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| Proteases as Selective Activators of Triggered Drug Release: A Potential Answer to the Problem of Biomaterial-Associated Infections? |
| Brendan F. Gilmore* |
| Bioactive Biomaterials and Infection Control Research Group, School of Pharmacy, Queen’s University Belfast, UK |
| *Corresponding author: |
Brendan F. Gilmore
Bioactive Biomaterials and Infection
Control Research Group
School of Pharmacy, Queen’s University Belfast, UK
E-mail: b.gilmore@qub.ac.uk |
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| Received July 25, 2012; Accepted July 28, 2012; Published July 31, 2012 |
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| Citation:Gilmore BF (2012) Proteases as Selective Activators of Triggered Drug
Release: A Potential Answer to the Problem of Biomaterial-Associated Infections?
J Biotechnol Biomater 2:e111. doi:10.4172/2155-952X.1000e111 |
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| Copyright: © 2012 Gilmore BF, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
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| It is estimated that at any given time, some 9% of patients in the
United Kingdom have a hospital acquired (nosocomial) infection,
usually bacterial in nature, which may add to the patients discomfort,
increase patient morbidity and mortality and result in increased length
and cost of hospital stay. A UK Department of Health commissioned
study estimated that nosocomial infections may be costing the
National Health Service as much as £1 billion a year, with the National
Audit Office estimating potential gross savings of £150 million per
year provided effective preventative strategies are employed. The
problem of medical device-related infection is one common to all
types of implanted, in-dwelling medical devices. Critically, at least
half of all cases of nosocomial infections associated with implanted
medical devices. A general characteristic of biofilm communities is
that they tend to exhibit significant recalcitrance to antibiotics and
antimicrobial challenge compared with planktonic bacteria of the
same species. The inaccessibility to antibiotics of the device-associated
biofilm and the resistance of biofilm communities to antibiotics
constitute the fundamental challenges in the prevention/eradication
of device-associated bacterial biofilm. A number of past approaches
such as immersion, coating and matrix loading to yield antimicrobialimpregnated
devices have achieved some level of short-term success.
However, major drawbacks have arisen, primarily as a result of less
than optimal drug release profiles and especially due to the release of
‘sub-inhibitory’ concentrations of antimicrobials into the immediate
milieu, believed to contribute to emergence of resistant organisms.
Furthermore, drug release (hence clinical success) of these devices was
facilitated by either the flow of biological fluids across the surface of
the device or immersion of the device in relatively large volumes of
biological fluids. Therefore such approaches are often limited by the
amount of antimicrobial agent which can be incorporated into the
polymer matrix and by the time over which therapeutically active
concentrations of the active agent are released and maintained, limiting
the lifetime of therapeutic activity of the device. Regardless of the class
antimicrobial agent, approaches which balance activity with appropriate
release to retard the emergence of resistance are urgently required to
adequately control device-related biofilm infections. Clearly, there is
enormous scope to minimise the risk of development of device-related
nosocomial infections and to improve the clinical outcomes of patients
hosting implanted medical devices. A logical direction for attention in
this regard must be the modification of the polymer surface/coating
of the medical device, either as a general approach or one tailored
specifically to the site of intended use and likely challenge pathogen(s). |
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| Proteases, also termed proteinases or peptidases, describe a
large and diverse group of proteins that cleave, or cut, or degrade
other proteins by hydrolyzing protein amide bonds (peptide bond
hydrolases). Five major families of proteases have been defined; the
serine proteases, cysteine proteases, aspartate proteases, metallo
proteases and threonine protease. A sixth protease family termed
the glutamic peptidases was not described until 2004. Under normal
conditions, the physiological roles of proteolytic enzymes are exquisitely
controlled and are central to a staggeringly diverse array of cellular and
physiological functions. However, a number of diseases (including
cancer, arthritis, diabetes and infections) are characterised by aberrant or uncontrolled proteolysis which often gives rise to the manifestation
of the disease. Upregulation of protease activity has been linked to a
wide variety of diseases including inflammation, microbial infection,
tumour invasion and angiogenesis. The multifarious roles of proteases
in diseases such as cancer, diabetes, neurological disorders, rheumatoid
arthritis, cardiovascular conditions and Alzheimer’s disease make them
major and exciting therapeutic targets. The potential of proteases as
therapeutic targets, disease markers, prodrug activators, and as targets
for potent and selective inhibitors for the modulation of the disease
process is immense. Thus, disease-associated proteases make attractive
targets for drug delivery system. Recent work in our group has sought
to harness pathogen associated proteases to coordinate drug release
with the presence of infectious microorganisms. |
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| The term “prodrug” was first introduced in 1958 to describe any
compound that undergoes biotransformation prior to elaboration
of their pharmacological effects [1]. Classically, release of the active
drug from the prodrug can be controlled via a chemical or enzymatic
biotransformation. The rationale behind prodrug design is to overcome
various limitations of parent drug, such as poor chemical stability, low
bioavailability, poor aqueous solubility and toxicity. Prodrug design
can also be used to improve targeting of drug action. Ideally, a prodrug
is inactive in its native form, but can be converted to its active form
when triggered. |
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| Numerous protease-triggered prodrugs have been developed,
while the majority have been developed for cancer therapeutics by
taking advantages of the selectivity and specificity of proteases that are
differentially active in the tumour microenvironment. Doxorubicin
has shown antitumor activity against human breast, ovarian, liver
and lung carcinomas. L-leucyl-doxorubicin is an early example of
amino acid prodrug developed in the 1980s [2]. A single amino acid
leucine was simply conjugated to the free amino group of doxorubicin
via the formation of an amide bond. It has been shown that L-leucyldoxorubicin
is converted into active doxorubicin intracellularly or
in the pericellular space by hydrolytic enzymes, with peptidases such
as cathepsin B having been suggested as the putative activator [3,4].
