Hester I. Bakker

Site-Specific Inhibition of Glomerulonephritis Progression by Targeted Delivery of Dexamethasone to Glomerular Endothelium

Glomerulonephritis represents a group of renal diseases with glomerular inflammation as a common pathologic finding. Because of the underlying immunologic character of these disorders, they are frequently treated with glucocorticoids and cytotoxic immunosuppressive agents. Although effective, use of these compounds has limitations as a result of toxicity and systemic side effects. In the current study, we tested the hypothesis that targeted delivery of dexamethasone (dexa) by immunoliposomes to activated glomerular endothelium decreases renal injury but prevents its systemic side effects. E-selectin was chosen as a target molecule based on its disease-specific expression on activated glomerular endothelium in a mouse anti-glomerular basement membrane glomerulonephritis. Site-selective delivery of AbEsel liposome-encapsulated dexamethasone strongly reduced glomerular proinflammatory gene expression without affecting blood glucose levels, a severe side effect of administration of free dexamethasone. Dexa-AbEsel liposomes reduced renal injury as shown by a reduction of blood urea nitrogen levels, decreased glomerular crescent formation, and down-regulation of disease-associated genes. Immunoliposomal drug delivery to glomerular endothelium presents a powerful new strategy for treatment of glomerulonephritis to sustain efficacy and prevent side effects of potent anti-inflammatory drugs.

Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener’s granulomatosis?

The majority of patients with Wegener’s granulomatosis (WG) are chronic nasal carriers of Staphylococcus aureus. Chronic nasal carriage of S. aureus is associated with an increased risk of developing a relapse of the disease. The mechanism by which this occurs is still unknown. We hypothesized that a cationic protein of S. aureus, staphylococcal acid phosphatase (SAcP), acts as a planted antigen and initiates glomerulonephritis and vasculitis in patients with WG. In order to test the hypothesis that SAcP can act as a planted antigen in WG, we studied the ability of SAcP to bind to human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells. We also studied whether this binding can be prevented by preincubation with an anionic protein, and whether binding of SAcP activates endothelial cells. We also evaluated whether antibodies in sera of patients with WG are able to bind to endothelial cell‐bound SAcP. The results show that SAcP can act as a planted antigen by binding to both types of endothelial cells in a concentration‐dependent manner. Binding of concentrations as low as 4 μg/ml can be detected on HUVEC within 5 min of incubation. Binding of SAcP to endothelial cells was charge‐dependent but did not activate endothelial cells. Finally, endothelial cell‐bound SAcP was recognized by sera of patients with WG. The data suggest a possible pathogenic role for SAcP by acting as a planted antigen thereby initiating glomerulonephritis and vasculitis in patients with WG.

The Antiproliferative Drug Doxorubicin Inhibits Liver Fibrosis in Bile Duct-Ligated Rats and Can Be Selectively Delivered to Hepatic Stellate Cells in Vivo

Hepatic stellate cell (HSC) proliferation is a key event in liver fibrosis; therefore, pharmacological intervention with antiproliferative drugs may result in antifibrotic effects. In this article, the antiproliferative effect of three cytostatic drugs was tested in cultured rat HSC. Subsequently, the antifibrotic potential of the most potent drug was evaluated in vivo. As a strategy to overcome drug-related toxicity, we additionally studied how to deliver this drug specifically to HSC by conjugating it to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA). We investigated the effect of cisplatin, chlorambucil, and doxorubicin (DOX) on 5-bromo-2′-deoxyuridine incorporation in cultured HSC and found DOX to be the most potent drug. Treatment of bile duct-ligated (BDL) rats with daily i.v. injections of 0.35 mg/kg DOX from day 3 to 10 after BDL reduced α-smooth muscle actin-stained area in liver sections from 8.5 ± 0.8 to 5.1 ± 0.9% (P < 0.01) and collagen-stained area from 13.1 ± 1.3 to 8.9 ± 1.5% (P < 0.05). DOX was coupled to M6PHSA, and the organ distribution of this construct (M6PHSA-DOX) was investigated. Twenty minutes after i.v. administration, 50 ± 6% of the dose was present in the livers, and colocalization of M6PHSA-DOX with HSC markers was observed. In addition, in vitro studies showed selective binding of M6PHSA-DOX to activated HSC. Moreover, M6PHSA-DOX strongly attenuated HSC proliferation in vitro, indicating that active drug is released after uptake of the conjugate. DOX inhibits liver fibrosis in BDL rats, and HSC-selective targeting of this drug is possible. This may offer perspectives for the application of antiproliferative drugs for antifibrotic purposes.

Design and Fungicidal Activity of Mucoadhesive Lactoferrin Tablets for the Treatment of Oropharyngeal Candidosis

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7.5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata. In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.

Synergy of bovine lactoferrin with the anti-cytomegalovirus drug cidofovir in vitro

UNLABELLED Human cytomegalovirus (HCMV) causes severe morbidity and mortality in immunocompromised patients. Treatment of HCMV infections with conventional antiviral drugs like ganciclovir and cidofovir has major drawbacks (i.e. serious side effects). Therefore, combination therapies using drugs with different antiviral mechanisms should be envisaged. Potential synergy between lactoferrin (LF), an antibacterial, antimycotic and antiviral protein, and the antiviral drugs acyclovir, ganciclovir, foscarnet and cidofovir was investigated, using an in vitro test system with the recombinant RC256 HCMV strain. RESULTS Combination of LF with acyclovir and foscarnet resulted in antagonism. When LF and ganciclovir were combined, neither synergy nor antagonism was observed. Strikingly, the combination of LF with cidofovir resulted in marked synergy. The synergistic effect could be explained by inhibition of two subsequent steps in the viral replication cycle: HCMV penetration into the target cells and intracellular synthesis of HCMV DNA. In conclusion, LF might be a potential candidate for combination therapy with cidofovir.

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.