Species Differences of Serum Albumins: I. Drug Binding Sites

Purpose. The purpose of this study was the classification and identification of drug binding sites on albumins from several species in order to understand species differences of both drug binding properties and drug interaction on protein binding. Methods. Binding properties and types of drug-drug interaction on the different albumins were examined using typical site I binding drugs, warfarin (WF) and phenylbutazone (PBZ), and site II binding drugs, ibuprofen (IP) and diazepam (DZ) on human albumin. Equilibrium dialysis was carried out for two drugs and the free concentrations of drugs were then treated using the methods of Kragh-Hansen (Mol. Pharmacol. 34. 160–171, (1988)). Results. Binding affinities of site I drugs to bovine, rabbit and rat albumins were reasonably similar to human albumin. However, interestingly, those to dog albumin were considerably smaller than human albumin. On the other hand, binding parameters of DZ to bovine, rabbit and rat albumins were apparently different from those of human albumin. These differences are best explained by microenvironmental changes in the binding sites resulting from change of size and/or hydrophobicity of the binding pocket, rather than a variation in amino acid residues. Conclusions. We will propose herein that mammalian serum albumins used in this study contain specific drug binding sites: Rabbit and rat albumins contain a drug binding site, corresponding to site I on human albumin, and dog albumin contains a specific drug binding site corresponding to site II on the human albumin molecule.     

Species Differences of Serum Albumins: III. Analysis of Structural Characteristics and Ligand Binding Properties During N-B Transitions

Purpose. The aim of this study was to investigate the characteristics of the structural transitions and changes in ligand binding properties of different albumins during the pH-dependent structural transition, often referred to as the N-B transition. Methods. Structural transitions were evaluated by means of spectrometry, differential scanning calorimetry and chemical modification. In addition, ligand binding properties were investigated using typical site-specific bound drugs (warfarin, phenylbutazone, ibuprofen and diazepam). Results. Conformational changes, including N-B transition, clearly occurred in albumins from all species used in this study. The conformational stabilities of all the albumins were clearly lost in the weakly alkaline pH range. This was probably the result of the destruction of salt bridges between domain I and domain III in the albumin molecule. In addition, the profiles of the ANS-induced fluorescence were different and could be classified into two patterns, suggesting that hydrophobic pockets in the albumin molecules were different for the different species. The data suggest that the amino acid residues responsible for the transitions were some of the His residues located in domain I. Further, the ligand binding properties of the albumins were slightly different but statistically significant. Conclusions. The overall mechanisms of the N-B transition may be similar for all the albumins, but its impact is considerably different among the species in terms of both structural characteristics and ligand binding properties. Furthermore, the transitions appear to be multi-step transitions.     

Simultaneous differential scanning calorimetry,X-ray diffraction and FTIR spectrometry in studies of ovalbumin denaturation

The new application of differential scanning calorimetry (DSC) and the susceptibility of ovalbumin to α-chymotrypsin gave a quantitative estimation of protein denaturation in solid ovalbumin. Solid ovalbumin in granules with 11% of water was heated at 100 °C in closed and nonclosed ampules. In order to compare effects of size and crystal structure, two proteins (bovine albumin and γ-globulin) were examined at similar conditions for the extent of denaturation. Ovalbumin and bovine albumin showed similar extents of denaturation, but γ-globulin, with a very different molecular mass, showed the maximal conformational changes. The enthalpy of denaturation was measured to elucidate the conformational changes in solid proteins. Its value was used for calculation of the degree of denaturation. The thermodynamic data associated with transition were calculated and the number of bonds broken during denaturation was determined. Intrinsic fluorescence was utilized in order to compare these two methods. Moreover, X-ray diffraction and FTIR spectrometry were applied to native and denatured proteins.     

Appraisal of anti-arthritic and nephroprotective potential of Cuscuta reflexa

Context: Cuscuta reflexa Roxb. (Cuscutaceae) has been used traditionally for treating sore knees and kidney problems, but its efficacy has not been scientifically examined in treating arthritis and nephrotoxicity.

Objective: Present study determines antiarthritic and nephroprotective potential of the aqueous methanolic extract of Cuscuta reflexa (AMECR).

Materials and methods: Antiarthritic activity of Cuscuta reflexa in formaldehyde and turpentine oil-induced rat arthritis models was appraised at 200, 400 and 600?mg/kg doses for 10 days and 6?h period, respectively, and in vitro protein denaturation (bovine serum albumin, egg albumin) inhibition was studied at 25–800?μg/mL concentration. The nephroprotective effect involved gentamicin-induced nephrotoxicity in rats at 200, 400 and 600?mg/kg doses.

