Biolabile Peptidyl Delivery Systems Toward Sequential Drug Release Elena Ragozin,1 Boris Redko,1,2 Elena Tuchinsky,3 Alex Rozovsky,1,2 Amnon Albeck,2 Flavio Grynszpan,1 Gary Gellerman1 1
Department of Biological Chemistry, Ariel University, Ariel 40700, Israel
2
Department of Chemistry, Bar-Ilan University, Ramat Gan 52900, Israel
3
Department of Molecular Biology, Ariel University, Ariel 40700, Israel
Received 14 September 2015; revised 22 November 2015; accepted 30 November 2015 Published online 11 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22794
ABSTRACT:
the past two calendar years by emailing the Biopolymers editorial office at [email protected].
Compact carriers for peptidyl delivery systems (PDSs) loaded with various drugs were synthesized using a simple and convenient solid phase organic synthesis strategy,
INTRODUCTION
including semi-orthogonal functional group protection
ver the last three decades, chemists, chemical engineers, and biologists have joined forces to create, improve, and optimize molecular systems for targeted drug delivery.1 The main idea behind this concept is to locally increase the concentration of a given drug by altering its pharmacokinetics and biodistribution while delivering the medicine at its desired site of action with minimal degradation.2 By doing this, the effect of the drug can be prolonged by slow release and side-effects can be reduced as a consequence of avoiding or diminishing the direct contact of the drug with undesired biological receptors. These drug delivery systems (DDSs) hold promise for expediting the implementation of personalized medicine, facilitating tailored treatments for a range of human ailments, like cancer, in a custom-tailored fashion. Today, several delivery systems are already available to patients. Some DDSs are based on noncovalent interactions, like stimuliresponsive encapsulating hydrogels, liposomes,3 and micelles. Nanoparticles loaded with anticancer drugs have been proposed to achieve passive accumulation through the enhanced permeability and retention effect (EPR),4–6 while substantially reducing side effects.7–9 Other systems are based on labile covalent bonds involving carrier–drug conjugates in a multi prodrug fashion. Once the carrier systems reach their predetermined target inside the body of the patient the labile covalent bonds get cleaved and drugs are released. In the case of dendrimers10–12 or polymer carriers,13,14 the drug load is released either through enzyme cleavage or simply by chemical hydrolysis of the labile linkers.15 Some of the most common functional groups that are amenable to prodrug design
schemes. Each attachment point of the compact carrier can thus be bound to an anticancer agent through a biodegradable covalent link. Chemo- and biostability experiments of a model peptidyl platform loaded with three different drugs revealed pH and liver homogenate (metabolic) dependent sequential release behavior. The versatility of this approach will serve to expedite the preparation of PDS libraries. This approach may prove useful for applications suitable for personalized medicine where multiple drug delivery is required in a sequential C 2015 Wiley Periodicals, Inc. and controlled fashion. V
Biopolymers (Pept Sci) 106: 119–132, 2016. Keywords: peptidyl delivery system; chemostability; biostability; drug delivery; multiple drug delivery; sequential drug release; prodrug
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from Additional Supporting Information may be found in the online version of this article. Correspondence to: Gary Gellerman; Department of Biological Chemistry, Ariel University, Ariel 40700, Israel; e-mail: [email protected] C 2015 Wiley Periodicals, Inc. V
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include esters, carbonates, carbamates, amides, imides, oximes, ketals, and phosphates (Fig. 1).16 When biodegradation of the carrier does not occur, the DDS can only be eliminated through renal secretion and in this case it is important to keep the polymer at a relatively low molecular weight range in order to avoid unwanted health complications.17 Biodegradable and bioerodible polymers have also been described.18 These carriers are particularly advantageous when they generate nontoxic degradation products that can be metabolized and readily eliminated from the body.19,20 In principle, it is also possible to control the kinetics of drug delivery by chemically adjusting the polymer/drug degradation rate. Unfortunately, some of the large polymer based DDS may induce an immune response in the patient. In addition, their chemical/physical properties are difficult to keep constant from batch-to-batch production.19,21 Polymer based DDSs