Recent approaches for reducing hemolytic activity of chemotherapeutic agents

Recent approaches for reducing hemolytic activity of chemotherapeutic agents

Journal of Controlled Release 211 (2015) 10–21 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevie...

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Journal of Controlled Release 211 (2015) 10–21

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

Recent approaches for reducing hemolytic activity of chemotherapeutic agents Gunjan Jeswani b, Amit Alexander a, Shailendra Saraf c, Swarnlata Saraf c, Azra Qureshi a, Ajazuddin a,⁎ a b c

Rungta College of Pharmaceutical Sciences and Research, Kohka, Kurud Road, Bhilai, Chhattisgarh 490024, India Faculty of Pharmaceutical Science, SSTC, Bhilai, Chhattisgarh, India University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh 492010, India

a r t i c l e

i n f o

Article history: Received 23 April 2015 Received in revised form 31 May 2015 Accepted 1 June 2015 Available online 3 June 2015 Keywords: Chemotherapeutic agent Hemolysis Polymer-drug conjugation Colloidal carriers

a b s t r a c t Drug induced hemolysis is a frequent complication associated with chemotherapy. It results from interaction of drug with erythrocyte membrane and leads to cell lysis. In recent past, various approaches were made to reduce drug-induced hemolysis, which includes drug polymer conjugation, drug delivery via colloidal carriers and hydrogels, co-administration of botanical agents and modification in molecular chemistry of drug molecules. The basic concept behind these strategies is to protect the red blood cells from membrane damaging effects of drugs. There are several examples of drug polymer conjugate that either are approved by Food and Drug Administration or are under clinical trial for delivering drugs with reduced toxicities. Likewise, colloidal carriers are also used successfully nowadays for the delivery of various chemotherapeutic agents like gemcitabine and amphotericin B with remarkable decrease in their hemolytic activity. Similarly, co-administration of botanical agents with drugs works as secondary system proving protection and strength to erythrocyte membranes. In addition to the above statement, interaction hindrance between RBC and drug molecule by molecular modification plays an important role in reducing hemolysis. This review predominantly describes the above recent approaches explored to achieve the reduced hemolytic activity of drugs especially chemotherapeutic agents. © 2015 Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . 1.1. Mechanism of hemolysis . . . . . . . . . 2. Recent avenues for reducing the hemolytic activity 2.1. Drug polymer conjugation . . . . . . . . 2.1.1. PEGylation . . . . . . . . . . . 2.2. In situ hydrogels . . . . . . . . . . . . . 2.3. Colloidal carriers . . . . . . . . . . . . . 2.3.1. Polymeric micelles . . . . . . . . 2.4. Microencapsulation . . . . . . . . . . . 2.4.1. Nanoemulsions . . . . . . . . . 2.5. Botanical agents . . . . . . . . . . . . . 2.5.1. Antioxidant mechanism . . . . . 2.5.2. Enzymatic reaction . . . . . . . 2.6. Molecular chemistry/modification . . . . . 2.6.1. Structural analogues . . . . . . . 2.6.2. Structural derivative . . . . . . . 3. Future prospects . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Novel Drug Development Laboratory, Department of Pharmaceutics, Rungta College of Pharmaceutical Sciences & Research, Kohka, Kurud Road, Bhilai, Chhattisgarh 490024, India. E-mail address: [email protected] (Ajazuddin).

http://dx.doi.org/10.1016/j.jconrel.2015.06.001 0168-3659/© 2015 Elsevier B.V. All rights reserved.

G. Jeswani et al. / Journal of Controlled Release 211 (2015) 10–21

1. Introduction Hemolysis is a term used to indicate the breakdown of erythrocyte membrane with release of hemoglobin into the plasma. There are several causes of hemolysis including immunologic abnormalities, antigen– antibody reactions, mechanical injury, certain infections, hereditary and acquired cell membrane disorders, G6PD1 deficiency, hemoglobinopathies (e.g., sickle cell diseases, thalassemia) and chemotherapeutic agents [1,2]. Hemolysis results in anemia and is a most significant drawback for chemotherapeutic bioactives, limiting their direct use in combating the microbial attack. Most of the chemotherapeutic bioactives viz., carboplatin, cisplatin and nonplatinum prescribed for treating different cancers, have side effects that cause myelosuppression, resulting in severe hemolytic anemia [3]. Myelosuppression is a situation in which there is a decrease in the capacity of the bone marrow to produce blood cells. Primary indications include abnormal paleness of the skin, jaundice, or yellowish texture of skin, eyes, and mouth, high fever, weakness, enlargement of the spleen and liver, tachycardia and heart murmur. Damaged stem cells cause reduction in the WBC2, platelet, and RBC3 counts. These in turn, cause vulnerability to infections and excessive bleeding thereof. However, the overall incidence of hemolytic anemia is limited and the chemotherapeutic agent induced hemolysis is likely boundless. Major clinical complication attributable to hemolytic effect is intravenous infusion at higher dose. As a result, practicing clinicians and healthcare administrators face many challenges in treating patients with chemotherapy-induced hemolytic anemia. The main motivation behind focusing on this topic is that researchers, with the focus on improving the quality of life for patients on chemotherapy have until now somehow overlooked the hemolytic effects and paid attention only to pain and loss of appetite. Intense anemia can lead to predisposition of heart, cardiovascular and lung diseases. Considering the significance, researchers have come up with novel techniques to prevent the undesired hemolytic effect of chemotherapeutic agents among them some of them are patented. A short summary of some of the important patents pertaining to the strategies for reduction of hemolysis is indicated in Table 1. Although, hemolysis is not a restricted effect of chemotherapeutic agents as discussed in the forthcoming section but limiting our search, this review mainly garners some of the recent feasible conceptualizations for reducing the hemolytic activity of chemotherapeutic agents. 1.1. Mechanism of hemolysis Although, the mechanism of hemolysis is unclear, various attempts have been made to elucidate the mechanism. In general, after administration through intravenous route blood components quickly coat the drug molecules [4]. This enables the RES4 to recognize the injected molecules. Macrophages also play a significant role in detection of particles/ molecules in the blood. As discussed by several researchers the size of nanoparticles greatly influences the clearance by RES. Bigger particles are easily cleared as compared to smaller ones of 150–300 nm size range, leading to the distinct changes in biodistribution properties [5,6]. Conversely, smaller particle avoid RES elimination as reported by Gref et al. [7]. Regarding amphiphilic compounds (chemical compounds containing both hydrophilic and hydrophobic properties), as observed the mechanism of hemolysis involves surfactant membrane interaction and membrane solubilization. Surfactant membrane interaction and membrane disruption may be the root cause of occurrence of hemolysis. Micelle formation also plays a significant role at this step. The molecular events generally take place in five distinct steps, (1) the cell surface 1 2 3 4