Results: Plant extract at 600?mg/kg significantly reduced paw oedema and joint swelling with maximal inhibition of 71.22% at the 6th hour for turpentine oil and 76.74% on 10th day for formaldehyde. Likewise, in vitro results corroborated significant concentration-dependent increase in percentage protection at 800?μg/mL against both bovine serum albumin (89.30%) and egg albumin (93.51%) denaturation. Similarly, 600?mg/kg dose showed maximum nephroprotection by reducing serum urea (41.400?±?0.510?mg/dL), uric acid (0.740?±?0.032?mg/dL), blood urea nitrogen (18.370?±?0.328), creatinine (3.267?±?0.076) and minimizing kidney weight gain (0.586?±?0.005) and histopathological alterations on 8th day. Furthermore, phytochemical and HPLC analysis revealed the presence of important phytoconstituents.

Discussion and conclusions: These results suggest that AMECR provides protection against arthritis and nephrotoxicity that might be due to the existence of phytoconstituents, thus supporting folkloric claim.     

Structural and ligand-binding properties of serum albumin species interacting with a biomembrane interface

In the process of drug development, preclinical testing using experimental animals is an important aspect, for verification of the efficacy and safety of a drug. Serum albumin is a major binding protein for endogenous and exogenous ligands and regulates their distribution in various tissues. In this study, the structural and drug-binding properties of albumins on a biomembrane surface were investigated using reverse micelles as a model membrane. In reverse micelles, the secondary structures of all albumins were found, to varying degrees, to be intermediate between the native and denatured states. The tertiary structures of human and bovine albumin were similar to those of the native and intermediate states, respectively, whereas those of the dog, rabbit, and rat were in a denatured state. Thus, bovine albumin is an appropriate model for studying structural changes in human albumin in a membrane-water phase. Binding studies also showed the presence of species difference in the change in binding capacity of albumins during their interaction with reverse micelles. Among the albumins, rat albumin appears to be a good model for the protein-mediated drug uptake of human albumin in a biomembrane environment. These findings are significant in terms of the appropriate extrapolation of pharmacokinetics and pharmacodynamics data in various animals to humans.     

Interleukin-1 Receptor (IL-1R) Liquid Formulation Development Using Differential Scanning Calorimetry

Purpose. To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC). Methods. Optimal pH conditions were determined by monitoring degradation products encountered during accelerated studies (at elevated temperatures) using SDS-PAGE. At the pH optimum, DSC screened for excipients that enhanced thermal stability by shifting the Tm to higher values. Using SEC the relationship between thermal unfolding and stability was investigated by considering if lower Tm's in the presence of preservatives correlated with degradation products at 37°C over time. The degree of aggregation relative to that of a control determined the level of stability achieved. Results. Circular dichroism (CD) measurements confirmed molecular modeling studies showing IL-1R to be about 39% -sheet. Two major transitions characterized the DSC data with Tm's observed near 47°C and 66°C. Among 21 excipients screened, NaCl exhibited the greatest stabilizing influences based on shifting the low temperature transition to 53°C. The low temperature transition was later found to comprise two transitions, yielding a total of three melting transitions for IL-1R. High Tm's arising from the presence of preservatives correlated with the order of stability (i.e., 0.065% phenol > 0.1% m-Cresol > 0.9% benzyl alcohol). Conclusions. The three melting transitions are consistent in origin with the cooperative unfolding of three unique immunoglobulin-like domains of IL-1R. Optimal stability was achieved in 20 mM sodium citrate at pH 6 with sufficient NaCl to attain the tonicity of human serum. A correlation between the predicted ranking of stability and the extent of aggregation was demonstrated using DSC.     

A comparison of drug binding sites on mammalian albumins.

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.     

In Vitro and in Vivo Properties of Recombinant Human Serum Albumin from Pichia Pastoris Purified by a Method of Short Processing Time

Purpose. Recombinant human serum albumin (rHSA), secreted by a Pichia pastorisexpression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. Methods. Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with ¹²⁵I-labeled albumin. Results. Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. Conclusion. The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.     

Albumin-induced changes of fluorescence spectra of 1-pyrenebutyric acid

Changes in the fluorescence spectra and quantum yield of 1-pyrenebutyric acid (1) induced by human, bovine, rabbit, and dog serum albumins and healthy adult serum, liver-disease patient serum, and renal-disease patient serum were compared at pH 7.4. Human serum albumin, healthy adult serum, and liver-disease patient serum caused marked quenching of the fluorescence of 1-pyrenebutyric acid. On the contrary, bovine, rabbit, and dog serum albumins increased the fluorescence intensity. Renal-disease patient serum also enhanced the intensity but caused quenching of the fluorescence after treatment with charcoal. Thus, 1-pyrenebutyric acid clearly shows interspecies differences in the environment or the structure of the binding site on serum albumin between humans and other animals. Furthermore, it is suggested that some substance which influences the character of the binding site for 1-pyrenebutyric acid exists in the serum of renal-disease patients.     

