Catharina Smit

Charge modification of plasma and milk proteins results in antiviral active compounds

Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti‐HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available εNH2 groups were anionized yielded the most potent anti‐HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti‐HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120–CD4 mediated virus/cell binding. The relative contribution of these effects to the anti‐HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti‐HIV activity but a clearly increased anti‐HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti‐HIV and anti‐HCMV activity, respectively. The N‐terminal portion of Lf appeared essential for its anti‐HCMV effect: N‐terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications. Copyright

Homing of negatively charged albumins to the lymphatic system: general implications for drug targeting to peripheral tissues and viral reservoirs.

The present study shows the lymphatic distribution of the negatively charged anti-HIV-1 agents succinylated or aconytilated human serum albumins (HSAs) in rats. Quantitation of blood and lymphatic concentrations of these proteins was performed through fluorescence detection of the fluorescein isothiocyanate (FITC)-labeled proteins. At several time points after i.v. injection, samples were taken from the cannulated thoracic duct and the carotid artery. Distribution of the negatively charged albumins (NCAs) to lymph was much more rapid than that of albumin itself and was dependent on the total net negative charge added to the protein: the half-life times of lymphatic equilibration were 15, 30, and 120 min for FITC-labeled aconytilated HSA, FITC-labeled succinylated HSA, and FITC-labeled HSA, respectively. Lymph to blood concentration ratios of the studied compounds obtained at steady state approached unity. In addition, the fluorescence in both body fluids was shown to represent unchanged labeled proteins. It was therefore inferred that the NCAs efficiently passed the endothelial barrier from blood to the interstitial compartment. Subsequently, we studied whether a specialized process was involved in the endothelial passage of the NCAs to the lymph. The following observations supported such a mechanism: a) preinjection of the scavenger receptor blockers polyinosinic- and formaldehyde-treated HSA reduced the transport from blood to the lymphatic compartment of FITC-labeled aconytilated HSA by more than 90%; b) the rate of lymphatic distribution was largely reduced when the body temperature of the rat was lowered to 28 degrees; and c) pre-administration of chloroquine resulted in a significant reduction in the lymphatic distribution of the NCAs. These data collectively indicate that a scavenger receptor-mediated process is involved in the transendothelial transport of NCAs. In situ localization in lymph nodes of the rat showed that FITC-labeled aconytilated and succinylated HSA are mainly present in the germinal center and parafollicular zones. The efficient distribution of these anionized proteins to the lymphatic system is of particular interest for HIV therapy, taking into account that replication of HIV mainly takes place in the lymphoid system. The observation that macromolecules, through charge modification, can extravasate through a receptor-mediated transcytotic process is potentially of major importance for the delivery of drugs with macromolecular carriers to cells not directly in contact with the blood.

Targeting of superoxide dismutase to the liver results in anti-inflammatory effects in rats with fibrotic livers

BACKGROUND/AIMS The rapid clearance from plasma and the limited uptake of superoxide dismutase (SOD) in the liver hampers the effectiveness of this enzyme in liver diseases. We therefore compared the pharmacokinetics and in vivo efficacy of SOD with two modified forms of this protein: SOD coupled to the copolymer DIVEMA and mannosylated-SOD. METHODS Reactive oxygen scavenging activity of SOD conjugates was tested in livers of bile duct ligated rats. Intrahepatic production of reactive oxygen species (ROS) and neutrophil infiltration were studied immunohistochemically and related to the organ and cellular distribution of radiolabeled SOD conjugates. RESULTS Native SOD was rapidly cleared from the circulation and accumulated in renal tubuli. The enzyme had no effect on the intrahepatic ROS production. Covalent attachment of SOD to DIVEMA yielded a polyanionic conjugate with a prolonged elimination half-life compared to native SOD. In contrast to native SOD, DIVEMA-SOD was taken up by the liver via scavenger receptors. Mannosylation of SOD (Man-SOD) resulted in a conjugate that was rapidly cleared from the blood. This Man-SOD was taken up by non-parenchymal liver cells. The pharmacokinetics of SOD and its derivatives were similar in normal and bile duct ligated rats. Efficacy studies with Man-SOD revealed only a slight decrease in intrahepatic ROS production. However, DIVEMA-SOD exhibited a potent inhibitory effect on ROS production in the liver. Nearly complete ROS-scavenging activity was observed in the portal areas. CONCLUSIONS Considering the prolonged half-life, the increased delivery of SOD to the target cells, and the concomitant increased effectiveness, application of DIVEMA-SOD seems a promising new approach to attenuate intrahepatic inflammatory processes.