Glucose-6-phosphate dehydrogenase. White blood cells. Red blood cells. Reticuloendothelial system.

11

absorbs drug/surfactant particles, (2) drug/surfactant gets inserted into the membrane, (3) the changes in arrangement of membrane begin, (4) permeability of membrane increases and (5) finally leads to lysis of membrane (Fig. 1) [8]. The required quantity of surfactant to solubilize a membrane increases with the beginning of formation of micelles. Critical micellar concentration correlates with the above statement [9]. As already, discussed emulsifiers or soaps lead to hemolysis by lowering the interfacial tension of bimolecular lipid film of erythrocytes. While non-polar part of detergent dissolves in oil phase, polar part remains in aqueous medium. As a result, lipid portion of cell membrane is pulled towards aqueous phase, which brings about lysis of cell. In a study, it has been manifested that the addition of an emulsifier with a high cloud point (phospholipids or non-ionic surfactant) like Synperonic® F-68(HLB approx. 29) resulted in an increase cloud point of the emulsifier mixture leading to higher resistance of the emulsifier film against breakdown. This leads to the formation of an extra layer covering the emulsifier mixed film. This further reduces the direct contact of emulsifier and cell membrane resulting into the reduced incidence of hemolysis. This can be a useful approach for parenteral application [10]. Osmotic swelling is another mechanism of hemolysis induced by drugs that enhance the permeability for small ions, which allows the erythrocyte swelling by water influx to balance the osmotic pressure of the cell. It results in physical rupture of RBCs and hemolysis [11]. Some therapeutic agents like saponins show limited therapeutic application due to hemolytic activity associated with them. The mechanism of action involves the interaction of saponins with lipid membrane of erythrocyte and results in the formation of pores in cell by forming insoluble complex with lipid; in addition, saponins interact with aquaporin and induce hemolysis by intake of water [12].

2. Recent avenues for reducing the hemolytic activity 2.1. Drug polymer conjugation The concept of drug–polymer conjugation is mostly exploited to enhance the bioavailability and aqueous solubility of less soluble drugs. Simultaneously, this also reveals the ability of drug to deliver in a precise manner [13]. For regulatory purposes, it is defined as New Chemical Entity. Marketed polymer–drug conjugates like Xyotax™ (PGA–paclitaxel), and Oncaspars® demonstrate the potentials of the technology. Polymeric drug conjugates technically consists of a drug, spacer and a polymeric backbone with targeting moiety. Some of the most popular polymers include polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide copolymers, pullulan, PGA, and poly(L-lysine).

2.1.1. PEGylation PEGylation is a process where polymer chains of polyethylene glycol are covalently attached to another molecule, normally a drug or any other bioactive moiety. PEGs5 are synthetic polymers, which are water insoluble and non-ionic. They have the inherent capacity as drug carriers because of their biocompatibility and heterogeneity. PEG is safe polymer with diminutive toxicity, and is normally removed from the body in intact form either by the kidneys or in the feces [14]. It has pronounced effects on biodistribution and pharmacokinetic by increasing blood circulation half-life, reducing the tissue distribution (RES and macrophage uptake). PEGylation is achieved through chemical procedures and enzymatic/genetic processes. Chemical procedure involves two basic steps where the primary step deals with derivatization and activation of PEG with linkers and the second step deals with the subsequent conjugation of these activated PEG moieties with bioactive. 5

Polyethylene glycol.

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Table 1 Important patent related to hemolysis. Patent publication number

Inventors

Patent description in brief

WO1993005791 A1

Paul B Weisz, Edward J Macarak

EP0558750 B1

Junzo 6–16 Shirogane 3-Chome InagawachoSeki, Hirofumi Nipponshinyaku Co. Ltd. Yamamoto InduJaveri, KaliappanadarNellaiappan

Use of an anionic oligosaccharide, such as polysulfated cyclodextrin, to achieve the desired reduction in hemolytic activity. Amphotericin B dispersion in water is prepared without the use of any type of aids which may precipitate on dilution and give result to hemolysis and other toxicities. Preparation of docetaxel liposomes through techniques using proteins, organic solvents, inorganic salts and heat that produce hemolytic and other side effects. Liposome having a lipid bilayer comprising a head group derivatized lipid.

