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Enhanced brain drug delivery: safely crossing the blood–brain barrier
Drug Discovery Today: Technologies
Vol. 9, No. 2 2012
Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Formulation technologies to overcome poor drug-like properties
Enhanced brain drug delivery: safely crossing the blood–brain barrier Pieter J. Gaillard*, Corine C. Visser, Chantal C.M. Appeldoorn, Jaap Rip to-BBB Technologies BV, Leiden, The Netherlands
The blood–brain barrier presents a significant hurdle in CNS drug development. Blood-to-brain delivery by effectively crossing this barrier allows therapeutics to
Section editors: Max Zeller – addc GmbH, Fuellinsdorf, Switzerland. Tom Alfredson – Gilead Sciences, Foster City, CA, USA.
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. Introduction Treatment of CNS-related disorders is challenging. Nearly all of the larger drugs and most of the smaller drugs are not able to reach the brain in high enough concentrations to exert an effect [1]. This is mainly due to the blood–brain barrier, a highly dynamic neuroprotective barrier, constituted by the endothelial cells of the capillaries in the brain. In short, blood–brain barrier function results from 3 layers of protection: a physical barrier (tight junctions prevent para-cellular transport), a transport barrier (specific transport mechanisms for in- and efflux, controlling trans-cellular transport) and a metabolic barrier (metabolizing enzymes prevent trans-cellular transport) [2]. Research to deliver drugs to the CNS is focusing on either locally circumventing the blood–brain barrier (through direct injections, convection enhanced delivery, i.e., using a positive pressure infusion, or intranasal delivery), or globally through the blood stream. The latter can be accomplished either by (transiently) opening the blood–brain barrier or *Corresponding author: P.J. Gaillard ([email protected]) 1740-6749/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2011.12.002
using endogenous transport mechanisms to enhance transport across the blood–brain barrier [3–5]. Using this vascular route is considered most promising for drug delivery throughout the entire brain due to the large surface area of the blood– brain barrier [6]. To ultimately ensure a safe route of delivery, endogenous transport mechanisms should better be used rather than (transient) opening of the neuroprotective blood–brain barrier [3].
Enhancing drug delivery Improving drug-like properties ‘Drug-like’ is defined by Lipinski as those compounds that have sufficiently acceptable ADME (absorption, distribution, metabolism and excretion) properties and sufficiently acceptable toxicity properties to survive through the completion of human phase I clinical trials [7]. Rules of thumb for size, lipophilicity and hydrogen bonding have been formulated to increase the likelihood for successful drug development [8– 10]. However, this rule does not take into account whether or not a molecule is a substrate for brain efflux transporters, such as P-glycoprotein (Pgp), which are present at the blood–brain barrier in high concentrations. Chemically modifying drugs to increase their blood–brain barrier permeability, often also results in an increased accumulation in peripheral tissues. Therefore, this altered tissue distribution usually does not result in an increased plasma concentration, while it does affect peripheral toxicity negatively [11]. Therefore, e155
Drug Discovery Today: Technologies | Formulation technologies to overcome poor drug-like properties
improving drug-like properties, such as solubility and permeability, in many cases does not improve brain drug delivery without an impairment of the safety requirements. In the past decades, more and more biological drugs, such as peptides, proteins and antibodies, have been developed. These are mostly not orally administered, however, optimal drug-like properties like solubility and permeability to enhance delivery to the site of action and limiting side-effects remain to be equally important for these larger molecules as it is for small molecules. These biological drugs, however, do not readily allow for the chemical modifications that are amendable for small molecules to improve these properties, making this class of drugs especially difficult to enter the brain.
Improving brain drug delivery To enhance drug delivery to the brain, several approaches can be used. Intranasal delivery is one of these methods, although this approach bears limiting issues for broad applicability, such as a small volume of administration, a poor permeability across the epithelial membrane and a limited diffusion throughout the whole brain [12]. More invasive methods, such as direct injections into the brain or ventricles (cerebrospinal fluid), thereby physically circumventing the blood– brain barrier, do allow for an efficient yet often too local delivery [13–15]. Convection-enhanced delivery will allow for a more diffuse spread throughout larger areas of the brain; however, safety and the invasiveness is still a great concern of this method [12]. Therefore, these methods have limited suitability for disorders of the CNS that are spread throughout the brain and warrant the development of less invasive and safer alternative approaches. The large surface area of the human blood–brain barrier and the fact that almost every neuron has its own brain capillary for oxygen and nutrient supply makes the vascular route a very promising one for drug delivery to the brain [6,11]. Roughly two methods have been described in the literature to enhance blood-to-brain drug delivery: disruption of the blood–brain barrier by osmotic imbalance or vasoactive compounds or physiological strategies aiming to use endogenous transport mechanisms. The first method has the disadvantage that neurons may be damaged (semi)-permanently due to unwanted blood components entering the
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brain [16]. By contrast, the physiological strategies have a large potential as discussed in several review papers (see e.g. [17]), but only if these can be optimized to deliver drugs to the brain within a therapeutic window. This has proven to be a formidable challenge over the recent years, since only two technologies have so far reached the clinical testing phase.
