Andreas Wunder

Albumin-Based Drug Delivery as Novel Therapeutic Approach for Rheumatoid Arthritis

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.

The loading rate determines tumor targeting properties of methotrexate albumin conjugates in rats

Albumin dominates the plasma proteins in man. Following our observation that albumin turnover in rodent tumors is markedly increased, we will present evidence that albumin can be employed as an efficient carrier for targeting cytostatic agents like methotrexate (MTX) into tumors. The considerable discrepancy in the molecular weight of MTX (454 Da) and albumin (about 67 000 Da) tempted researchers to load multiple drug molecules on one carrier molecule. It was supposed that the optimal therapeutic efficacy of MTX protein conjugates could be achieved by increasing the number of the molecules of MTX attached to the carrier. In this paper we will show that only loading rates of close to 1 mol of the cytostatic drug MTX/mol of albumin offer optimal conditions for targeting MTX-albumin conjugates into rodent tumors. Conjugates bearing 5, 7,10 and 20 molecules of MTX on average showed considerable alterations in the HPLC profiles of the conjugates compared to albumin. Conjugates carrying 5-20 mol MTX, tagged with a residualizing radiolabel, were efficiently trapped by the liver before reaching the tumor. The tumor uptake rates of these conjugates declined dramatically with an increasing molecular load of the cytotoxic drug linked to albumin. Competition experiments with maleylated bovine serum albumin and fucoidan revealed that scavenger receptors present on the cells of the liver monocyte macrophage system were involved in this process. For further preclinical and clinical studies, we chose MTX-albumin conjugates, derivatized at a molar ratio of 1:1. These conjugates enjoy the same favorable tumor targeting properties like albumin, e.g. high tumor uptake rates, low liver uptake rates and a very long biological half-life

Near-infrared fluorescent probes for imaging vascular pathophysiology

Light in the near-infrared (NIR) region between 700–900 nm can penetrate deep into living tissue, thereby offering a unique opportunity to use near-infrared fluorescence (NIRF) imaging techniques to detect and visualize fluorescent probes in-vivo. In the past few years, many novel NIR fluorescent probes have been designed, synthesized and studied in a variety of disease conditions. Recent research has focused primarily on the class of cyanines dyes as non-specific agents and as part of specific NIR fluorescent probes. The publications reviewed herein discuss the characteristics of cyanine dyes and their conjugates and present examples for the application of these probes for imaging vascular pathophysiology.

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats.

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111ln]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/ kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.

Near-infrared fluorescence imaging with fluorescently labeled albumin: a novel method for non-invasive optical imaging of blood-brain barrier impairment after focal cerebral ischemia in mice.

Impairment of the blood-brain barrier (BBB) after cerebral ischemia leads to extravasation of plasma constituents into the brain parenchyma. We describe a novel method using non-invasive near-infrared fluorescence (NIRF) imaging and bovine serum albumin labeled with a NIRF dye (NIRF-BSA) to detect BBB impairment after middle cerebral artery occlusion (MCAO) in mice. We first explored the time course of BBB impairment after transient MCAO using Evans blue (EB), which binds to plasma albumin in vivo. An initial BBB impairment was observed at 4-8h and a second impairment at 12-16h after reperfusion. No EB extravasation was detected at 8-12h. Non-invasive NIRF imaging with NIRF-BSA confirmed biphasic BBB impairment. Upon co-injection of NIRF-BSA with EB we found a strong correlation between the detected NIRF signal and the amount of extravasated EB (r=0.857, P=0.00178). When MCAO mice received NIRF-BSA together with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA), T1-weighted images showed Gd-DTPA enhancement at all times while NIRF imaging showed biphasic BBB impairment. In conclusion, NIRF-BSA is a suitable marker of plasma albumin extravasation in the mouse brain. Non-invasive NIRF imaging with NIRF-BSA is a useful tool to study BBB integrity in preclinical models of central nervous system pathology.

In vivo near-infrared fluorescence imaging of matrix metalloproteinase activity after cerebral ischemia

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of cerebral ischemia. In this study, we explored whether MMP activity can be visualized by noninvasive near-infrared fluorescence (NIRF) imaging using an MMP-activatable probe in a mouse model of stroke. C57BI6 mice were subjected to transient middle cerebral artery occlusion (MCAO) or sham operation. Noninvasive NIRF imaging was performed 24 h after probe injection, and target-to-background ratios (TBRs) between the two hemispheres were determined. TBRs were significantly higher in MCAO mice injected with the MMP-activatable probe than in sham-operated mice and in MCAO mice that were injected with the nonactivatable probe as controls. Treatment with an MMP inhibitor resulted in significantly lower TBRs and lesion volumes compared to injection of vehicle. To test the contribution of MMP-9 to the fluorescence signal, MMP9-deficient (MMP9−/-) mice and wild-type controls were subjected to MCAO of different durations to attain comparable lesion volumes. TBRs were significantly lower in MMP9−/- mice, suggesting a substantial contribution of MMP-9 activity to the signal. Our study shows that MMP activity after cerebral ischemia can be imaged noninvasively with NIRF using an MMP-activatable probe, which might be a useful tool to study MMP activity in the pathophysiology of the disease.