US20110070295 A1 EP0785773 B1 US5880123 A

Imran Ahmad, Suresh K. Bhatia, Andrew S. Janoff, Eric Mayhew Timothy Harrison

US6413537 B1

Glen S. Kwon, Bong K. Yoo

US20040072990 A1

Jya-Wei Cheng, Shiou-RuTzeng

US4968675 A CA2207652 C

Ching-Chiang Su, Teresa Harshman Timothy Harrison

WO2008029282 A2

Olivier Dorchies, Jiping Liu, William L Rocco

Reducing the hemolytic effects of an amphiphilic compound, this comprises formulating compound with a non-ionic surface-active agent. Masking an amphiphilic compound by the formation of micelles through the surface active agent prevents its damaging interaction with erythrocyte cell membranes. Novel formulation of nystatin for parenteral administration to treat systemic fungal infections. The formulation avoids the toxicity and solubility problems of prior nystatin formulations. Reducing the toxicity of nystatin that appears to be related to its low solubility with the help of dispersing agent (poloxamers, 10 to 98%), cosolvent (0 to 1%). Antimicrobial peptides related to cyclic and short peptides (less than 10 amino acid residues) with unique patterns of aromatic and cationic residues that perform a wide range of antimicrobial activities but display low hemolysis. A pharmaceutical formulation which permits injection of an amino-steroid drug without hemolysis. Use of a non-ionic surface-active agent or an emulsion for the reduction of the hemolytic effects of an amphiphilic compound. A pharmaceutically acceptable aqueous formulation comprising (2R)-2-[4-(7-bromo-2-quinolyloxy)phenoxy] propanoic acid or a pharmaceutically acceptable salt thereof, a physiologically acceptable cyclodextrin, and at least one solubility-enhancing agent.

Recently, we reported PEGylation of melphalan in order to overcome its major problem of solubility and hemolytic activity. It is a drug of choice for the treatment of cancer related to the breast, bone marrow and ovaries. A polymeric prodrug was fabricated by conjugating linear methoxy poly(ethylene glycol) of molecular weights 2000 and 5000 Da with the drug. The prepared conjugates demonstrate significant increase in their aqueous solubility and reduce their inherent hemolytic activity. The length of PEG molecule also affects the hemolytic activity of melphalan as PEG-5000 is found to be better as compared to PEG-2000 in reducing the hemolytic activity (Fig. 2) [15]. Jing X et al. [56] in 2014 also reported the reduced hemolytic activity of paclitaxel exploiting relevant strategy. 2.2. In situ hydrogels Recently, Alexander et al. have highlighted the significance of loading a PEGylated conjugate into an in situ thermogelling hydrogel [16]. Previously, they have underlined the importance of the drug–polymer conjugation using melphalan as a model drug. They had prepared a conjugate with linear M-PEG6 of 2000 and 5000 Da. However, the studies had shown that drug–polymer conjugation has significantly reduced the hemolytic activity when conjugated with PEG 5000. The polymeric prodrug was formed because of an ester linkage between the polymer and the drug. The effectiveness of the conjugates can only be justified when it is loaded to any delivery system. Hence, to validate the practical feasibility of the work, Alexander et al. successfully loaded the melphalan conjugate into the MMW7 chitosan/glycerophosphate disodium salt (C/GP) based smart thermosensitive injectable hydrogel. Chitosan based injectable hydrogels can deliver the drug to the targeted area in the presence of any polyols such as glycerol, ethylene glycol, and sorbitol. These polyols retain the chitosan in solution form by forming a cover of water around chitosan chain in acidic solution. The increase in pH value of chitosan solution is due to collection of GP. This further reduces the electrostatic repulsion among the chitosan molecules, leading to an increase in the hydrogen bonding of chitosan interchain. The prepared 6 7

Methoxy poly(ethylene glycol). Medium molecular weight.

hydrogels do not exhibit any alteration in the hemolytic activity. This reflects that the presence of the chitosan does not alter the hemolytic activity of the conjugate. Moreover, 80% of drug release was found during 100 h form injectable hydrogels containing thermo sensitive polymers impregnated with the C/GP (Fig. 3). Thus, the chitosan based in situ forming thermosensitive hydrogels could be an ideal carrier for the delivery of the PEGylated drugs especially with higher molecular weight. 2.3. Colloidal carriers Drug delivery via colloidal carriers has been used since the last two decades as an efficient and safe mode of achieving targeted and controlled delivery of bioactives. Vesicular carriers or particulates of 25 nm–1 μm size range are known as colloidal carriers. Vesicles are prepared by surrounding liquid phase with amphiphilic molecules into concentric layers [17]. The same approach of micelle formation by selfassembly of amphiphilic molecules extends to vesicles, emulsions and microemulsions. Liposomes, niosomes, sphinosomes, transferosomes, pharmacosomes, colloidosomes, herbosomes and cubosomes are the different novel approaches used for delivering the drugs through colloidal system. 2.3.1. Polymeric micelles Polymeric micelles offer excellent biocompatibility and prolonged circulation time and provide targeted and controlled drug delivery with reduced toxicity [18]. Their structure is that of nanoscopic core/ shell which is composed of block copolymers having hydrophilic and hydrophobic units. The hydrophobic unit constitutes the core and the hydrophilic unit becomes the shell, combined known as micelles. The constituents can be rearranged in a different manner resulting in the formation of diblock copolymers, triblock copolymers, and grafted copolymers. As compared to the low molecular weight conventional systems composed of low molecular weight surfactants, polymeric micelles have added the advantage of a) biological compatibility, b) controlled drug release, c) high loading capacity, and d) enhanced stability [13]. The three most widely studied block copolymer classes are poly(propylene oxide), poly(L-amino acid)s and poly(ester)s.

