Reduction of advanced liver fibrosis by short-term targeted delivery of an angiotensin receptor blocker to hepatic stellate cells in rats.

UNLABELLED: There is no effective therapy for advanced liver fibrosis. Angiotensin type 1 (AT1) receptor blockers attenuate liver fibrogenesis, yet their efficacy in reversing advanced fibrosis is unknown. We investigated whether the specific delivery of an AT1 receptor blocker to activated hepatic stellate cells (HSCs) reduces established liver fibrosis. We used a platinum-based linker to develop a conjugate of the AT1 receptor blocker losartan and the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin (losartan-M6PHSA). An average of seven losartan molecules were successfully coupled to M6PHSA. Rats with advanced liver fibrosis due to prolonged bile duct ligation or carbon tetrachloride administration were treated with daily doses of saline, losartan-M6PHSA, M6PHSA or oral losartan during 3 days. Computer-based morphometric quantification of inflammatory cells (CD43), myofibroblasts (smooth muscle alpha-actin [alpha-SMA]) and collagen deposition (Sirius red and hydroxyproline content) were measured. Hepatic expression of procollagen alpha2(I) and genes involved in fibrogenesis was assessed by quantitative polymerase chain reaction. Losartan-M6PHSA accumulated in the fibrotic livers and colocalized with HSCs, as assessed by immunostaining of anti-HSA and anti-alpha-SMA. Losartan-M6PHSA, but not oral losartan, reduced collagen deposition, accumulation of myofibroblasts, inflammation and procollagen alpha2(I) gene expression. Losartan-M6PHSA did not affect metalloproteinase type 2 and 9 activity and did not cause apoptosis of activated HSCs. CONCLUSION: Short-term treatment with HSC-targeted losartan markedly reduces advanced liver fibrosis. This approach may provide a novel means to treat chronic liver diseases.

RGD-avidin-biotin pretargeting to alpha v beta 3 integrin enhances the proapoptotic activity of TNF alpha related apoptosis inducing ligand (TRAIL).

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL's potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin-biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity.

Organ- and cell-type specific delivery of kinase inhibitors: a novel approach in the development of targeted drugs.

During the past years, we have explored the cellular delivery of kinase inhibitors. Kinase inhibitors have selectivity for specific kinases but they lack cellular selectivity. This is exemplified by recent reports on cardiotoxicity of kinase inhibitors used in cancer treatment. We postulate that targeted cellular delivery of kinase inhibitors can improve their safety/toxicity profiles, as will be exemplified by recent published studies. Cell specific delivery of therapeutics is a quickly growing area of investigation. This innovative strategy employs carrier molecules that bind to receptors exposed on the surface of cell types involved in disease processes. Binding and receptor mediated internalization of the carrier facilitates local accumulation of the product in target cells. Upon systemic administration, this may create local drug depots in specific organs, while other tissues are avoided, thus favoring enhanced localized drug efficacy and reduced side-effects. Synthesis of targeted kinase inhibitor-carrier conjugates was achieved using a new approach, in which kinase inhibitors were bound to a platinum(II) atom, the so-called Universal Linkage System (ULS). We review this novel linkage chemistry and demonstrate the applicability of ULS for drug targeting approaches aiming at angiogenic endothelial cells, hepatic stellate cells, and kidney tubular cells. We will review important issues like drug release mechanism, safety of the linker, and pharmacokinetics of the products in animals. Finally, we review the pharmacological efficacy of the cellular targeted drug conjugates in experimental animal models, especially in renal and liver fibrosis models.

Improved efficacy of alphavbeta3-targeted albumin conjugates by conjugation of a novel auristatin derivative.

Cellular handling of drug delivery preparations en route to the lysosomal compartment has been extensively studied, but little is known about cellular handling of drugs subsequent to their release from the delivery system. We studied a series of closely related drug targeting conjugates, consisting of albumins equipped with alpha vbeta 3-selective RGD-peptide homing ligands, PEG stealth domains, and either the antitubulin agent monomethyl auristatin E (MMAE) or a new F-variant (MMAF). Since MMAF has a C-terminal charge, this compound is potentially less prone to passive redistribution after its release from the carrier. We demonstrate that RGD-peptide-equipped albumin conjugates with MMAF were indeed more potent than MMAE conjugates, in killing both alpha vbeta 3-positive tumor cells and proliferating endothelial cells. Efficacy increased more in tumor cells than in endothelial cells, suggesting different drug redistribution behavior for the two cell types. Binding affinity and uptake of the conjugate and the cellular handling of released drug contributed to the final efficacy of drug-carrier conjugates, highlighting the importance of all aspects to be carefully considered in the design of targeted drug delivery preparations.

Local inhibition of liver fibrosis by specific delivery of a platelet-derived growth factor kinase inhibitor to hepatic stellate cells.