usually include a targeting moiety which leads the carrier-drug conjugate to its biological target.22 Tumors, where different cancer cells co-exist at various stages of their cell cycle, are difficult targets for single drugs and could be best addressed by the use of drug cocktails.23,24 Administration of such cocktails is often implemented at the maximum tolerated dose (MTD) of each drug, which is associated with serious side effects.25 DDSs are well suited to overcome the above mentioned problems. For instance, the use of liposomes for the delivery of various anticancer drug cocktails has been reported.3 Bahadur and Peisheng designed a nanococktail delivery system based on pH and redox sensitive nano-gels, which release their payload of paclitaxel (PTX) and doxorubicin (DOX) after entering cancer cells via avb5 integrin mediated endocytosis.26 Steffensen and Simanek described poly(block) polymers that can be covalently loaded with different drugs through chemical manipulation involving selective removal of protecting groups.27 Their results are impressive, but the solution phase synthetic approach remains too laborious for practical applications. Although many drug delivery approaches have been examined, the development of DDSs carrying different drugs loaded onto one single scaffold still challenges medicinal chemists. Here we propose the development of multidrug compact carrier systems using amino acid building blocks. Peptidyl compact carriers present several significant advantages, albeit currently limited by relatively low drug loading. These systems may be rationally designed and prepared by solid phase organic synthesis (SPOS) methods in a reproducible way. For the current task, we adopted a synthetic strategy comprising a targeting peptide sequence and a short oligopeptide carrier loaded with a combination of up to three units of the same or different drugs. This approach can obviously be expanded to allow
FIGURE 1 Some useful biolabile covalent linkers.
higher drug loading by extending the number of amino acid units in the carrier oligopeptide. The assembly of all the components of this peptidyl delivery system (PDS) can be performed on a resin support. This approach facilitates straightforward sequence modifications toward the improvement of targeting and biodistribution, stability, and controlled release of the drugs, improved solubility, as well as lowering the immunogenicity of the PDSs. Combinations of these compact PDSs, bearing an assortment of drugs to be released at different but controlled rates, are suitable and attractive for personalized medicinal purposes, which in the future will undoubtedly lead the fight against diseases like cancer. Recently several Lys-based nontoxic and biocompatible pHresponsive nanoparticle architectures for drug delivery were reported.28 Wang and co-workers synthesized a small lactosaminated L-lysine (Lac-Lys) drug delivery carrier to inhibit human hepatitis B virus (HBV) gene expression in liver cells.29 Moreover, multiple antigen peptides (MAPs) were applied in multiple copies (typically 4, 8, or 16) on a small multivalent Lys based core.30–33 In the past, we successfully showed that drug resistance to the drug chlorambucil (CLB) in murine leukemic cells can be overcome by the conjugation of a small biodegradable dendrimer bearing up to four CLB units to a targeting 13mer peptide.34 We have shown the drug release profile and the biological activity of a DDS, including a cyclic somatostatin backbone analog as a targeting unit.35 More importantly, we also reported on the SPOS and the biological activity of a compact DDS, consisting of a targeting 14mer myelin basic protein (MBP) sequence linked to a single Lys amino acid carrier connected to one molecule of CLB and one molecule of camptothecin (CAMP) via hydrolysable covalent bonds.36 Biopolymers (Peptide Science)
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that can be exploited to form biolabile covalent bonds, as shown in Figures 1 and 2. Thus, we concentrated our efforts on the optimization of an Fmoc based protocol compatible with the assembly of compact peptidyl carriers, including preactivation, orthogonal protection/deprotection, and coupling methods. We investigated the sequential release of different drugs from a di-Lys compact carrier and characterized the cytotoxic activity of the system in a biologically relevant milieu. In our present studies, we were not concerned by the selectivity imparted by the targeting peptide, and safely assumed that it can be incorporated to the PDS synthesis, as we have successfully done in the past.34,36
EXPERIMENTAL FIGURE 2 Selected anticancer drugs and drug candidates suitable for the described PDSs.