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Fig. 1. Different mechanism of red blood cell (RBC) hemolysis.

Falamarzian and Lavasanifar in 2010 studied various analogues of MePEO-b-PCL8 co-polymer family with different substitutes on the PCL9 block (benzyl and cholesteryl substituent on PCL, abbreviated as MePEO-b-PBCL10 and MePEO-b-PChCL11. These were synthesized to overcome the shortcomings of aqueous solubility and dose-dependent AmphotericinB (AmB) toxicity towards mammalian erythrocyte cell. Nevertheless, its effective antifungal and antiparasitic properties within narrow therapeutic window are much more significant than its toxicity [19]. In comparison with other analogues, hemolytic activity of AmB significantly reduced. The order followed was MePEO-b-PChCL(7%) b MePEO-b-PCL(15%) b MePEO-b-PPaCL(40%) b MePEO-b-PBCL(60%) b Fungizone® (90%) at 30 μg/ml AmB concentrations, respectively (Fig. 4). Other than the statement above, mentioned polymeric PEO-bPHSA12 micelles are also very effective in enhancing the solubility of AmB. The formulation was found to reduce the hemolytic activity of AmB by controlling its release pattern from micelle which reduces the complete exposure of drug into systemic circulation (Lavasanifar et al., 2001).

2.4. Microencapsulation Microencapsulation is a technique where in tiny droplets of liquid or solid material particles surrounded are coated with a continuous film of polymeric material. A few classes of biodegradable polymers used in microcapsule preparation include polyesters, polyanhydrides, poly(ortho esters), polyphosphazenes and polysaccharides [24]. Recently, Dolores et al. [57] suggested a novel microcapsule formulation of Amphotericin B (AmB) for reducing the hemolytic activity and compared it to the commercial formulation of AmBisome® (a liposomal-marketed formulation of AmB). Formulation containing 5 mg/ml of free-dimeric Amphotericin B was prepared as a colloidal dispersion with sodium deoxycholate, dibasic sodium phosphate and monobasic sodium phosphate. Similarly, another formulation containing Amphotericin in free-poly-aggregated form was prepared. MP (microencapsulated poly-aggregated AmB) and MD (microencapsulated dimer AmB) were prepared from free-poly-aggregated AmB or freedimeric AmB, respectively. The aggregation of AmB results in progressive increase in the particle size of parenterally administered AmB. The surface area per unit mass reduced due to aggregation, which 8 9 10 11 12

Methoxy poly(ethylene oxide)-b-poly(ε-caprolactone). Polycaprolactone. Methoxy poly(ethylene oxide)-b-(benzyl) poly(ε-caprolactone). Methoxy poly(ethylene oxide)-b-(cholesteryl) poly(ε-caprolactone). Poly(ethylene oxide)-block-poly(N-hexyl stearate L-aspartamide).

further lowers the hemolytic activity. Thus, aggregation and altered particle size of the different poly-aggregated AmB formulations play the major protective role in microencapsulation. In general, aggregation leads to increased particle size accompanied with smaller surface area and therefore reduced hemolytic adverse effects [25] (Fig. 5a). They also found that mild heating of Fungizone® (free AmB) also results in aggregation of AmB particles. Thus, they compare the hemolytic reduction caused by heat induced aggregation (HF) with the other formulated pH controlled aggregation preparation of AmB i.e. AmBisome®, FP and MP and found significant difference in the percent hemolytic reduction between heat induced and pH controlled aggregation (Fig. 5b). 2.4.1. Nanoemulsions Nanoemulsions also known as submicron emulsions increase the bioavailability of drugs (primarily belonging to classes II and IV of biopharmaceutical classification system) by presenting and maintaining the drug in a dissolved state, at the molecular level [26]. Lipid emulsions protect the drugs from RES by incorporating the hydrophobic drug into the inert lipid phase. These formulations are prepared by blending oily organic compounds with aqueous hydrophilic compounds under extreme pressure. In respect to the capacity of forming amplified extravascular structure, which can dissolve large quantities of insoluble drugs nanoemulsions, are the ideal carriers for systemic transport. Nevertheless, they also offer biocompatibility and protection from hydrolysis and enzymatic degradation [27]. The reduction in hemolytic activity of lytic agent is reported by the use of lipid emulsion. Lipid emulsions are used to improve the solubility of poorly aqueous soluble drugs. The phospholipid used in the formulation creates a hydrophobic barrier on the surface that causes reduction in hemolytic activity. In several formulation of lipid emulsion, the lytic agent was intercalated in the lipoidal core. As a consequence interaction of lytic agent with the biomembrane was reduced leading to less hemolytic activity. Hemolysis evaluation of a novel polyethylene glycol mediated intravenous injectable lipid nanoemulsion of paclitaxel demonstrated that lipid nanoemulsion could significantly reduce extraction of RES organs and improve intravenous injection safety including irritation, hemolysis and acute toxicity. In a recent study, on comparing the results of hemolysis with Taxol®, extremely low hemolysis of two-vial formulation of paclitaxel loaded lipid nanoemulsion was found, indicating that two-vial formulation of paclitaxel loaded lipid nanoemulsion could effectively protect hemolysis [28]. Recently, Mayson and team have reported the work based on the microemulsion loaded with Gemcitabine, which is a deoxycytidine analogue, used for cancer therapy. It mainly acts by inhibiting the DNA synthesis in cancerous cells that results in apoptosis of cancer cells. In anticancer preparations, gemcitabine is used in large dose because it is degraded by plasma

