prostaglandin I2 or endothelium-derived relaxing factor may be reflected by various renal vascular, tubular, and systemic homeostatic events related to the renin-angiotensin system.

Effects of atrial natriuretic peptide on renal medullary prostaglandin E2 production.

Like arachidonic acid (AA) and bradykinin (BK), the intrarenal administration of atrial natriuretic peptide (ANP) has been shown to increase the urinary excretion of prostaglandin E2 (PGE2). In the present study, the direct in vitro effects of ANP on PGE2 production were compared with those of AA and BK. Canine renal inner medullary slices were preincubated for 30 min and washed in aerated Krebs-Ringer buffer (37 degrees C). During the final incubation period, with the use of varied concentrations of AA, BK, or ANP in Krebs-Ringer buffer, samples were obtained at 0 and 30 min to be used for radioimmunoassay of PGE2. Although the rate of PGE2 production was significantly increased 11-fold with AA and threefold with BK, it was unaffected by four different doses of ANP (10(-5) to 10(-11) M). Furthermore, the production of PGE2 during basal and stimulated (BK or AA) conditions was significantly blocked by indomethacin but not by ANP. These results indicate that ANP had no direct stimulatory or inhibitory effect on the medullary production of PGE2.

Effect of atriopeptin II on contrast medium-induced changes in renal blood flow.

Volume expansion has been shown to attenuate the vaso-constrictive effects of contrast medium. Since endogenous circulating levels of atrial natriuretic peptides are elevated during volume expansion and since atrial natriuretic peptides have been shown to blunt or block the vasoconstrictive effects of various pharmacologic agents, we examined the effect of atriopeptin II infusion on contrast medium-induced alterations in renal blood flow. Transient changes in renal blood flow were measured with an electromagnetic flow probe following bolus injections of the contrast medium, sodium meglumine diatrizoate (Renovist II, 2 ml/4 sec), into the renal arteries of dogs. Under control conditions (that is, saline vehicle), the bolus of contrast medium caused an initial 15 to 21% increase in renal blood flow at 14 to 21 seconds, followed by a subsequent 5 to 13% decrease in renal blood flow at 43 to 59 seconds after injection. Infusion of atriopeptin II (0.05 pg/kg/min) into the renal artery did not alter arterial blood pressure, glomerular filtration rate, or baseline renal blood flow, but did produce increases in urine flow rate and clearance of sodium. The infusion of atriopeptin II attenuated the contrast medium-induced reduction in RBF, but the infusion of a threshold dose of bradykinin (1 ng/kg/min) had no effect. These results suggest that low doses of atriopeptin II can selectively attenuate the vasoconstrictive effects of contrast medium on renal blood flow without influencing systemic hemodynamics.

Comparison of the effects of calcium antagonists and converting enzyme inhibitors on renal function under normal and hypertensive conditions.

Calcium antagonists decrease the ability of the kidney to autoregulate renal blood flow (RBF) and glomerular filtration rate (GFR). Therefore, when afferent renovascular resistance is elevated, as in essential hypertension, there is a resultant increase in RBF and GFR with the administration of calcium antagonists. These agents also induce a marked natriuresis because of direct tubular action through unknown mechanisms. The natriuresis can be dissociated from renal and systemic hemodynamic actions, indicating that the decreased sodium reabsorption could override other compensatory mechanisms explaining the absence of sodium retention during the treatment. The renal effects of converting enzyme inhibitors (CEIs) can be explained by the reduction of intrarenal formation in angiotensin II. Because the activation of the renin-angiotensin system is mainly responsible for inducing sodium retention during a decrease in systemic blood pressure, CEIs could have a protecting effect without disturbing other homeostatic mechanisms. CEIs decrease efferent glomerular resistance, reducing capillary pressure and thereby reducing GFR. This effect is not translated in sodium retention because the reduction of GFR is mild during captopril administration in kidneys with normal or increased renal perfusion pressure. At low renal perfusion pressure, the reduced glomerular afferent vasoconstriction can compromise GFR, leading to renal insufficiency. Although these situations are not likely to be encountered during the treatment of uncomplicated essential hypertension, in severe hypertension with hypertrophy of pre-glomerular vessels, glomerular perfusion may decrease. Combination therapy of calcium antagonists and CEIs has been reported to be an effective treatment of severe hypertension. Currently, little information is available on the manner in which renal function is affected by simultaneous administration of both drugs.

Estimation of tissue volume from serial tomographic sections. A statistical random marking method.

Computer operator-interactive planimetric methods that have been employed to measure volume of a selected tissue in serial computed tomographic sections are time consuming, and the accuracy of the measurement is limited by the operator's skill in outlining the tissue. We introduce an alternate method that is 5 to 6 times faster than planimetry and is independent of the operator's outlining dexterity. This method involves the random marking of voxels in a three-dimensional array of known size and subsequent identification of the proportion of those voxels that are within the tissue of interest. Tissue of the kidney and left ventricular myocardium were imaged in situ with a high speed, volume scanning, computerized x-ray tomographic imaging system. Volumetric measurements made by the random marking method correlated highly (r = 0.99) with measurements made by planimetry and with postmortem weight. These data indicate that the random marking method provides a rapid and accurate means of estimating tissue volume in serial tomographic sections.