10 key development criteria for brain drug delivery To improve the development of safe and effective treatments for patients with devastating brain diseases using endogenous transporter mechanisms at the blood–brain barrier, we have formulated 10 key development criteria [3]. These criteria are focusing on three main topics: (1) targeting the blood–brain barrier, (2) drug carrier systems and (3) drug development from lab to clinic (Table 1). The first and second topics are both essential for improving drug-like properties of CNS drugs. With regard to blood–brain barrier targeting, we have listed criteria that go beyond receptor-specific binding, that is, safety of the selected receptor and targeting ligand, as well as applicability in both acute and chronic conditions is important to consider when selecting a ligand/receptor combination for clinical development. The right selection of a drug carrier system is important to improve the drug-like properties, and improve the pharmacokinetic profile of an intravenously administered drug. Finally, low cost and straightforward manufacturing, activity in all animal models and strong intellectual property (IP) protection are essential for a rapid and cost-effective drug development. In the paragraphs below, each of the 10 key development criteria will be highlighted by using recent examples from literature, as well as from our own work.
Targeting the blood–brain barrier In the past years, blood-to-brain drug delivery has been focusing on several ligands/receptors, among others on antibodies against the transferrin receptor (OX26/8D3/human chimeric anti-murine TfR antibody; see e.g. [18,19]) or the insulin receptor (HIRMAb; see e.g. [20,21]), peptide or protein ligands for the low density lipoprotein receptor-related protein (LRP) receptors (p97/melanotransferrin [22], receptorassociated protein (RAP) [23], apolipoproteins [24] and angiopep-2 [25,26]), the diphtheria toxin receptor (CRM197; [27])
Table 1. 10 key development criteria for blood-to-brain drug delivery Targeting the blood–brain barrier
Drug carriers
Drug development from lab to clinic
1. Proven inherently safe receptor biology in humans
6. No modification of active ingredient
8. Low costs & straightforward manufacturing
2. Safe and human applicable ligand
7. Able to carry various classes of molecules
9. Activity in all animal models
3. Receptor specific binding
10. Strong IP protection
4. Applicable for acute and chronic indications 5. Favourable pharmacokinetics
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Table 2. Overview of the suitability of brain drug delivery systems according to the 10 key development criteria Technology
EPiC Technology
G-Technology
‘Trojan Horse’ a
‘Anti-TfR Ab’
Transcend
anti-TfR-anti-BACE1
P97-anti tumor drugs
Lead Product
ANG1005/GRN1005
2B3-101
AGT182
Development phase
Completed clinical phase I/II
Initiated clinical phase I/II
Preclinical
Research
Research
Short description
Direct conjugate between angiopep-2 and 3 molecules paclitaxel
Glutathione pegylated liposomal doxorubicin
Fusion protein between HIRMAb and an enzyme
Heavy chain hetero-dimerization of 2 antibodies
Direct conjugates between p97 and drugs
10 key development criteria
U
2. Safe and human applicable ligand
U
U
?
3. Receptor specific binding
U
U
U
U
U
4. Applicable for acute/chronic indications
U/
U
U/
5. Favourable pharmacokinetics
6. No modification of active ingredient
U/
U/
U
U
?
7. Able to carry various classes of molecules
U
8. Low costs & straightforward manufacturing
U
U
?
9. Activity in all animal models
U
U
U
10. Strong IP protection
U
U
U
U
U
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Company website
http://www.angiochem.com/
http://www.tobbb.com/
http://www.armagen.com/
http://www.gene.com/
http://www.bioasis.ca/
Key references
[25,26]
[30]
[21]
[19]
[22]
a Armagen has multiple fusion proteins between HIRMAb and other proteins. In this table we refer to the most recent publication, which was on a fusion protein of HIRMAb and the enzyme iduronate-2 sulfatase (IDS) for MPS Type II (Hunter’s syndrome), see http://www.armagen.com/products.php.
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1. Proven inherently safe receptor biology in humans
Drug Discovery Today: Technologies | Formulation technologies to overcome poor drug-like properties
or the glutathione transporter (glutathione; [3,28–30]). Of these, angiopep-2 conjugated to paclitaxel (ANG1005/ GRN1005; Table 2) and glutathione pegylated liposomal doxorubicin (2B3-101; Table 2) have been or are currently being investigated in clinical studies (see http://www. clinicaltrials.gov/; ANG1005: NCT00539344 and NCT00539383; 2B3-101: NCT01386580). The transferrin, insulin and LRP receptors all have important biological, scavenger and signaling functions. When using these receptors for blood-to-brain drug delivery, this might interfere with their endogenous function, resulting in safety issues that need careful monitoring and thereby adds risk to the successful clinical development of technologies based thereon. For the LRP receptor, the recently conducted phase I studies with ANG1005 (recently renamed as GRN1005) have shown no safety issues in patients with brain metastases or glioma [25,26], indicating that for these indications the LRP receptor can be safely targeted. Targeting to the diphtheria toxin receptor can be considered safe, as this receptor does not have an endogenous transport function at the blood–brain barrier. However, because this receptor is the membrane-bound precursor of heparin-binding epidermal growth factor (HB-EGF), prolonged targeting might influence the biological function of this growth factor. Of these receptors, the glutathione transporter seems to have the best safety profile. Physiological plasma and brain concentrations of glutathione are in the millimolar range, while the currently used concentrations for drug targeting are still in the micromolar range. Therefore, targeting to the glutathione receptor is not probably competing with its physiological function. Receptor-specific binding is a criterion that is met by all of the ligands discussed in this review. However, the safety of these ligands is not always self-evident. Antibodies or ligands that are not of human origin could cause an immunogenic effect [31]. Studies with HIRMAb fusion proteins have shown an immune response in rhesus monkeys [32,33], a finding that requires further research, especially for chronic administration of these fusion proteins. CRM197, a ligand for the diphtheria toxin receptor, and glutathione are already in clinical use; CRM197 is used as a carrier protein in human vaccines and as an inhibitor of tumor growth by scavenging the soluble form of HB-EGF. Glutathione has FDA GRAS (generally regarded as safe) status and is used as supportive therapy in chemotherapy and as a food supplement. In a GLP preclinical study in rodents, empty glutathione pegylated liposomes were considered safe, as there were no treatment-related changes in the brain after repeated exposure. Furthermore, no effect on neurobehavior was found in a modified Irwin test in rats [34]. Finally, angiopep-2 is considered safe, especially in acute indications, due to the fact that the peptide sequence is derived from a protein of human e158
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origin [35]. In the phase I clinical studies, angiopep-2 conjugated with paclitaxel was indeed considered tolerable for patients with brain tumors [25,26]. The diseased state could affect efficacy as well as the safety of targeted drug delivery systems as receptor levels and their role might vary during the course of the disease [36]. In addition, for chronic administration a different safety profile of the targeted drug delivery system is warranted compared to acute administration. Glutathione seems to have the best safety profile for prolonged administration, while enough data are not yet available to determine the long-term safety of the other receptor–ligand systems [3].