Comparative pharmacokinetic, immunologic and hematologic studies on the anti-HIV-1/2 compounds aconitylated and succinylated HSA

Charge modification by succinylation or cis-aconitylation of the terminal epsilon NH2 functions of the amino acid lysine in human serum albumin, resulted in polyanionic compounds with an anti-HIV-1 activity in the low nanomolar concentration range. After iv injections in rats of the negatively charged albumins (NCAs), a dose dependent elimination pattern was observed indicating a saturable eliminations pathway. The Michaelis-Menten parameters Vmax and K(m) were 62 +/- 8 micrograms.min-1.kg-1 and 16 +/- 4 micrograms.ml-1 (Clintr 3.9 +/- 1.1 ml.min-1.kg-1) and 74 +/- 6 micrograms.min-1.kg-1 and 11 +/- 2 micrograms.ml-1 (Clintr 6.7 +/- 1.2 ml.min-1.kg-1) for aconitylated-HSA (Aco-HSA) and succinylated-HSA (Suc-HSA) respectively, using 125I-labelled proteins. The volume of distribution (V) of both compounds was approximately 60 ml.kg-1. Coadministration of poly-inosinic acid and formaldehyde treated albumin showed a marked inhibition of blood clearance indicating that the compounds are mainly cleared from the bloodstream by scavenger receptors on liver and spleen endothelial cells and macrophages. The Michaelis-Menten constant K(m) was remarkably higher when the hydrophobic flurophore fluorescein was covalently linked to the protein, indicating that the affinity for the scavenger receptors is largely decreased by FiTC conjugation. The latter observation may have implications for the kinetic behavior of drug-carrier preparations if antiviral drugs like AZT or PMEA are linked to these intrinsic active carriers. In contrast to other polyanionic compounds like heparins and dextran sulfate, these NCAs did not exhibit acute toxicity and had no effect on blood coagulation. They neither had an effect on the lymphocyte proliferation. Studies on immunogenicity of the homologous derivatized albumins in rats did not show a significant response. The present pharmacokinetic and toxicologic data of Suc-HSA and Aco-HSA show that both compounds are interesting preparations for studies in SIV infected monkeys and AIDS patients.

Pharmacokinetics of Succinylated Serum Albumin in Wistar Rats and Cynomolgus Monkeys: Implications for Dosage Regimens in the Therapy of HIV Infection

Succinylation of serum albumin produces a negatively charged protein with a potent anti-HIV-1/2 and anti-SIVmac activity. The in vitro IC50 values of Suc-HSA against the respective primate lentiviruses are in the low nanomolar concentration range. Succinylated homologous serum albumin was given intravenously at four different doses, ranging from 0.1 to 10 mg/kg to Wistar rats and Cynomolgus monkeys. Plasma samples were assayed for their drug content using iodinated proteins. The pharmacokinetics parameters were calculated by a single compartment model, taking into account a potential saturable elimination process. In rats as well as in monkeys succinylated serum albumin clearly showed dose-dependent kinetics. The rate of Suc-SSA elimination from the bloodstream in macaques could be described by a Vmax of 11.7 ± 0.2 µg/min kg-(1) and a K. of 0.40 ± 0.06 µg/mL (5.5 nM). The elimination of Suc-RSA in rats was characterized by a 10-fold higher V. of 112 ± 29 µg/min kg(-1) and a much higher K. of 25 ± 9 µg/mL (340 nM). The volume of distribution was about the plasma volume for both species. In rats, no significant differences were found between the kinetic parameters of Suc-RSA, Suc-HSA, or Suc-SSA. Histochemical staining of tissue sections obtained from the liver, spleen, kidneys, and different lymph nodes showed that endothelial cells and macrophages from the liver and spleen are involved in the clearance of the negatively charged albumins. Since replication of HIV mainly takes place in the lymphoid tissue, uptake of succinylated albumin in this system may imply an interesting therapeutic aspect of the negatively charged albumins.

The antiviral protein human lactoferrin is distributed in the body to Cytomegalovirus (CMV) infection-prone cells and tissues

AbstractPurpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. Methods and Results. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels, was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs, mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. Conclusions. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.

The metabolic fate of the Anti-HIV active drug carrier succinylated human serum albumin after intravenous administration in rats.

The pharmacokinetics and metabolic fate of the intrinsically active (anti-HIV) drug carrier succinylated human serum albumin (Suc-HSA) was studied in rats. Suc-HSA was prepared by derivatizing HSA with 1,4-[14C]-succinic anhydride, a modification by which all available εNH2-groups in HSA were converted into carboxylic groups. After iv injections of 0.3, 1.0, 3.0 and 10.0 mg/kg in freely moving rats, Suc-HSA showed a dose dependent elimination pattern, indicating a saturable elimination pathway. The Michaelis-Menten parameters Vmax and Km were 98.7 μg.min−1.kg−1 and 8.5 μg.ml−1 respectively. The kinetics of Suc-HSA was influenced by anaesthesia. In anaesthetised animals, Vmax and Km were found to be 26.9 μg.min−1.kg−1 and 0.26 μg.ml−1, respectively. This implies an intrinsic clearance of 100ml.min−1.kg−1, which is about 10-fold higher as compared to 12 ml.min−1.kg−1 in freely moving animals. Intravenous administration of a sub-saturable dose of 3.0mg.kg−11,4-[14C]-Suc-HSA to freely moving rats resulted in a biphasic elimination with an initial t1/2 of 20 min and a terminal t1/2 of 40 hrs. Excretion of metabolites in urine and faeces lasted for at least 48 hours. About 70% of the radioactive dose was excreted in urine, whereas maximally 2% was detected in faeces. Suc-HSA was degraded to its individual amino acids including succinylated lysine (the only radioactive product formed). Succinylated lysine was not further metabolised and mainly excreted via the urine. Immunohistochemical staining showed that even after 48 hrs Suc-HSA could be detected in livers. Together with the urinary excretion patterns, this points to a gradual degradation of Suc-HSA.