The present work expands on our encouraging results and demonstrates the flexibility of our approach by generating an oligopeptide compact carrier bearing several units of the same or different anticancer drugs. To that end, we optimized a short and efficient SPOS method for the rapid generation of dipeptide carriers loaded with various combinations of up to three anticancer drugs. For practical reasons we decided not to include the peptide targeting sequence to the synthesis of the currently presented peptidyl carriers. By extrapolation, this strategy can be expanded to admit higher drug loading on lager oligopeptide carriers. In this context we chose several anticancer drugs working through different anti-proliferative mechanisms (Fig. 2): 1. Chlorambucil (CLB) is a nitrogen mustard DNA alkylating agent, used as standard chemotherapy treatment for chronic lymphocytic leukemia (CLL). Repeated use of CLB is known to induce drug resistance in CLL patients.37,38 2. Azatoxin (AZA) is an epipodophyllotoxin–ellipticine hybrid with nonintercalative DNA topoisomerase II (Topo II) inhibitory activity,39,40 which failed in clinical trials, but presented promising results in in vivo testing. 3. Camptothecin (CAMP) is a potent topoisomerase I (Topo I) inhibitor, showing strong antitumor activity both in vitro and in vivo. However, it suffers from poor water solubility and therefore is not orally available.41 4. 3-(9-acridinylamino)25-hydroxymethylaniline (AHMA) is a potential (Topo II)-mediated anticancer 9-aminoacridine analog. It presents enhanced antitumor efficacy against solid tumors in vivo, but it failed in clinical trials.42 A common key feature shared by the selected active compounds resides in the presence of builtin functional groups Biopolymers (Peptide Science)
Materials CAMP and CLB were purchased from Tzamal Laboratories, Petah Tikva, Israel. All cell lines were cultured in an RPMI medium supplemented with glutamine, 10% fetal bovine serum and with penicillin and streptomycin (100 IU/mL of each). The growth medium was supplemented with antibiotics and 2 mM glutamine. The cell culture growth medium and all of its additives were purchased from Biological Industries, BetHa’emek, Israel. All cell cultures were grown at a 378C incubator in an environment containing 6% CO2. The cytotoxicity of the materials was determined by measuring the mitochondrial enzyme activity, using a commercial XTT assay kit (Biological Industries, Bet-Ha’emek, Israel). All samples contained DMSO at final concentration
Department of Biological Chemistry, Ariel University, Ariel 40700, Israel
2
Department of Chemistry, Bar-Ilan University, Ramat Gan 52900, Israel
3
Department of Molecular Biology, Ariel University, Ariel 40700, Israel
Received 14 September 2015; revised 22 November 2015; accepted 30 November 2015 Published online 11 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22794
ABSTRACT:
the past two calendar years by emailing the Biopolymers editorial office at [email protected].
Compact carriers for peptidyl delivery systems (PDSs) loaded with various drugs were synthesized using a simple and convenient solid phase organic synthesis strategy,
INTRODUCTION
including semi-orthogonal functional group protection
ver the last three decades, chemists, chemical engineers, and biologists have joined forces to create, improve, and optimize molecular systems for targeted drug delivery.1 The main idea behind this concept is to locally increase the concentration of a given drug by altering its pharmacokinetics and biodistribution while delivering the medicine at its desired site of action with minimal degradation.2 By doing this, the effect of the drug can be prolonged by slow release and side-effects can be reduced as a consequence of avoiding or diminishing the direct contact of the drug with undesired biological receptors. These drug delivery systems (DDSs) hold promise for expediting the implementation of personalized medicine, facilitating tailored treatments for a range of human ailments, like cancer, in a custom-tailored fashion. Today, several delivery systems are already available to patients. Some DDSs are based on noncovalent interactions, like stimuliresponsive encapsulating hydrogels, liposomes,3 and micelles. Nanoparticles loaded with anticancer drugs have been proposed to achieve passive accumulation through the enhanced permeability and retention effect (EPR),4–6 while substantially reducing side effects.7–9 Other systems are based on labile covalent bonds involving carrier–drug conjugates in a multi prodrug fashion. Once the carrier systems reach their predetermined target inside the body of the patient the labile covalent bonds get cleaved and drugs are released. In the case of dendrimers10–12 or polymer carriers,13,14 the drug load is released either through enzyme cleavage or simply by chemical hydrolysis of the labile linkers.15 Some of the most common functional groups that are amenable to prodrug design
schemes. Each attachment point of the compact carrier can thus be bound to an anticancer agent through a biodegradable covalent link. Chemo- and biostability experiments of a model peptidyl platform loaded with three different drugs revealed pH and liver homogenate (metabolic) dependent sequential release behavior. The versatility of this approach will serve to expedite the preparation of PDS libraries. This approach may prove useful for applications suitable for personalized medicine where multiple drug delivery is required in a sequential C 2015 Wiley Periodicals, Inc. and controlled fashion. V
Biopolymers (Pept Sci) 106: 119–132, 2016. Keywords: peptidyl delivery system; chemostability; biostability; drug delivery; multiple drug delivery; sequential drug release; prodrug
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from Additional Supporting Information may be found in the online version of this article. Correspondence to: Gary Gellerman; Department of Biological Chemistry, Ariel University, Ariel 40700, Israel; e-mail: [email protected] C 2015 Wiley Periodicals, Inc. V
PeptideScience Volume 106 / Number 1
O
119
120
Ragozin et al.