Drug carrier systems For CNS drug delivery, like delivery to any other tissue, peripheral pharmacokinetics, elimination and biodistribution, including binding to plasma proteins or (enzymatic) degradation of the drug in blood plays an important role in the overall efficacy of the system [37]. By taking this into account when selecting a suitable drug carrier system, these properties can be largely influenced. Direct conjugates or fusion proteins of drug and targeting ligand often have a similar pharmacokinetic profile to the non-conjugated drug. Indeed, the half-life of angiopep-2 conjugated with paclitaxel is about 4 hours [26]. By contrast, drug carriers like liposomes or nanoparticles can be pegylated, which results in a longer half-life in plasma, up to several days [3]. Another benefit of liposomes is their ability to encapsulate both large and small molecules, as well as hydrophilic and lipophilic compounds without modification of the active ingredient. Using a direct conjugation method, some drugs loose their activity upon modification. For example, doxorubicin conjugation to angiopep-2 required a cleavable bond as doxorubicin lost its antiproliferative activity when conjugated via a noncleavable linker [38]. Also, as with chemical conjugates, fusion proteins between therapeutic human enzymes and human targeting proteins proved in many occasions to be functionally less active (e.g. the enzyme activity of paraoxonase-1 (PON-1) conjugated to HIRMAb and was only 25% of the native PON-1 [39]) or even inactive or immunogenic (unpublished results from The Sanfilippo Syndrome Medical Research Foundation, Inc. (SSMRF)). An additional benefit of incorporation into liposomes can be the improved solubility of low soluble drugs. Coimbra et al. [40] have used a passive encapsulation method for several natural compounds; these were either encapsulated in the aqueous core or in the lipid bilayer. Encapsulation can, therefore, never be higher than the solubility in the selected solvents. By contrast, active or remote loading methods, in which the molecule is precipitated in the core of the liposomes, can result in a higher concentration of the end product. In general, remote loading is used for amphiphatic weak bases or acids with reasonable solubility in aqueous solvents
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Drug Discovery Today: Technologies | Formulation technologies to overcome poor drug-like properties
[41]. Recently, we have further optimized this method so that active loading can now also be used for drugs with a low solubility by combining it with the use of solubilizers, such as cyclodextrins or PEG (unpublished results).
Drug development from lab to clinic It may seem obvious to focus on low costs and straightforward manufacturing methods; however, given the long list of literature reports and the limited number of drug delivery systems in clinical research, it is not completely redundant to mention these points. By applying to-BBB’s G-Technology to already marketed pegylated liposomal doxorubicin, manufacturing and development costs have been very costeffective, especially because of the comparability and predictability between the new and existing methodologies. For other products based on the G-Technology it may already be less straightforward as the choice of lipids and other constituents, as well as the production method might vary, and the active ingredient (i.e., encapsulated drug) will often drive the costs of goods. Angiochem has chosen for a chemical linkage of paclitaxel to angiopep-2. For clinical use it is important to obtain a consistent product, especially when more than 1 possible binding site is present. As there are 3 possible conjugation sites in angiopep-2, 3 molecules of paclitaxel have been conjugated to 1 angiopep-2 molecule. Obviously, for subsequent products this again needs to be optimized and validated as well. Drug development is further facilitated if already established models for early proof-of-concept and safety studies can be selected. For the HIRMAb, animal testing can, therefore, only be done in Old World primates that is more costly, time consuming and obviously has generated less comparative data. Likewise, CRM197 cannot be used in rats and mice due to a mutation in the transport receptor in these species, making this a difficult ligand to validate in routine preclinical models as well [3]. Last but not least, a strong IP protection is necessary to obtain and justify the large cash investments that are necessary to develop any targeted drug delivery system into a successful new treatment option for CNS disorders. Together with the long drug development time lines, this also calls for a speedy development route to maximize the return of investment within the patent life of a drug delivery technology.