The binding of salicylate to plasma protein from several animal species

A comparison has been made of the binding of salicylate to plasma from man, green monkey, rabbit, rat, dog, and guinea-pig. Since the total protein content of normal, citrated, pooled plasmas of these species was not identical, comparisons were made with samples adjusted to the same total protein concentration. The % binding to plasmas of 5·3% total protein at 50 μg of drug/ml was found to be: man, 72%; green monkey, 70%; guinea pig, 64%; rabbit, 64%; rat, 47%; dog, 45%. Results for 150 μg and 500 μg of salicylate/ml are also reported. The albumin: globulin ratio as determined by electrophoresis varied widely among the species. Fractionation of the plasmas and comparison of binding, using 3% solutions of the various albumin fractions in buffer, indicated that the low binding of dog and rat plasmas was due primarily to the low binding affinity of the albumins. Adequate characterization of plasma samples is needed when comparative binding studies are made.     

Effects of Additives on Heat Denaturation of rhDNase in Solutions

Purpose. To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. Methods. Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T_m) and enthalpy (H_m) of rhDNase. The effects of denaturants (guanidine HC1 and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6–7. Results. The T_m and H_m of rhDNase in pure water were measured as 67.4 °C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca²⁺ stabilizes rhDNase against thermal denaturation and elevates T_m and H_m while Mg²⁺, Mn²⁺ and Zn²⁺ destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T_m and H_m. All destabilization of rhDNase can be prevented by adding Ca²⁺ to the solutions. Conclusions. CaCl₂ and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HC1 destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca²⁺ the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.     

THERMAL TRANSITIONS OF ACYLATED RIBONUCLEASE

The amino groups of ribonuclease have been specifically acylated using five N-acyl-succinimides in which the RCO-moieties varied in hydrocarbon chain length. The effects of these groups on the optical rotatory dispersion, circular dichroism, enzymic activity and stability to thermal denaturation have been determined. Thermal transitions, measured by changes in specific rotation at 365 nm, obeyed the criteria for a two-state process both before and after the acylation treatments. The transitions were fully reversible under the conditions of measurement and the observed standard enthalpies were constant over at least 80% of the transitions. The effect of acetylation was to decrease the transition temperature by 7°. Longer hydrocarbon chains decreased this further but only by a fraction of a degree per extra -CH₂- group. The calculation of thermodynamic quantities is limited by the precision with which enthalpies can be measured but indicates that the standard free energy for the transition process decreases by about 270 cal per acetyl group and a further 20 cal per additional -CH₂- group. The major source of destabilisation on acetylation appears to be the introduction of eleven hydrophobic CH₃ groups per protein molecule rather than the increase in net negative charge. The thermodynamic data permit inferences to be drawn about changes in the nature of the side chain environment on thermal denaturation.     

Species Differences in Stereoselective Hydrolase Activity in Intestinal Mucosa

Purpose. The aim of this study is to investigate species differences in the stereoselective hydrolysis for propranolol ester prodrugs in mammalian intestinal mucosa and Caco-2 cells. Methods. Hydrolase activities for propranolol prodrugs and p-nitro-phenylacetate in man (age: 51–71 years), the beagle dog (age: 4 years) and Wistar rat (age: 8 weeks) intestinal mucosa, and also in Caco-2 cells (passage between 60–70) were estimated by determining the rate of production of propranolol and p-nitrophenol, respectively. Results. The hydrolase activities for both propranolol prodrugs and p-nitrophenylacetate were in the order of man > rat >> Caco-2 cells > dog for intestinal microsomes, and rat > Caco-2 cells = man > dog for intestinal cytosol. Dog microsomes showed stereoselective hydrolysis for propranolol prodrugs, but not those from human or rat. Interestingly, both subcellular fractions of Caco-2 cells showed remarkable R-enantioselectivity except acetyl propranolol. Enzyme kinetic experiments for each enantiomer of butyryl propranolol in microsomes revealed that dog possesses both low and high affinity hydrolases. Both Km and Vmax values in rat were largest among examined microsomes, while Vmax/Km was largest in man. Finally, it was shown that the carboxylesterases might contribute to the hydrolysis of propranolol prodrug in all species by inhibition experiments. Conclusions. The hydrolase activities for propranolol prodrugs and p-nitrophenylacetate in intestinal mucosa showed great species differences and those in human intestine were closer to those of rat intestine than dog intestine or Caco-2 cells.     