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Fig. 2. Comparison of percent hemolysis of pure melphalan and its PEGylated conjugate. Adapted and reprinted with permission from Ajazuddin et al. (2013).

deaminase. The high dose of this drug results in very serious adverse effects. In order to improve the efficacy of gemcitabine as anticancer drug, Mayson et al. prepared microemulsion of gemcitabine. They primarily prepared three different formulations having different surfactant:oil ratios (0.4:1), (0.8:1) and (3:1). When gemcitabine was incorporated in

given microemulsion in similar concentration, the formulations were characterized as ME1, ME2 and ME3. All microemulsion preparations were subjected for hemolytic evaluation and it was found in this study that microemulsion of gemcitabine has extensive ability to reduce hemolysis as compared to conventional gemcitabine solution. It was also

Fig. 3. Melphalan release profile from chitosan solution with and without glycerophosphate [16].

Fig. 4. Comparison in hemolytic activity of AmB nanocarrier formulations and its commercial formulation (Fungizone) in rat red blood cells. Adapted and reprinted with permission from Falamarzian and Lavasanifar (2010).

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Fig. 5. Effect on the hemolysis reduction based on the aggregation state of the microencapsulated (with albumin) AmB formulations compared to the free AmB formulations. MP (microencapsulated poly-aggregated AmB) MD (microencapsulated dimer AmB). Adapted and reprinted with permission from Dolores et al. (2013).

inflammatory, antitumor, and radical scavenging properties [33]. The multifunctional benefits of natural and botanical agents provide opportunities to develop strategies for prevention of drug related toxicities. Some of the plants with the potential of reducing hemolytic effects have been explored by coadministration of extracts, but many such remain unexplored and can be investigated in the near future. Lists of plants with antihemolytic effects are depicted in Table 4. (See Tables 2 and 3.) (See Table 5.)

Fig. 6. The percent hemolytic activity of microemulsion preparations without gemcitabine (M1, M2 and M3) and with gemcitabine (M1-D, M2-D and M3-D). Adapted and reprinted with permission from Mayson et al. (2014).

found that the formulation having the lowest ratio of surfactant:oil showed the lowest hemolytic activity as compared to other formulations (Fig. 6) [29]. 2.5. Botanical agents Botanical agents have always gathered much attention because of their multi-faceted actions like antioxidant, phytoprotection, anti-

2.5.1. Antioxidant mechanism A large number of plant extracts possess strong H-donating activity. Hence they become antioxidants to a large extent. Phytochemical classes responsible for antioxidative activity include phenolic acids (gallic, protocatechuic, caffeic, and rosmarinic acids), phenolic diterpenes (carnosol, carnosicacid, rosmanol, and rosmadial), flavonoids (quercetin, catechin, naringenin, and kaempferol), volatile oils (eugenol, carvacrol, thymol, and menthol) and pigments (anthocyanin and anthocyanidin). These act as effective chelating agents which contribute H ions to oxygen radicals. This results in decrease in oxidation [34]. Systems/compounds delaying auto-oxidation are called antioxidants. They inhibit the formation of free radicals or interrupt propagation of free radicals. The following are the mechanism of action: (1) removing peroxidation species, (2) decompose lipid peroxides, (3) chelating metal ions, (4) not permitting formation of peroxides, (5) interrupting auto oxidation chain reaction, and (6) decreasing confined oxygen concentration [34].

Table 2 Polymeric micellar formulations with reduced hemolytic activity. Drug

Clinical use

AmB

Leishmaniasis AmB polymeric micelles of poly(ethylene oxide)-block-poly(benzyl-L-aspartate)

AmB

AmB

Ethaselen

Paclitaxel

Formulation

Hemolytic effect (Percentage of hemolysis)

Reason for reduction in hemolytic activity

0% hemolysis at 3.0

Controlled release of drug from poly(ethylene [19] oxide)-block-poly(benzyl-L-aspartate)micelles.

mg/ml of AmB 0% hemolysis up to 15 μg/ml

Leishmaniasis AmB polymeric micelles of poly(ethylene oxide)-block-poly(N-hexyl stearate L-aspartamide) (PEO-b-PHSA) Systemioc AmB polymeric micelles of poly(ethylene 32% hemolysis at 12 mycosis oxide)-block-poly(εcaprolacatone-co-trimethylcarbonate) μg/ml (1%) and 9% (1% and 10%) hemolysis (10%) Anticancer Ethaselen polymeric micelles of monomethoxy poly(ethylene glycol) and poly(lactide) Anticancer

Doxorubicin Anticancer

Transferrin/PEG/O-carboxymethyl chitosan/fatty acid/paclitxel micelles (TPOCFP)

Doxorubicin loaded MPEG-b-PCL Micelles

With TPOCFP 5 (5 mg paclitaxel) and TPOCFP 10 (10 mg paclitaxel) hemolysis was less than 5% 0% hemolysis at 100 μg/ml

Reference

Micelles of PEO-b-PHSA decrease the hemolytic [20] effect by gradually releasing the nontoxic unaggregated state of unimers. The polymer disaggregates the drug and there [21] is reduced concentration of free drug. In injecting micelle of amphiphilic copolymer, the polymer prevents the interaction of ethaselen with blood components. Combination of attributes associated with PEGylation, trasferin, fatty acid resulted to low hemolytic activity.

