Corine C. Visser

Enhanced brain drug delivery: safely crossing the blood–brain barrier

The blood–brain barrier presents a significant hurdle in CNS drug development. Blood-to-brain delivery by effectively crossing this barrier allows therapeutics to reach a large area of the brain. Over the past decades several drug delivery technologies have been developed, some more successful than others, which we hold against 10 key development criteria. Adhering to these criteria will allow a more successful development of therapeutics for patients with devastating brain diseases.

Targeted delivery across the blood–brain barrier

The safest and most effective way of targeting drugs to the entire brain is via delivery systems directed at endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective in animal models including primates, but no clinical trials have been performed so far. This review focuses on the well-characterised transferrin and insulin receptor-targeted systems, as well as on the more recently described systems that use the low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor). The possibilities and limitations of these systems are compared and their future for human application is discussed.

Validation of the Transferrin Receptor for Drug Targeting to Brain Capillary Endothelial Cells In Vitro

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood–brain barrier (BBB) and its concentration in biological media can be easily quantified. Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 μg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37°C. This was approximately 3-fold higher compared to binding at 4°C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37°C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed. In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.

Characterization and Modulation of the Transferrin Receptor on Brain Capillary Endothelial Cells

AbstractPurpose. The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of 125I-transferrin (125I-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide (LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain. Methods. Experiments were performed with primary cultured bovine BCECs that were incubated with 125I-Tf at 4°C (to determine binding) or at 37°C (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (Kd, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (Bmax). Results. On BCECs, the TfR is expressed extracellularly (Bmax of 0.13 fmol/μg cell protein), but also has a large intracellular pool (total Bmax of 1.37 fmol/μg cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4°C, a Kd of 2.38 μg/ml was found, whereas the Kd at 37°C was 5.03 μg/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/μg cell protein, respectively, whereas it had no influence on Kd. Bmax at 37°C after DFO preincubation was also increased from 0.90 to 2.31 fmol/μg cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation. Conclusions. The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the blood-brain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.

Targeted blood-to-brain drug delivery --10 key development criteria.

Drug delivery to the brain remains challenging due to the presence of the blood-brain barrier. In this review, 10 key development criteria are presented that are important for successful drug development to treat CNS diseases by targeted drug delivery systems. Although several routes of delivery are being investigated, such as intranasal delivery, direct injections into the brain or CSF, and transient opening of the blood-brain barrier, the focus of this review is on physiological strategies aiming to target endogenous transport mechanisms. Examples from literature, focusing on targeted drug delivery systems that are being commercially developed, will be discussed to illustrate the 10 key development criteria. The first four criteria apply to the targeting of the blood-brain barrier: (1) a proven inherently safe receptor biology, (2) a safe and human applicable ligand, (3) receptor specific binding, and (4) applicable for acute and chronic indications. Next to an efficient and safe targeting strategy, as captured in key criteria 1 to 4, a favorable pharmacokinetic profile is also important (key criterion 5). With regard to the drug carriers, two criteria are important: (6) no modification of active ingredient and (7) able to carry various classes of molecules. The final three criteria apply to the development of a drug from lab to clinic: (8) low costs and straightforward manufacturing, (9) activity in all animal models, and (10) strong intellectual property (IP) protection. Adhering to these 10 key development criteria will allow for a successful brain drug development.

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe3+), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe3+). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe3+ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes. Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe3+ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself. In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe3+ from Tf. This method can be applied to other metal-containing homing devices as well.

Blood-to-Brain Drug Delivery Using Nanocarriers

Brain and nervous system disorders represent a large, unmet medical need affecting two billion people worldwide; a number that is expected to grow with increasing life expectancy and the expanding global population. CNS drug development is hampered by the restricted transport of drug candidates across the blood-brain barrier (BBB). We will discuss blood-to-brain drug delivery strategies that make use of nanocarriers, like liposomes, albumin nanoparticles, and polymeric nanoparticles. The focus will be on the key pharmaceutical, pharmacological, and regulatory aspects towards the clinical development of nanocarriers. Clinical development of treatments employing nanocarriers is not as straightforward as for a single active moiety; therefore, we will highlight the issues that should be considered when translating basic research towards clinical development. Although it is still unrealistic to expect a magic bullet for exclusive CNS drug delivery, much progress has been made towards successful development of novel treatments for patients with devastating brain diseases.

Upcycling drugs for brain-related diseases: a sustainable future for targeted drug delivery

With increasing drug-development costs, drug-development time, the patent cliff and soaring healthcare costs, the pharmaceutical industry needs developments for a more sustainable future. to-BBB is contributing to this sustainable development by ‘upcycling’ drugs that are already on the market. For the pharmaceutical industry this upcycling of marketed drugs will be very attractive, as this will allow sustained market share of drugs that are coming off patent. Upcycling drugs for new or alternative purposes is beneficial and sustainable in general, as this reduces development costs, development time and the use of resources for development considerably. Examples include to-BBB’s formulation for treating brain tumors, 2B3–101, and a formulation aimed at targeting neuroinflammation, 2B3–201.

Recent Advances and Trends in the Brain Delivery of Small Molecule Based Cancer Therapies

Current treatment of brain cancer is hampered by few effective treatment options and characterized by low survival rates. Some advanced drug delivery systems carrying small molecules are currently being developed and may have a major positive impact in the overall outcomes of the treatment regimens. We will discuss the benefits of liposomal formulations, the most commonly used nanoparticles for drug delivery. Liposomal formulation of chemotherapeutics can improve the efficacy and reduce the side effects of the chemotherapy by improving the drug release, pharmacokinetic and biodistribution profile. Uptake of these nanoparticles into the brain is however limited due to the presence of a blood–brain and blood–tumor barrier. By combining safe targeting ligands and receptors with well-known and safe nanocarrier technologies, brain-targeted liposomes may overcome these barriers and result in new and effective chemotherapeutic drugs for clinical use. Examples and practical issues involved in liposomal formulation of chemotherapeutics are discussed.