The 10 key development criteria in practice When holding the most prominent drug delivery technologies against the 10 key development criteria, it becomes apparent that the technologies that indeed have been approved for clinical research adhere to most, if not all, of the 10 key development criteria (Table 2). Efficacy and brain uptake of the technologies mentioned in Table 2 have been shown in preclinical models, and for ANG1005/GRN1005 in clinical research as well. However, the biggest discriminator
between the lead products in clinical research and the ones in preclinical or research phase is their safety. In two phase I clinical studies it was shown that ANG1005/GRN1005 is safe and well tolerated. No evidence of CNS toxicity and/or immunogenicity was observed [25,26]. In an extensive GLP preclinical safety study, no treatment-related changes in brain were found after repeated 2B3-101 exposure. Also, no major differences in the safety profiles of 2B3-101 and pegylated liposomal doxorubicin (Doxil1/Caelyx1) were observed [34]. In a modified Irwin test, no neurobehavioral effects of 2B3-101 were noted. The EPiC Technology and the G-Technology use safe and human applicable ligands, where only the G-Technology targets a proven inherently safe receptor biology in humans. Both the Trojan horse technology from Armagen Technologies and the technology developed by Genentech use antibodies for targeting. Antibodies with a high affinity can bind permanently to the receptor, thereby interrupting or interfering with the physiological function of the receptor [42]. Genentech researchers have developed a new low affinity antibody against the rodent transferrin receptor, which may offer a better safety profile; however, this has to date not been investigated in humans [19]. The Transcend technology uses an endogenous human protein as targeting ligand, called p97, which targets the LRP receptors, a class of multifunctional scavenger and signaling receptors. While endogenous ligands are undoubtedly safe, the safety of binding to the LRP receptors is difficult to predict in human application, taking its complex signaling function into consideration [11]. With regard to the drug carriers associated with the technologies in Table 2, the G-Technology is the most versatile as both large and small molecules, as well as hydrophilic and lipophilic compounds can be encapsulated in the liposomes without modification of the active ingredient [3]. The other technologies discussed in this review make use of chemical conjugation, fusion proteins or heavy chain heterodimerization of 2 antibodies. For all these latter techniques, a modification of the drug is necessary, which could potentially result in a loss of activity and/or immunogenicity against the newly formed conjugate/fusion protein. Furthermore, for unmodified drugs that are released from liposomes, one can continue to use and compare available data on the drug itself, which makes the drug development process more predictable as compared to when it would be the case for structurally modified drugs. The use of pegylated liposomes also increases the half-life of the drugs. This prolonged circulation in plasma can allow sufficient time to reach and maintain presence of the drug at the target site. Finally, adhering to the criteria related to drug development from lab to clinic will not only speed up the process, but will also reduce the cost. In the end this could make the difference for developing a successful product. www.drugdiscoverytoday.com
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Conclusion In 2008 Neuroinsights reported that the worldwide economic burden caused by CNS diseases has reached over $2 trillion a year, and it is likely that this burden will continue to rise with the increasingly ageing population [43]. Treatments for many CNS diseases are not yet widely available, mainly due to the presence of the blood–brain barrier and [therefore] lack of validated targets for these CNS diseases. Although there are many routes for drug delivery to the brain, we believe that the treatment of brain diseases will be best achieved by safely enhancing the blood-to-brain delivery of drugs. Adhering to the 10 key development criteria presented in this review will allow a faster and more successful development of treatments for patients with devastating brain diseases.
19 20
21
22 23
24
25 26
Conflict of interest All authors are employees of to-BBB Technologies BV. Dr. Gaillard is also a shareholder of the company.
27 28
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Yu, Y.J. et al. (2011) Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci. Transl. Med. 3, 84ra44 Boado, R.J. et al. (2007) Humanization of anti-human insulin receptor antibody for drug targeting across the human blood–brain barrier. Biotechnol. Bioeng. 96, 381–391 Lu, J.Z. et al. (2011) Expression in CHO cells and pharmacokinetics and brain uptake in the Rhesus monkey of an IgG-iduronate-2-sulfatase fusion protein. Biotechnol. Bioeng. 108, 1954–1964 Karkan, D. et al. (2008) A unique carrier for delivery of therapeutic compounds beyond the blood–brain barrier. PLoS One 3, e2469 Prince, W.S. et al. (2004) Lipoprotein receptor binding, cellular uptake, and lysosomal delivery of fusions between the receptor-associated protein (RAP) and alpha-L-iduronidase or acid alpha-glucosidase. J. Biol. Chem. 279, 35037–35046 Gelperina, S. et al. (2010) Drug delivery to the brain using surfactantcoated poly(lactide-co-glycolide) nanoparticles: influence of the formulation parameters. Eur. J. Pharm. Biopharm. 74, 157–163 Drappatz, J. et al. (2010) ANG1005: results of a phase I study in patients with recurrent malignant glioma. J. Clin. Oncol. 28 (abstract 2009) Sarantopoulos, J. et al. (2010) ANG1005: results of a phase I study in patients with advanced solid tumors and brain metastases. J. Clin. Oncol. 28 (abstract 2556) Gaillard, P.J. et al. (2005) Diphtheria toxin receptor-targeted brain drug delivery. Int. Congr. Series 1277, 185–198 Geldenhuys, W. et al. (2011) Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers. J. Drug Target. 