Visualizing cell death in experimental focal cerebral ischemia: promises, problems, and perspectives

One of the hallmarks of stroke pathophysiology is the widespread death of many different types of brain cells. As our understanding of the complex disease that is stroke has grown, it is now generally accepted that various different mechanisms can result in cell damage and eventual death. A plethora of techniques is available to identify various pathological features of cell death in stroke; each has its own drawbacks and pitfalls, and most are unable to distinguish between different types of cell death, which partially explains the widespread misuse of many terms. The purpose of this review is to summarize the standard histopathological and immunohistochemical techniques used to identify various pathological features of stroke. We then discuss how these methods should be properly interpreted on the basis of what they are showing, as well as advantages and disadvantages that require consideration. As there is much interest in the visualization of stroke using noninvasive imaging strategies, we also specifically discuss how these techniques can be interpreted within the context of cell death.

Magnetic resonance imaging of the pancreas and pancreatic tumors in a mouse orthotopic model of human cancer

Pancreatic adenocarcinoma has a rising incidence and a very poor survival rate. To develop new treatment strategies, extensive research is performed on animal models of pancreatic cancer. Orthotopic pancreatic tumors models, where the tumor is implanted into the pancreas, resemble the human disease more closely than subcutaneous tumor models, yet are difficult to monitor. In our study we report a magnetic resonance imaging (MRI) approach to visualize the pancreas in mice and to monitor orthotopically implanted pancreatic tumors. An MRI scanner was used to image normal murine pancreas and the pancreas of mice implanted with a human pancreatic adenocarcinoma cell line. Gadolinium (Gd)‐DTPA‐enhanced T1‐ and T2‐weighted standard sequences were used with the objective to identify the pancreas and to monitor the growth of orthotopic tumors during 30 days. The pancreas as well as the implanted tumors could be easily identified using MRI. On T2‐weighted images, the implanted tumors were easily visualized at the implantation side with high signal intensity. After application of a contrast agent, the tumors showed an enhancement. Heterogeneities within the tumor could be delineated, corresponding to histology, and the size of the tumor could be measured precisely. MR serves as a noninvasive high‐resolution image modality to monitor murine pancreas as well as size, growth and even areas of heterogeneity in orthotopic pancreatic tumors.

Non-invasive visualization of CNS inflammation with nuclear and optical imaging

Inflammation is crucially involved in many diseases of the CNS. Immune cells may attack the CNS, as in multiple sclerosis, and therefore be responsible for primary damage. Immune cells may also be activated by injury to the CNS, as for example in stroke or brain trauma, secondarily enhancing lesion growth. In general, CNS inflammation involves a complex interplay of pro- and anti-inflammatory cells and molecules. The blood-brain barrier loses its integrity, plasma proteins leak into the CNS parenchyma, followed by invasion of blood-borne immune cells, and activation of resident microglial cells and astrocytes. However, inflammation not only exacerbates CNS disease, it is also indispensable in containment and resolution of tissue damage, as well as repair and regeneration. The time course and the contribution of inflammatory processes to the pathophysiology of the disease depend on several factors including the type of injury and the time point after injury, and can exhibit a high individual variability. Imaging technologies that enable specific visualization of these inflammatory processes non-invasively are therefore highly desirable. They provide powerful tools to further evaluate the contribution of specific processes to the pathophysiology of CNS disease. Moreover, these technologies may be valuable in detecting and assessing disease progression, in stratifying patients for therapy, and in monitoring therapy. Among the existing non-invasive imaging methods to visualize neuroinflammation in the CNS, we here review the current status of nuclear and optical imaging techniques, with particular emphasis on the sensitivity, specificity, as well as the limitations of these approaches.

Towards noninvasive molecular fluorescence imaging of the human brain

Fluorescence molecular brain imaging is a new modality allowing the detection of specific contrast agents down to very low concentration ranges (picomolar) in disease models. Here we demonstrate a first noninvasive application of fluorescence imaging in the human brain, where concentrations down to about 100 nM of a nonspecific dye were detected. We argue that due to its high sensitivity, optical molecular imaging of the brain is feasible, which – together with its bedside applicability – makes it a promising technique for use in patients.