[22]

Continuous slow release for a long period.

[23]

[22]

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Table 3 Colloidal carrier for reducing hemolytic activity. Drug/lytic Clinical agent use

Formulation

Hemolytic effect (Percentage of hemolysis)

Reason for reduction in hemolytic activity

Reference

Chitosan

Polymer

Chitosan emulsions with lactic acid

2% hemolysis with 0.5% chitosan in lipid emulsions with Lactic acid

[30]

Sodium oleate

Emulsifier

Solution/dispersion of sodium oleate with the surface active agent

Paclitaxel

Anticancer Lipid nanoemulsion of PEG400 conjugated paclitaxel Anticancer Two Cremophor-free microemulsions, lecithin:butanol:myvacetoil:water (LBMW) and capmul:myvacet oil:water (CMW) Anticancer Microemulsion with reduced Cremophor EL

A decrease from 100% to 5% was observed in hemolytic activity of sodium oleate with increase of emulsifier concentration from 0 to 5% (w/w) –

Very less amount of acid used in the preparation of chitosan lipid emulsion, so no additional acid present to cause hemolysis Inclusion of lytic agent into the core of emulsifier at the interface. As a result the direct contact of the lytic agent with the erythrocytic membrane was decreased Paclitaxel quickly released from nonoemulsion thus preventing uptake by macrophages Optimizing the lower surfactant: RBCs ratio for microemulsion of paclitaxel

Paclitaxel

Paclitaxel

The formulation:blood ratio at which 50% hemolysis (H50) occurs was 0.12 and 0.055 for LBMW and CMW containing 1 mg/ml showed 0% hemolysis at 37 C

Reduced amount of Cremophor EL containing paclitaxel microemulsion

[10]

[28] [31]

[32]

Table 4 Plants with antihemolytic activity. Hemolysis reducing agent

Category

Mechanism

Reference

Clinacanthus nutans Silybum marianum

Antioxidant Favonolignan

[40] [41]

Maytenus royleanus Psidium guajava

Antioxidant Antioxidant

Free radical scavenging activity Interact with lipoperoxyl radicals and spare alpha tocopherol molecules, incorporation into these lipid bilayers of the cell membrane leading to reduced hemolysis Scavenging of hydroxyl radicals Radical scavenging activity

In the last decade, studies have shed some light on the antihemolytic effects of natural products or their constituents [36–38]. Since red blood cell membrane contains high amount of polyunsaturated fatty acids, they are vulnerable to oxidative stress therefore protection through antioxidants can be an effective approach especially to anemia patients where ROS13 production is higher than normal [39]. In continuation, the protective effect of morin was affirmed on 5-Fluorouracil (5-FU) induced toxicity on erythrocytes of Sprague–Dawley rats. 5-FU commonly causes hematotoxicity due to decrease in platelet count, RBC count and myelosuppression. It belongs to chemotherapeutic drugs and reportedly elevates membrane lipid peroxidation due to free radical induced oxidative stress. Lipid peroxidation leads to erythrocyte membrane permeability thus results in hemolysis. Morin (3,5,7,2′,4′pentahydroxyflavone) is a major component isolated from herbs and fruits of the Moraceae family. Morin is also an active component of guava and almond (Prunusdulcis). Previous studies suggest that it elicits oxidative defense against ROS induced oxidative stress, antiinflammatory, and anti-proliferative effects in vivo and in vitro [40]. Oral administration of morin with 5-FU significantly reduces the hemolysis as shown in Fig. 7. In addition, erythrocytes co-treated with 5-FU and morin showed increased resistance to hypotonic solution. This effect may partly be due to the protective effects of morin, quenching of superoxide radicals effectively and scavenging of hydroxyl, and peroxyl radicals, by reducing the level of lipid peroxidation [35]. (+)-Catechin, from Actinidiaargutaplanch (Actinidiaceae) also has protective effect against 5-FU-induced myelosuppression [36]. The above results reveal that the stimulating herbal medicines and plant metabolites quench the ROS in animal models. This may be useful for improving hemolytic effects in clinical trials.

2.5.2. Enzymatic reaction Reducing the hemolytic activity of chemotherapeutic agents using enzymatic mechanism of botanicals can be considered as an attractive 13

Reactive oxygen species.

[42] [43]