include esters, carbonates, carbamates, amides, imides, oximes, ketals, and phosphates (Fig. 1).16 When biodegradation of the carrier does not occur, the DDS can only be eliminated through renal secretion and in this case it is important to keep the polymer at a relatively low molecular weight range in order to avoid unwanted health complications.17 Biodegradable and bioerodible polymers have also been described.18 These carriers are particularly advantageous when they generate nontoxic degradation products that can be metabolized and readily eliminated from the body.19,20 In principle, it is also possible to control the kinetics of drug delivery by chemically adjusting the polymer/drug degradation rate. Unfortunately, some of the large polymer based DDS may induce an immune response in the patient. In addition, their chemical/physical properties are difficult to keep constant from batch-to-batch production.19,21 Polymer based DDSs usually include a targeting moiety which leads the carrier-drug conjugate to its biological target.22 Tumors, where different cancer cells co-exist at various stages of their cell cycle, are difficult targets for single drugs and could be best addressed by the use of drug cocktails.23,24 Administration of such cocktails is often implemented at the maximum tolerated dose (MTD) of each drug, which is associated with serious side effects.25 DDSs are well suited to overcome the above mentioned problems. For instance, the use of liposomes for the delivery of various anticancer drug cocktails has been reported.3 Bahadur and Peisheng designed a nanococktail delivery system based on pH and redox sensitive nano-gels, which release their payload of paclitaxel (PTX) and doxorubicin (DOX) after entering cancer cells via avb5 integrin mediated endocytosis.26 Steffensen and Simanek described poly(block) polymers that can be covalently loaded with different drugs through chemical manipulation involving selective removal of protecting groups.27 Their results are impressive, but the solution phase synthetic approach remains too laborious for practical applications. Although many drug delivery approaches have been examined, the development of DDSs carrying different drugs loaded onto one single scaffold still challenges medicinal chemists. Here we propose the development of multidrug compact carrier systems using amino acid building blocks. Peptidyl compact carriers present several significant advantages, albeit currently limited by relatively low drug loading. These systems may be rationally designed and prepared by solid phase organic synthesis (SPOS) methods in a reproducible way. For the current task, we adopted a synthetic strategy comprising a targeting peptide sequence and a short oligopeptide carrier loaded with a combination of up to three units of the same or different drugs. This approach can obviously be expanded to allow
FIGURE 1 Some useful biolabile covalent linkers.