19, 837– 845 More, S.S. and Vince, R. (2008) Design, synthesis and biological evaluation of glutathione peptidomimetics as components of anti-Parkinson prodrugs. J. Med. Chem. 51, 4581–4588 van Tellingen, O. et al. (2010) GSH-conjugation improves efficacy of Doxil against intracranial xenografts. Neuro-Oncology 12 (suppl 3), iii34 Jones, A.R. and Shusta, E.V. (2007) Blood–brain barrier transport of therapeutics via receptor-mediation. Pharm. Res. 24, 1759–1771 Boado, R.J. et al. (2009) AGT-181: expression in CHO cells and pharmacokinetics, safety, and plasma iduronidase enzyme activity in Rhesus monkeys. J. Biotechnol. 144, 135–141 Pardridge, W.M. and Boado, R.J. (2009) Pharmacokinetics and safety in rhesus monkeys of a monoclonal antibody-GDNF fusion protein for targeted blood–brain barrier delivery. Pharm. Res. 26, 2227–2236 Gaillard, P.J. et al. (2011) Development of glutathione pegylated liposomal doxorubicin (2B3-101) for the treatment of brain metastases from breast cancer. Presented at: 4th European Conference for Clinical Nanomedicine Basel, Switzerland, 23–25 May, 2011 Demeule, M. et al. (2008) Identification and design of peptides as a new drug delivery system for the brain. J. Pharmacol. Exp. Ther. 324, 1064–1072 Rip, J. et al. (2009) Differential receptor-mediated drug targeting to the diseased brain. Expert Opin. Drug Deliv. 6, 227–237 Banks, W.A. (2008) Delivery of peptides to the brain: emphasis on therapeutic development. Biopolymers 90, 589–594 Che, C. et al. (2010) New Angiopep-modified doxorubicin (ANG1007) and etoposide (ANG1009) chemotherapeutics with increased brain penetration. J. Med. Chem. 53, 2814–2824 Boado, R.J. et al. (2008) IgG-paraoxonase-1 fusion protein for targeted drug delivery across the human blood–brain barrier. Mol. Pharm. 5, 1037–1043 Coimbra, M. et al. (2011) Improving solubility and chemical stability of natural compounds for medicinal use by incorporation into liposomes. Int. J. Pharm. 416, 433–442 Zucker, D. et al. (2009) Liposome drugs’ loading efficiency: a working model based on loading conditions and drug’s physicochemical properties. J. Control. Release 139, 73–80 Gosk, S. et al. (2004) Targeting anti-transferrin receptor antibody (OX26) and OX26-conjugated liposomes to brain capillary endothelial cells using in situ perfusion. J. Cereb. Blood Flow Metab. 24, 1193–1204 The Neurotechnology Industry 2008 Report published by NeuroInsights
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Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Formulation technologies to overcome poor drug-like properties
Enhanced brain drug delivery: safely crossing the blood–brain barrier Pieter J. Gaillard*, Corine C. Visser, Chantal C.M. Appeldoorn, Jaap Rip to-BBB Technologies BV, Leiden, The Netherlands
The blood–brain barrier presents a significant hurdle in CNS drug development. Blood-to-brain delivery by effectively crossing this barrier allows therapeutics to
Section editors: Max Zeller – addc GmbH, Fuellinsdorf, Switzerland. Tom Alfredson – Gilead Sciences, Foster City, CA, USA.
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. Introduction Treatment of CNS-related disorders is challenging. Nearly all of the larger drugs and most of the smaller drugs are not able to reach the brain in high enough concentrations to exert an effect [1]. This is mainly due to the blood–brain barrier, a highly dynamic neuroprotective barrier, constituted by the endothelial cells of the capillaries in the brain. In short, blood–brain barrier function results from 3 layers of protection: a physical barrier (tight junctions prevent para-cellular transport), a transport barrier (specific transport mechanisms for in- and efflux, controlling trans-cellular transport) and a metabolic barrier (metabolizing enzymes prevent trans-cellular transport) [2]. Research to deliver drugs to the CNS is focusing on either locally circumventing the blood–brain barrier (through direct injections, convection enhanced delivery, i.e., using a positive pressure infusion, or intranasal delivery), or globally through the blood stream. The latter can be accomplished either by (transiently) opening the blood–brain barrier or *Corresponding author: P.J. Gaillard ([email protected]) 1740-6749/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2011.12.002
using endogenous transport mechanisms to enhance transport across the blood–brain barrier [3–5]. Using this vascular route is considered most promising for drug delivery throughout the entire brain due to the large surface area of the blood– brain barrier [6]. To ultimately ensure a safe route of delivery, endogenous transport mechanisms should better be used rather than (transient) opening of the neuroprotective blood–brain barrier [3].
Enhancing drug delivery Improving drug-like properties ‘Drug-like’ is defined by Lipinski as those compounds that have sufficiently acceptable ADME (absorption, distribution, metabolism and excretion) properties and sufficiently acceptable toxicity properties to survive through the completion of human phase I clinical trials [7]. Rules of thumb for size, lipophilicity and hydrogen bonding have been formulated to increase the likelihood for successful drug development [8– 10]. However, this rule does not take into account whether or not a molecule is a substrate for brain efflux transporters, such as P-glycoprotein (Pgp), which are present at the blood–brain barrier in high concentrations. Chemically modifying drugs to increase their blood–brain barrier permeability, often also results in an increased accumulation in peripheral tissues. Therefore, this altered tissue distribution usually does not result in an increased plasma concentration, while it does affect peripheral toxicity negatively [11]. Therefore, e155
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improving drug-like properties, such as solubility and permeability, in many cases does not improve brain drug delivery without an impairment of the safety requirements. In the past decades, more and more biological drugs, such as peptides, proteins and antibodies, have been developed. These are mostly not orally administered, however, optimal drug-like properties like solubility and permeability to enhance delivery to the site of action and limiting side-effects remain to be equally important for these larger molecules as it is for small molecules. These biological drugs, however, do not readily allow for the chemical modifications that are amendable for small molecules to improve these properties, making this class of drugs especially difficult to enter the brain.