strategy. This is demonstrated by incubating the pneumolysin with aqueous extracts of garlic and pure allicin (active component of garlic). One prepared freshly and the other previously prepared and stored at 4 °C for not less than 10 days. Pneumolysin is an extremely important factor contributing to the pathogenesis of Streptococcus pneumoniae. Immunization with pneumolysin toxoid or antipneumolysin antibody protects from lethal damage by virulent pneumococci [37]. The results of the study showed that allicin and garlic extracts save erythrocytes from the lytic effect of pneumolysin. Pre-incubation of whole bacterial cell to approximately 2 μM/ml of allicin completely inhibit pneumolysin hemolytic action. In comparison less concentration of fresh garlic extract is required for the inhibition of pneumolysin hemolytic activity than allicin. On the other hand bacterial cell membrane is readily infiltrated by allicin. The activity of allicin depends on concentration and time required (Fig. 8). Pneumolysin consists of single polypeptide chain. The cytotoxic effects of pneumolysin are inhibited by transition of cysteine residues to disulfides [38]. With reduced hemolytic effect pneumolysin can be used as candidate for the investigation of pneumococcal infections [39]. 2.6. Molecular chemistry/modification 2.6.1. Structural analogues Analogues are often prepared to achieve an improved biological activity profile, with a greater potency. In this approach, one drug is selected as a lead compound and its structural analogues are prepared. The chemical structure and main biological activity of such an analogue are often similar to the lead drug. It may differ in one or more atoms, functional groups, or substructures. Gramicidin is a crystalline, water insoluble antibiotic, used chiefly in treating local infections caused by gram-positive organisms. Therapeutic use of gramicidin is limited to external application. It accelerates hemolysis at a concentration which is lower than what is needed for bacteria cell death. The research shows that the introduction of hydrophobic group along with amino group reduces the hemolytic activity of gramicidin without affecting the antibiotic activity [44].

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Table 5 Chemical modification for reducing hemolytic activity. Drug/lytic agent

Clinical use

Formulation

Hemolytic effect (Percentage of hemolysis)

Cationic antimicrobial peptides

Antimicrobial

Insertion of reduced amide bond Ψ [CH2NH] into a helical peptide of antibacterial nature

Showed 70% hemolysis up to 200

Tetrasubstituted olefins

Anticancer

Ferrocenyl derivatives of tetrasubstituted olefins

Bacterial agglutinating peptide

Antimicrobial

Neomysin

Antimicrobial

Dopamine

Gratisin

Antimicrobial in cosmetic and antioxidant Antimicrobial

Three amino acid residues in GL13NH2 (bacteria agglutinating peptide) were replaced by lysine residues to produce GL13K Neomycin phenol(chloroxylenol) conjugate Dopamine ester using lipase

Gramicidin

Antimicrobial

Diosin

Antitumor

Alkylphospholipids

Anticancer

a

Gratisin analogue with Ser residue in place of Pro5,5′ Gramicidin analogue D-Ala residues in place of D-Tyr6,6 residues 6′O-alkylated dioscin derivatives

Erucylphosphocholine and homocholine analog of erufosine

Reason for reduction in hemolytic activity

Reference

Reduction in electrostatic interactions [49] and hydrophobic interactions between the cationic antimicrobial peptides and lipoidal biomembranes 2-Ferrocynyl-1,1-diphenyl-but-1-ene Alteration in the affinity for erythrocyte [48] and ferrocenophane showed very membrane by modification in chemical low hemolysis at an upper structure concentration of 250 μg/ml GL13K 1 mg/ml caused minor Its structural similarity to human parotid [50] hemolysis secretary protein that reduced hemolysis μg/ml

0.75% hemolysis at 100 μg/ml Dopamine propionate and Dopamine sterate were not hemolytic upto 1000 μg/ml 20% hemolytic activity at 100 μg/Ma Less than 20% hemolytic activity at 100 μ g/Ma –



Alteration in the size and shape of hydrophobic domain Presence of smaller hydrophobic moieties Additional positive charge reduces hemolytic activity Hydrophilicity reduces hemolytic activity Alteration in the chemistry by 6″ O-monosubstitution lowers hemolytic activity Preferentially form lamellar structures in aqueous solutions instead of micelles

[51] [8]

[52] [53] [54]

[55]

Data obtained from the graphical result.

Structures of eight (1–8) synthesized compounds are shown in Fig. 9. Compounds 6, 7, and 8 were simply cis and trans substituted structural conformations. Hemolytic activity tested on sheep erythrocytes indicates that compounds 4 and 5 showed the lowest hemolytic activity compared to gramicidin S as shown in Fig. 10. Their hemolytic activity was greatly affected by the diastereomeric difference and hydrophobic hydrophilic balance of (Lys) n-CO (CH2)6CH3 moiety on amino group of Pro5 (4β-NH2). 2.6.2. Structural derivative Among the several proposed mechanisms of action for anticancer drugs the interaction with biological membrane has been largely favored [45]. A small alteration in chemistry of chemotherapeutic agents influences its affinity to erythrocyte membranes. Tamoxifen is

a synthetic antiestrogenic drug utilized in the chemotherapy of cancerous tissues of the breast. Adverse effects of tamoxifen include hemolytic anemia. Its cytotoxic effects are due to the release of peripheral proteins of membrane cytoskeleton and cytosol proteins. This leads to permanent disturbance in shape of blood cells and reduced mechanical strength [46]. Tamoxifen has been proved to be three times more hemolytic than hydroxytamoxifen because tamoxifen strongly partitions into biomembrane and changes the framework of erythrocyte [47]. Tamoxifen (12.5 μM) induces complete hemolysis after 1 h whereas hydroxytamoxifen produced complete hemolysis at only 35 μM after 1.5 h of incubation. Moreover, hydroxytamoxifen induced hemolysis can be prevented by α-tocopherol and α-tocopherol acetate which seal the membrane permeability paths resulting into structural stabilization (Fig. 11). Protection by α-tocopherol acetate indicates that hemolysis may also occur in deficiency of oxygen [11]. Thus, it is more important to provide mechanical stability to erythrocytes to prevent drug induced anemia. Similarly, five ferrocenyl derivatives of tetrasubstituted olefins showed low hemolytic activity and better antiproliferative activity [48]. 3. Future prospects

Fig. 7. Morin and hemolysis marker (% of hemolysis, CoHb and hemin) levels in the blood of control and experimental animals. Each value expressed as mean ± SD for six rats in each group. Statistical significance at P b 0.05. a: 5-FU vs. Control, b: 5-FU vs. Morin + 5-FU. Adapted and reprinted with permission from Nandha kumar Ramadass et al. (2012).