However, the single amino acid prodrugs are not stable in circulation.
For example, pharmacokinetic studies of leucyl-doxorubicin have
revealed that this prodrug was quickly converted into doxorubicin
in plasma [4]. A more stable prodrug of doxorubicin, CPI-0004Na
(N-succinyl-β-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin), was developed to improve the stability of prodrug in blood [5]. The
incorporation of the tetrapeptide structure improves in vivo stability and
selective cleavage by extracellular peptidases secreted by tumour cells
via a multi-step activation process [6]. N-succinyl-β-alanyl-L-leucyl-
L-alanyl-L-leucyl-doxorubicin was initially cleaved extracellularly into
L-alanyl-L-leucyl-doxorubicin by thimet oligopeptidase (TOP), a thioldependent
metallo-oligopeptidase. L-alanyl-L-leucyl-doxorubicin was
then cleaved into L-leucyl-doxorubicin and finally liberated as active
doxorubicin. |
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| In a recent study, Huang et al. [7] utilized the highly selective
expression and the specific proteolytic activity of fibroblast activation
protein-α (FAP-α) to construct a tumor targeting of FAP-α-based
doxorubicin prodrug (Z-Gly-Pro-doxorubicin). It was found that
FAP-α selective expressed on the surface of tumour-associated
fibroblasts while not detected in normal adult tissues except tissues
of healing wound [8]. FAP-α is a type-II transmembrance serine
protease and has been reported to specifically show endopeptidase
activity against N-blocked peptide base substrates, such as Z-Gly-Pro-
AMC [9,10]. Based on this knowledge, Z-Gly-Pro-doxorubicin was
developed for targeting delivery of doxorubicin. The studies revealed
that doxorubicin was released from Z-Gly-Pro-doxorubicin when
incubated with either FAP-α or tumour homogenate of FAP-α positive
tumour (4T1 tumour), while showed highly stability in mouse plasma
and various tissue homogenates. |
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| Matrix metalloprotease (MMP-2 and MMP-9) was also reported
to be exploited in the activation of acetyl L-prolyl-L-leucyl-glycyl-Lleucyl-
doxorubicin prodrug [6]. P3-P1’ sequence acetyl L-prolyl-Lleucyl-
glycyl-L-leucine is a recognized MMP substrate and cleavage
occurred at Gly-Leu bond to generate leucyl-doxorubicin. In addition
to the protease mentioned in this review so far, other proteases such
as plasmin, carboxypeptidase, enkephalinase, prostate-specific antigen,
thrombin, urokinase have been widely used in protease-responsive
prodrug design [11,12]. |
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| According to the above reports, several prerequisities are essential
for the development of protease-activated prodrug. (i) thorough
understanding of the role of protease in disease development; (ii) over
expression of protease in disease-associated environment, while low or
no hydrolytic activity in non-diseased environment; (iii) identification
of peptide sequence with high affinity of targeted protease for selective
and rapid activation of the prodrug. Longer specific peptide motifs are better than single amino acid to achieve better protease selectivity.
Schematic representation of protease-activated prodrug is shown in
Figure 1. |
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|
Figure 1: Schematic representation of protease-activated prodrug. The prodrug is composed of an N terminal blocked protease cleavable peptide sequence
and a potent therapeutic compound (drug). Upon addition of the targeted protease, the enzyme cleaves the prodrug at the peptide cleavage site, leading to
the release of peptide fragments and therapeutic agents. |
|
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| As mentioned above, prodrugs have been developed to enhance the
stability, solubility or to improve targeting delivery of the therapeutic
agent. Polymeric prodrugs provide additional opportunities for
extended and targeted drug delivery. Conjugation of a therapeutic
agent with a polymer leads to a polymeric prodrug. Drug release from
polymer can be achieved by several different stimuli such as light, pH,
temperature, electric/magnetic/sonic field and enzymatic action [13].