Improving brain drug delivery To enhance drug delivery to the brain, several approaches can be used. Intranasal delivery is one of these methods, although this approach bears limiting issues for broad applicability, such as a small volume of administration, a poor permeability across the epithelial membrane and a limited diffusion throughout the whole brain [12]. More invasive methods, such as direct injections into the brain or ventricles (cerebrospinal fluid), thereby physically circumventing the blood– brain barrier, do allow for an efficient yet often too local delivery [13–15]. Convection-enhanced delivery will allow for a more diffuse spread throughout larger areas of the brain; however, safety and the invasiveness is still a great concern of this method [12]. Therefore, these methods have limited suitability for disorders of the CNS that are spread throughout the brain and warrant the development of less invasive and safer alternative approaches. The large surface area of the human blood–brain barrier and the fact that almost every neuron has its own brain capillary for oxygen and nutrient supply makes the vascular route a very promising one for drug delivery to the brain [6,11]. Roughly two methods have been described in the literature to enhance blood-to-brain drug delivery: disruption of the blood–brain barrier by osmotic imbalance or vasoactive compounds or physiological strategies aiming to use endogenous transport mechanisms. The first method has the disadvantage that neurons may be damaged (semi)-permanently due to unwanted blood components entering the
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brain [16]. By contrast, the physiological strategies have a large potential as discussed in several review papers (see e.g. [17]), but only if these can be optimized to deliver drugs to the brain within a therapeutic window. This has proven to be a formidable challenge over the recent years, since only two technologies have so far reached the clinical testing phase.
10 key development criteria for brain drug delivery To improve the development of safe and effective treatments for patients with devastating brain diseases using endogenous transporter mechanisms at the blood–brain barrier, we have formulated 10 key development criteria [3]. These criteria are focusing on three main topics: (1) targeting the blood–brain barrier, (2) drug carrier systems and (3) drug development from lab to clinic (Table 1). The first and second topics are both essential for improving drug-like properties of CNS drugs. With regard to blood–brain barrier targeting, we have listed criteria that go beyond receptor-specific binding, that is, safety of the selected receptor and targeting ligand, as well as applicability in both acute and chronic conditions is important to consider when selecting a ligand/receptor combination for clinical development. The right selection of a drug carrier system is important to improve the drug-like properties, and improve the pharmacokinetic profile of an intravenously administered drug. Finally, low cost and straightforward manufacturing, activity in all animal models and strong intellectual property (IP) protection are essential for a rapid and cost-effective drug development. In the paragraphs below, each of the 10 key development criteria will be highlighted by using recent examples from literature, as well as from our own work.
Targeting the blood–brain barrier In the past years, blood-to-brain drug delivery has been focusing on several ligands/receptors, among others on antibodies against the transferrin receptor (OX26/8D3/human chimeric anti-murine TfR antibody; see e.g. [18,19]) or the insulin receptor (HIRMAb; see e.g. [20,21]), peptide or protein ligands for the low density lipoprotein receptor-related protein (LRP) receptors (p97/melanotransferrin [22], receptorassociated protein (RAP) [23], apolipoproteins [24] and angiopep-2 [25,26]), the diphtheria toxin receptor (CRM197; [27])
Table 1. 10 key development criteria for blood-to-brain drug delivery Targeting the blood–brain barrier
Drug carriers
Drug development from lab to clinic
1. Proven inherently safe receptor biology in humans
6. No modification of active ingredient
8. Low costs & straightforward manufacturing
2. Safe and human applicable ligand
7. Able to carry various classes of molecules
9. Activity in all animal models
3. Receptor specific binding
10. Strong IP protection
4. Applicable for acute and chronic indications 5. Favourable pharmacokinetics
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Table 2. Overview of the suitability of brain drug delivery systems according to the 10 key development criteria Technology
EPiC Technology
G-Technology
‘Trojan Horse’ a
‘Anti-TfR Ab’
Transcend
anti-TfR-anti-BACE1
P97-anti tumor drugs
Lead Product
ANG1005/GRN1005
2B3-101
AGT182
Development phase
Completed clinical phase I/II
Initiated clinical phase I/II
Preclinical
Research
Research
Short description
Direct conjugate between angiopep-2 and 3 molecules paclitaxel
Glutathione pegylated liposomal doxorubicin
Fusion protein between HIRMAb and an enzyme
Heavy chain hetero-dimerization of 2 antibodies
Direct conjugates between p97 and drugs
10 key development criteria
U
2. Safe and human applicable ligand
U
U
?
3. Receptor specific binding
U
U
U
U
U
4. Applicable for acute/chronic indications
U/
U
U/
5. Favourable pharmacokinetics
6. No modification of active ingredient
U/
U/
U
U
?
7. Able to carry various classes of molecules
U
8. Low costs & straightforward manufacturing
U
U
?
9. Activity in all animal models
U
U
U
10. Strong IP protection
U
U
U
U
U
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Company website
http://www.angiochem.com/
http://www.tobbb.com/
http://www.armagen.com/
http://www.gene.com/
http://www.bioasis.ca/
Key references
[25,26]
[30]
[21]
[19]
[22]
a Armagen has multiple fusion proteins between HIRMAb and other proteins. In this table we refer to the most recent publication, which was on a fusion protein of HIRMAb and the enzyme iduronate-2 sulfatase (IDS) for MPS Type II (Hunter’s syndrome), see http://www.armagen.com/products.php.