Reversibility of Heat-Induced Denaturation of the Recombinant Human Megakaryocyte Growth and Development Factor

Purpose. The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF). Methods. Changes in the far U V CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37°C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined. Results. In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4°C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37°C in imidazole than in phosphate, in both the presence and absence of sucrose. Conclusions. These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.     

Circular dichroism studies on the interaction of four structurally related long-acting sulfonamides with human and bovine serum albumin

The interaction of four sulfonamides with bovine and human serum albumin (BSA and HSA) was investigated by means of circular dichroism and u.v. absorbance measurements. In the case of all sulfonamides, extrinsic Cotton effects could be found for the interaction with BSA and HSA. The anisotropy factors of the electronic transitions in the p-amino benzenesulfonic acid and in the heterocyclic moieties of the drugs do not differ, therefore it is concluded that both parts of the sulfonamide molecules, the p-amino benzenesulfonic acid part and the heterocyclic ring are bound to the albumin surface. The circular dichroism measurements especially reveal some differences of the interaction of the sulfonamides with both albumins.     

Interdomain Interactions in the Chimeric Protein Toxin sCD4(178)-PE40: A Differential Scanning Calorimetry (DSC) Study

The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC). sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40). sCD4-PE40 undergoes two DSC transitions. An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46°C in buffered saline at pH 6.5. An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures. Both transitions are irreversible. DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5. Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities. sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40. PE40 denaturation occurs at the same temperatures as sCD4-PE40. These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component. These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40). Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.Brian E. Collins: B.E.C.'s contribution was made while he was a summer intern at The Upjohn Company.     

Further Evidence Regarding the Important Role of Chlorine Atoms of Aripiprazole on Binding to the Site II Area of Human Albumin

Previously, we reported on the high-affinity binding of aripiprazole (ARP), an antipsychotic drug, to human albumin and the role of the chlorine atom of ARP on this binding. In this study, we investigated the binding mode of ARP to human albumin in detail using ARP derivatives and several animal-derived albumins. ARP bound strongly to human and dog albumin. The circular dichroism (CD) spectra of ARP bound to human and dog albumin were also similar. Deschloro-ARP bound less strongly to all of the albumin species compared to ARP, and the shapes of CD spectra were similar for all albumin species. CD spectra of dimethyl-ARP, for which chlorine atoms were substituted methyl groups, were quite similar to that of deschloro-ARP. In displacement experiments, competitive binding was observed between ARP and deschloro-ARP. These results suggest that the chlorine atoms in ARP are involved in the binding modes of ARP for human and dog albumins, whereas ARP and deschloro-ARP appear to share the same binding region in site II. The aforementioned results imply that compounds having a chlorine atom bind more strongly to plasma proteins, resulting in a long blood retention time. Therefore, findings reported here may provide the basically useful data for drug design.     

Phosphotriesterase activity identified in purified serum albumins

The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates. Received: 10 September 1997 / Accepted: 17 November 1997     

Biotransformation of sultopride in man and several animal species

1. The biotransformation of sultopride has been investigated in rat, rabbit, dog and man.

2. In man sultopride was metabolically stable, and about 90% of an oral dose was excreted in urine unchanged and 4% as oxo-sultopride.

3. Rat, rabbit and dog metabolized sultopride more extensively and excreted less than 40% of an oral dose of ¹⁴C-sultopride in urine.

4. Four similar metabolites were excreted by the three animal species but the relative portions differed. The major radioactive component in rat urine was O-desmethyl sultopride, whereas oxo-sultopride and O-desmethyl sultopride were the major urinary metabolites in rabbit. Dog formed N-desethyl sultopride and oxo-sultopride as major urinary metabolites.

5. The male rat excreted smaller amounts of unchanged sultopride in urine than did the female rat.

6. The unchanged sultopride excreted in rat urine was increased slightly by repeated administration.     

Prolonged Circulation of Recombinant Human Granulocyte–Colony Stimulating Factor by Covalent Linkage to Albumin Through a Heterobifunctional Polyethylene Glycol

Purpose. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t_1/2) of rhG-CSF. Methods. Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 µg/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA. Results. The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 ± 0.121 ml/mm/kg for rhG-CSF to 0.172 ± 0.013 ml/min/kgfor RSA-PEG-rhG-CSF and 0.141 ± 0.005 ml/mm/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37°C decreased five fold. Conclusions. The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.