Liver fibrosis is characterized by excessive proliferation and activation of hepatic stellate cells (HSC), a process in which platelet-derived growth factor (PDGF) plays an important role. Inhibition of liver fibrosis via specific delivery of a PDGF kinase inhibitor to HSC might therefore be an attractive strategy. The HSC-selective carrier mannose-6-phosphate modified human serum albumin (M6PHSA) was equipped with a tyrosine kinase inhibitor, 4-chloro-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (PAP19) (an imatinib derivative), by means of the platinum-based universal linkage system (ULS). The antifibrotic activity of PAP19-M6PHSA was evaluated in culture-activated rat HSC and precision-cut liver slices from fibrotic rats. After 24-h incubation, both free inhibitor PAP19 and PAP19-M6PHSA showed potent activity, as determined by quantitative reverse transcription-polymerase chain reaction analysis of alpha-smooth muscle actin (alphaSMA) and procollagen 1a1. Next, we examined the organ distribution and antifibrotic activity of PAP19-M6PHSA in bile duct-ligated (BDL) rats. Male Wistar rats at day 10 after BDL were administered a single dose of PAP19-M6PHSA and sacrificed at 2 h, 1 day, or 2 days afterward. The accumulation of PAP19-M6PHSA in the liver was quantified by high-performance liquid chromatography analysis (30% of the injected dose at 2 h) and detected in the liver by staining of the carrier. Liver drug levels were sustained at 24 and 48 h after the single dose. Furthermore, PAP19-M6PHSA reduced collagen deposition (Sirius red staining) and alphaSMA staining of activated HSC at these time points in comparison with saline-treated rats. We therefore conclude that delivery of a PDGF-kinase inhibitor to HSC is a promising technology to attenuate liver fibrogenesis.

Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels.

Induction of apoptosis in endothelial cells is considered an attractive strategy to therapeutically interfere with a solid tumor's blood supply. In the present paper, we constructed cytotoxic conjugates that specifically target angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the potent antimitotic agent monomethyl-auristatin-E (MMAE) via a lysosomal cleavable linker to human serum albumin (HSA) and further equipped this drug-albumin conjugate with cyclic c(RGDfK) peptides for multivalent interaction with alphavbeta3-integrin. The RGD-peptides were conjugated via either an extended poly(ethylene glycol) linker or a short alkyl linker. The resulting drug-targeting conjugates RGDPEG-MMAE-HSA and RGD-MMAE-HSA demonstrated high binding affinity and specificity for alphavbeta3-integrin expressing human umbilical vein endothelial cells (HUVEC). Both types of conjugates were internalized by endothelial cells and killed the target cells at low nM concentrations. Furthermore, we observed RGD-dependent binding of the conjugates to C26 carcinoma. Upon i.v. administration to C26-tumor bearing mice, both drug-targeting conjugates displayed excellent tumor homing properties. Our results demonstrate that RGD-modified albumins are suitable carriers for cell selective intracellular delivery of cytotoxic compounds, and further studies will be conducted to assess the antivascular and tumor inhibitory potential of RGDPEG-MMAE-HSA and RGD-MMAE-HSA.

Delivery of the p38 MAPkinase inhibitor SB202190 to angiogenic endothelial cells: development of novel RGD-equipped and PEGylated drug-albumin conjugates using platinum(II)-based drug linker technology.

Endothelial cells play an important role in inflammatory disorders, as they control the recruitment of leukocytes into inflamed tissue and the formation of new blood vessels. Activation of p38MAP kinase results in the production of proinflammatory cytokines and the expression of adhesion molecules. P38MAP kinase inhibitors are therefore considered important candidates for the treatment of inflammatory disorders. In the present study, we propose a novel strategy to counteract these processes by delivery of the p38MAP kinase inhibitor SB202190 into angiogenic endothelial cells. A drug-targeting conjugate was developed by conjugation of SB202190 to human serum albumin (HSA) using a novel platinum-based linker. Specificity for angiogenic endothelial cells was introduced by conjugation of cyclic RGD-peptides via bifunctional polyethylene glycol linkers. The final products contained an average of nine SB202190 and six RGDPEG groups per albumin. The platinum-based linker displayed high stability in buffers and culture medium, but released SB202190 slowly upon competition with sulfur-containing ligands like glutathione. RGDPEG-SB-HSA bound to alpha(v3)-integrin expressing endothelial cells (human umbilical cord vein endothelial cells) with low nanomolar affinity and was subsequently internalized. When HUVEC were treated with TNF to induce inflammatory events, pretreatment with RGDPEG-SB-HSA partially inhibited proinflammatory gene expression (IL-8, E-selectin; 30% inhibition) and secretion of cytokines (IL-8, 34% inhibition). We conclude that the developed RGDPEG-SB-HSA conjugates provide a novel means to counteract inflammation disorders such as rheumatoid arthritis.

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.

RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature.

During the past decade, RGD-peptides have become a popular tool for the targeting of drugs and imaging agents to alphavbeta3-integrin expressing tumour vasculature. RGD-peptides have been introduced by recombinant means into therapeutic proteins and viruses. Chemical means have been applied to couple RGD-peptides and RGD-mimetics to liposomes, polymers, peptides, small molecule drugs and radiotracers. Some of these products show impressive results in preclinical animal models and a RGD targeted radiotracer has already successfully been tested in humans for the visualization of alphavbeta3-integrin, which demonstrates the feasibility of this approach. This review will summarize the structural requirements for RGD-peptides and RGD-mimetics as ligands for alphavbeta3. We will show how they have been introduced in the various types of constructs by chemical and recombinant techniques. The importance of multivalent RGD-constructs for high affinity binding and internalization will be highlighted. Furthermore the in vitro and in vivo efficacy of RGD-targeted therapeutics and diagnostics reported in recent years will be reviewed.