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1. Proven inherently safe receptor biology in humans
Drug Discovery Today: Technologies | Formulation technologies to overcome poor drug-like properties
or the glutathione transporter (glutathione; [3,28–30]). Of these, angiopep-2 conjugated to paclitaxel (ANG1005/ GRN1005; Table 2) and glutathione pegylated liposomal doxorubicin (2B3-101; Table 2) have been or are currently being investigated in clinical studies (see http://www. clinicaltrials.gov/; ANG1005: NCT00539344 and NCT00539383; 2B3-101: NCT01386580). The transferrin, insulin and LRP receptors all have important biological, scavenger and signaling functions. When using these receptors for blood-to-brain drug delivery, this might interfere with their endogenous function, resulting in safety issues that need careful monitoring and thereby adds risk to the successful clinical development of technologies based thereon. For the LRP receptor, the recently conducted phase I studies with ANG1005 (recently renamed as GRN1005) have shown no safety issues in patients with brain metastases or glioma [25,26], indicating that for these indications the LRP receptor can be safely targeted. Targeting to the diphtheria toxin receptor can be considered safe, as this receptor does not have an endogenous transport function at the blood–brain barrier. However, because this receptor is the membrane-bound precursor of heparin-binding epidermal growth factor (HB-EGF), prolonged targeting might influence the biological function of this growth factor. Of these receptors, the glutathione transporter seems to have the best safety profile. Physiological plasma and brain concentrations of glutathione are in the millimolar range, while the currently used concentrations for drug targeting are still in the micromolar range. Therefore, targeting to the glutathione receptor is not probably competing with its physiological function. Receptor-specific binding is a criterion that is met by all of the ligands discussed in this review. However, the safety of these ligands is not always self-evident. Antibodies or ligands that are not of human origin could cause an immunogenic effect [31]. Studies with HIRMAb fusion proteins have shown an immune response in rhesus monkeys [32,33], a finding that requires further research, especially for chronic administration of these fusion proteins. CRM197, a ligand for the diphtheria toxin receptor, and glutathione are already in clinical use; CRM197 is used as a carrier protein in human vaccines and as an inhibitor of tumor growth by scavenging the soluble form of HB-EGF. Glutathione has FDA GRAS (generally regarded as safe) status and is used as supportive therapy in chemotherapy and as a food supplement. In a GLP preclinical study in rodents, empty glutathione pegylated liposomes were considered safe, as there were no treatment-related changes in the brain after repeated exposure. Furthermore, no effect on neurobehavior was found in a modified Irwin test in rats [34]. Finally, angiopep-2 is considered safe, especially in acute indications, due to the fact that the peptide sequence is derived from a protein of human e158
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origin [35]. In the phase I clinical studies, angiopep-2 conjugated with paclitaxel was indeed considered tolerable for patients with brain tumors [25,26]. The diseased state could affect efficacy as well as the safety of targeted drug delivery systems as receptor levels and their role might vary during the course of the disease [36]. In addition, for chronic administration a different safety profile of the targeted drug delivery system is warranted compared to acute administration. Glutathione seems to have the best safety profile for prolonged administration, while enough data are not yet available to determine the long-term safety of the other receptor–ligand systems [3].
Drug carrier systems For CNS drug delivery, like delivery to any other tissue, peripheral pharmacokinetics, elimination and biodistribution, including binding to plasma proteins or (enzymatic) degradation of the drug in blood plays an important role in the overall efficacy of the system [37]. By taking this into account when selecting a suitable drug carrier system, these properties can be largely influenced. Direct conjugates or fusion proteins of drug and targeting ligand often have a similar pharmacokinetic profile to the non-conjugated drug. Indeed, the half-life of angiopep-2 conjugated with paclitaxel is about 4 hours [26]. By contrast, drug carriers like liposomes or nanoparticles can be pegylated, which results in a longer half-life in plasma, up to several days [3]. Another benefit of liposomes is their ability to encapsulate both large and small molecules, as well as hydrophilic and lipophilic compounds without modification of the active ingredient. Using a direct conjugation method, some drugs loose their activity upon modification. For example, doxorubicin conjugation to angiopep-2 required a cleavable bond as doxorubicin lost its antiproliferative activity when conjugated via a noncleavable linker [38]. Also, as with chemical conjugates, fusion proteins between therapeutic human enzymes and human targeting proteins proved in many occasions to be functionally less active (e.g. the enzyme activity of paraoxonase-1 (PON-1) conjugated to HIRMAb and was only 25% of the native PON-1 [39]) or even inactive or immunogenic (unpublished results from The Sanfilippo Syndrome Medical Research Foundation, Inc. (SSMRF)). An additional benefit of incorporation into liposomes can be the improved solubility of low soluble drugs. Coimbra et al. [40] have used a passive encapsulation method for several natural compounds; these were either encapsulated in the aqueous core or in the lipid bilayer. Encapsulation can, therefore, never be higher than the solubility in the selected solvents. By contrast, active or remote loading methods, in which the molecule is precipitated in the core of the liposomes, can result in a higher concentration of the end product. In general, remote loading is used for amphiphatic weak bases or acids with reasonable solubility in aqueous solvents
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Drug Discovery Today: Technologies | Formulation technologies to overcome poor drug-like properties
[41]. Recently, we have further optimized this method so that active loading can now also be used for drugs with a low solubility by combining it with the use of solubilizers, such as cyclodextrins or PEG (unpublished results).