Biodegradability, biocompatibility and reduced unwanted effects are major requirements of an ideal drug delivery. Hemolysis is the major problem associated with most of the chemotherapeutic agents. Research over the past few years proposes that many trials have been conducted to develop a drug with high therapeutic efficacy and minimum hemolytic effects. In process, various attempts have been made to shed light on the mechanism but some concepts still need to be elucidated, as in the significance of particle size of drug or carrier on biodistribution properties and its subsequent effect on hemolysis. Therefore, more detailed studies are required to get deeper understanding of mechanism and impact of size on clearance, so that the approaches can be directed towards specific causes of hemolysis and its prevention. Although colloidal delivery system due to their unique physicochemical properties (nano particle size, large surface area to mass

18

G. Jeswani et al. / Journal of Controlled Release 211 (2015) 10–21

Fig. 8. Prohibition of pneumolysin hemolytic activity in bacterial cell lysate by: (a) Pure allicin, (b) Old aqueous garlic extract, and (c) fresh aqueous garlic extracts. Adapted and reprinted with permission from M. Arzanlou et al. (2011).

ratio, controllable kinetics, modifiable structure and target specificity) facilitates the administration of chemotherapeutic agents with reduced toxicity, still it suffers from various limitations. These limitations include restricted entrapment efficiency and loading capacity, the complication of the physical state, stability problems during long term storage or administration conditions and unwanted effects of surfactants. To overcome technological drawbacks of this system as well as improving the effectiveness of the drugs novel delivery systems need to be designed and old ones need to be tailored according to the respective drug associated hematological problems. Low drug loading limitation of PEG-drug conjugates can be overcome through more branched and multi-arm conjugates like dendrimers. Considering the limitations in the use of PEG and other synthetic polymers biodegradable and smart polymers for conjugation need to be addressed shortly. In order to

achieve success, numerous polymeric delivery systems have gained much attention for targeting chemotherapeutic agents. In order to overcome the elimination process by RES resealed erythrocytes owing to their immuno-invasive properties can also be used for delivery of chemotherapeutic bioactive agents. Nevertheless, many such systems have not been explored until recently e.g. polyphosphazene and hydroxypropyle methacrylamide derivatives. However, drug delivery route is an important aspect responsible for treatment results. Parenterals have always been an uncomfortable and expensive route of administration, thus an affordable and efficient oral administration is the need of the hour for the delivery of polymeric conjugates. The time dependent release of drug is directly proportional to linker efficiency therefore there are considerable opportunities for

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Fig. 9. Primary structure of gramicidin (GS) and its analogues 1–8. Adapted and reprinted with permission from Makoto Tamaki et al. (2012).

Fig. 10. Dose dependence curve of percent hemolysis against sheep erythrocytes induced by gramicidin (GS) and its analogues 1–5. Adapted and reprinted with permission from Makoto Tamaki et al. (2012).

further enhancements with respect to linker design. Similarly, the use of bioenhancers paves the path for herbal approach towards drug induced hemolysis problem. Recently, several works are taking place for the enhancement of bioavailability of drugs by the use of bioenhancers. Most of them not only enhance the bioavailability of drug but also help in the reduction of toxicities like hemolytic activity associated with therapy. Taking example from previous studies as described in the previous section where coadministration of suitable plant product leads to significant reduction in hemolysis more such agents can be investigated.

The discussed formulation strategies were successful in reducing the hemolytic effect of chemotherapeutic agents. Thus, we suggest on the basis of discussed examples that these strategies can be taken to clinical trial level for development of ideal pharmaceuticals with reduced toxicity and improved quality of life. Lastly, whenever possible, all the approaches must compare with the same drug to evaluate their true potential. In our own personal opinion and evident based published facts, polymer conjugation remains to be the optimized delivery system for the reduction of the hemolytic effect. Moreover, new emerging strategies exploit the delivery of the drug in to the body. There are many potential tools in drug targeting strategy, which reduced hemolytic side effects, especially when dealing with the conjugated drug. Among these approaches, some could be utilized for the loading of the drug polymer conjugate into a PEGylated liposome; PLGA based nanoparticles and nanosuspensions; nanocapsules; stimuli triggered smart hydrogels; esterase-activatable prodrug micelles; boronic acid moieties in poly(amido amine)s; prodrug approach; mannosylated solid lipid nanoparticles; human erythrocytes; and endosomolytic diblock pHresponsive copolymer carrier for the intracellular delivery. Thus, exploitation of these novel strategies could represent potent and safe delivery systems, which had better control on the release of the drug, and at the same time, it will assure lower hemolytic side effects thereof. 4. Conclusion Hemolysis is the most detrimental side effect of chemotherapeutics. Although a number of clinical studies have been initiated for some of the above-discussed strategies with selected categories of drugs, to date no

Fig. 11. (A) Protection of α-tocopherol, (B) protection of α-tocopherol acetate against hydroxytamoxifen induced hemolysis. Adapted and reprinted with permission from M.M Cruz Silva et al. (2001).

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