higher drug loading by extending the number of amino acid units in the carrier oligopeptide. The assembly of all the components of this peptidyl delivery system (PDS) can be performed on a resin support. This approach facilitates straightforward sequence modifications toward the improvement of targeting and biodistribution, stability, and controlled release of the drugs, improved solubility, as well as lowering the immunogenicity of the PDSs. Combinations of these compact PDSs, bearing an assortment of drugs to be released at different but controlled rates, are suitable and attractive for personalized medicinal purposes, which in the future will undoubtedly lead the fight against diseases like cancer. Recently several Lys-based nontoxic and biocompatible pHresponsive nanoparticle architectures for drug delivery were reported.28 Wang and co-workers synthesized a small lactosaminated L-lysine (Lac-Lys) drug delivery carrier to inhibit human hepatitis B virus (HBV) gene expression in liver cells.29 Moreover, multiple antigen peptides (MAPs) were applied in multiple copies (typically 4, 8, or 16) on a small multivalent Lys based core.30–33 In the past, we successfully showed that drug resistance to the drug chlorambucil (CLB) in murine leukemic cells can be overcome by the conjugation of a small biodegradable dendrimer bearing up to four CLB units to a targeting 13mer peptide.34 We have shown the drug release profile and the biological activity of a DDS, including a cyclic somatostatin backbone analog as a targeting unit.35 More importantly, we also reported on the SPOS and the biological activity of a compact DDS, consisting of a targeting 14mer myelin basic protein (MBP) sequence linked to a single Lys amino acid carrier connected to one molecule of CLB and one molecule of camptothecin (CAMP) via hydrolysable covalent bonds.36 Biopolymers (Peptide Science)
Biolabile Peptidyl Delivery Systems
121
that can be exploited to form biolabile covalent bonds, as shown in Figures 1 and 2. Thus, we concentrated our efforts on the optimization of an Fmoc based protocol compatible with the assembly of compact peptidyl carriers, including preactivation, orthogonal protection/deprotection, and coupling methods. We investigated the sequential release of different drugs from a di-Lys compact carrier and characterized the cytotoxic activity of the system in a biologically relevant milieu. In our present studies, we were not concerned by the selectivity imparted by the targeting peptide, and safely assumed that it can be incorporated to the PDS synthesis, as we have successfully done in the past.34,36
EXPERIMENTAL FIGURE 2 Selected anticancer drugs and drug candidates suitable for the described PDSs.
The present work expands on our encouraging results and demonstrates the flexibility of our approach by generating an oligopeptide compact carrier bearing several units of the same or different anticancer drugs. To that end, we optimized a short and efficient SPOS method for the rapid generation of dipeptide carriers loaded with various combinations of up to three anticancer drugs. For practical reasons we decided not to include the peptide targeting sequence to the synthesis of the currently presented peptidyl carriers. By extrapolation, this strategy can be expanded to admit higher drug loading on lager oligopeptide carriers. In this context we chose several anticancer drugs working through different anti-proliferative mechanisms (Fig. 2): 1. Chlorambucil (CLB) is a nitrogen mustard DNA alkylating agent, used as standard chemotherapy treatment for chronic lymphocytic leukemia (CLL). Repeated use of CLB is known to induce drug resistance in CLL patients.37,38 2. Azatoxin (AZA) is an epipodophyllotoxin–ellipticine hybrid with nonintercalative DNA topoisomerase II (Topo II) inhibitory activity,39,40 which failed in clinical trials, but presented promising results in in vivo testing. 3. Camptothecin (CAMP) is a potent topoisomerase I (Topo I) inhibitor, showing strong antitumor activity both in vitro and in vivo. However, it suffers from poor water solubility and therefore is not orally available.41 4. 3-(9-acridinylamino)25-hydroxymethylaniline (AHMA) is a potential (Topo II)-mediated anticancer 9-aminoacridine analog. It presents enhanced antitumor efficacy against solid tumors in vivo, but it failed in clinical trials.42 A common key feature shared by the selected active compounds resides in the presence of builtin functional groups Biopolymers (Peptide Science)
Materials CAMP and CLB were purchased from Tzamal Laboratories, Petah Tikva, Israel. All cell lines were cultured in an RPMI medium supplemented with glutamine, 10% fetal bovine serum and with penicillin and streptomycin (100 IU/mL of each). The growth medium was supplemented with antibiotics and 2 mM glutamine. The cell culture growth medium and all of its additives were purchased from Biological Industries, BetHa’emek, Israel. All cell cultures were grown at a 378C incubator in an environment containing 6% CO2. The cytotoxicity of the materials was determined by measuring the mitochondrial enzyme activity, using a commercial XTT assay kit (Biological Industries, Bet-Ha’emek, Israel). All samples contained DMSO at final concentration