Drug development from lab to clinic It may seem obvious to focus on low costs and straightforward manufacturing methods; however, given the long list of literature reports and the limited number of drug delivery systems in clinical research, it is not completely redundant to mention these points. By applying to-BBB’s G-Technology to already marketed pegylated liposomal doxorubicin, manufacturing and development costs have been very costeffective, especially because of the comparability and predictability between the new and existing methodologies. For other products based on the G-Technology it may already be less straightforward as the choice of lipids and other constituents, as well as the production method might vary, and the active ingredient (i.e., encapsulated drug) will often drive the costs of goods. Angiochem has chosen for a chemical linkage of paclitaxel to angiopep-2. For clinical use it is important to obtain a consistent product, especially when more than 1 possible binding site is present. As there are 3 possible conjugation sites in angiopep-2, 3 molecules of paclitaxel have been conjugated to 1 angiopep-2 molecule. Obviously, for subsequent products this again needs to be optimized and validated as well. Drug development is further facilitated if already established models for early proof-of-concept and safety studies can be selected. For the HIRMAb, animal testing can, therefore, only be done in Old World primates that is more costly, time consuming and obviously has generated less comparative data. Likewise, CRM197 cannot be used in rats and mice due to a mutation in the transport receptor in these species, making this a difficult ligand to validate in routine preclinical models as well [3]. Last but not least, a strong IP protection is necessary to obtain and justify the large cash investments that are necessary to develop any targeted drug delivery system into a successful new treatment option for CNS disorders. Together with the long drug development time lines, this also calls for a speedy development route to maximize the return of investment within the patent life of a drug delivery technology.
The 10 key development criteria in practice When holding the most prominent drug delivery technologies against the 10 key development criteria, it becomes apparent that the technologies that indeed have been approved for clinical research adhere to most, if not all, of the 10 key development criteria (Table 2). Efficacy and brain uptake of the technologies mentioned in Table 2 have been shown in preclinical models, and for ANG1005/GRN1005 in clinical research as well. However, the biggest discriminator
between the lead products in clinical research and the ones in preclinical or research phase is their safety. In two phase I clinical studies it was shown that ANG1005/GRN1005 is safe and well tolerated. No evidence of CNS toxicity and/or immunogenicity was observed [25,26]. In an extensive GLP preclinical safety study, no treatment-related changes in brain were found after repeated 2B3-101 exposure. Also, no major differences in the safety profiles of 2B3-101 and pegylated liposomal doxorubicin (Doxil1/Caelyx1) were observed [34]. In a modified Irwin test, no neurobehavioral effects of 2B3-101 were noted. The EPiC Technology and the G-Technology use safe and human applicable ligands, where only the G-Technology targets a proven inherently safe receptor biology in humans. Both the Trojan horse technology from Armagen Technologies and the technology developed by Genentech use antibodies for targeting. Antibodies with a high affinity can bind permanently to the receptor, thereby interrupting or interfering with the physiological function of the receptor [42]. Genentech researchers have developed a new low affinity antibody against the rodent transferrin receptor, which may offer a better safety profile; however, this has to date not been investigated in humans [19]. The Transcend technology uses an endogenous human protein as targeting ligand, called p97, which targets the LRP receptors, a class of multifunctional scavenger and signaling receptors. While endogenous ligands are undoubtedly safe, the safety of binding to the LRP receptors is difficult to predict in human application, taking its complex signaling function into consideration [11]. With regard to the drug carriers associated with the technologies in Table 2, the G-Technology is the most versatile as both large and small molecules, as well as hydrophilic and lipophilic compounds can be encapsulated in the liposomes without modification of the active ingredient [3]. The other technologies discussed in this review make use of chemical conjugation, fusion proteins or heavy chain heterodimerization of 2 antibodies. For all these latter techniques, a modification of the drug is necessary, which could potentially result in a loss of activity and/or immunogenicity against the newly formed conjugate/fusion protein. Furthermore, for unmodified drugs that are released from liposomes, one can continue to use and compare available data on the drug itself, which makes the drug development process more predictable as compared to when it would be the case for structurally modified drugs. The use of pegylated liposomes also increases the half-life of the drugs. This prolonged circulation in plasma can allow sufficient time to reach and maintain presence of the drug at the target site. Finally, adhering to the criteria related to drug development from lab to clinic will not only speed up the process, but will also reduce the cost. In the end this could make the difference for developing a successful product. www.drugdiscoverytoday.com
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Conclusion In 2008 Neuroinsights reported that the worldwide economic burden caused by CNS diseases has reached over $2 trillion a year, and it is likely that this burden will continue to rise with the increasingly ageing population [43]. Treatments for many CNS diseases are not yet widely available, mainly due to the presence of the blood–brain barrier and [therefore] lack of validated targets for these CNS diseases. Although there are many routes for drug delivery to the brain, we believe that the treatment of brain diseases will be best achieved by safely enhancing the blood-to-brain delivery of drugs. Adhering to the 10 key development criteria presented in this review will allow a faster and more successful development of treatments for patients with devastating brain diseases.
19 20
21
22 23
24
25 26
Conflict of interest All authors are employees of to-BBB Technologies BV. Dr. Gaillard is also a shareholder of the company.
27 28
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