Of these, the protease-based stimuli strategies are particular interesting.
Typically, protease-sensitive polymers are composed of a proteasesensitive
substrate and another component that directs or controls
interactions that lead to macroscopic transitions [14]. The release of
therapeutic agent from the polymer is triggered by over-expressed
disease-associated proteases (Figure 2). |
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|
Figure 2: Schematic representation of protease-activated polymeric prodrug. |
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| The protease-based stimuli strategies have been extensively
developed for controlled drug release and drug delivery. For example,
various conjugates of 5-fluorouracil (5-FU) and dextran, poly (ethylene
glycol) (PEG) or poly [N5-(2-hydroxyethyl)-L-glutamine] (PHEG)
were developed and their ability to liberate 5-FU in the presence of
tumor-associated enzymes including collagenase type IV, cathepsin
B and cathepsin D were investigated [15]. 5-FU was attached to the
polymer at the C-terminal glycine residue of peptide spacers. The
conjugate were reported to be able to effect a site-specific release of
5-FU in the presence of collagenase and cathepsin B and the conjugates
P-Gly-Phe-Gly-Gly(FU)OEt (P = dextran/PEG/PHEG) were found to
be the best substrates for cathepsin B. |
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| In another study, Tanihara et al. [16] developed an antimicrobial
release system triggered by thrombin activity. In this system, an
insoluble polymer-drug conjugate was constructed by conjugating
gentamicin to poly(vinyl alcohol) hydrogel via a thrombin-sensitive
peptide linker (Gly-(D)-Phe-Pro-Arg-Gly-Phe-Pro-Ala-Gly-Gly).
The conjugate selectively released gentamicin when incubated with
thrombin-expressed Staphylococcus aureus infected wound fluid,
whereas no biologically active gentamicin detected when incubated
with non-infected wound fluid. Tauro and Gemeinhart [17] developed
a MMPs-specific hydrogel-based delivery system for selective and
local delivery of cisplatin. Cisplatin complexed to an MMP substrate
(Cys-Gly-Leu-Asp-Asp) was incorporated into poly (ethylene glycol) diacrylate (PEGDA) hydrogel. Cisplatin release was controlled based
on the activity of MMPs expressed in U-87 MG cell (a malignant
glioma cell line) culture. |
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| Recently, work in our group has led to the development hydrogel
medical device coatings bearing PEGylated peptidyl prodrugs of
conventional antibiotics which are substrates for the Staphylococcus
aureus serine protease virulence factor V8. In the presence of S. aureus expressing V8 protease, the protease activatable antibiotic-peptide
conjugate is cleaved, releasing sufficient concentrations of antibiotic
to effectively prevent adhesion and biofilm formation (unpublished
data), thus validating this approach to controlling bacterial adhesion
to material surfaces. |
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| In summary, a wide range of proteases have been validated as
specific triggers for controlling drug release. Numerous prodrugs
and polymeric prodrug conjugates have been developed based on the
selective activity of disease-associated proteases to achieve controlled
drug release and targeted drug delivery. Smart protease-triggered drug
release systems may provide a potential answer to numerous diseases
characterised by aberrant proteolysis and, importantly, chronic biofilm
and device associated infections where drug release is coordinated with
the presence of protease-expressing pathogens. |
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| References |
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- Albert A (1958) Chemical aspects of selective toxicity. Nature 182: 421-422.
- Baurain R, Masquelier M, Deprez-De Campeneere D, Trouet A (1980) Amino acid and dipeptide derivatives of daunorubicin. 2. Cellular pharmacology and antitumor activity on L1210 leukemic cells in vitro and in vivo. J Med Chem 23: 1171-1174.
- Trouet A, Masquelier M, Baurain R, Deprez-De Campeneere D (1982) A covalent linkage between daunorubicin and proteins that is stable in serum and reversible by lysosomal hydrolases, as required for a lysosomotropic drug-carrier conjugate: in vitro and in vivo studies. Proc Natl Acad Sci U S A 79: 626-629.
- Breistol K, Hendriks HR, O. Fodstad (1999) Superior therapeutic efficacy of N-L-leucyl-doxorubicin versus doxorubicin in human melanoma xenografts correlates with higher tumour concentrations of free drug. Eur J Cancer35: 1143-1149.
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- Scanlan MJ, Raj BK, Calvo B, Garin-Chesa P, Sanz-Moncasi MP, et al. (1994) Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc Natl Acad Sci U S A 91: 5657-5661.
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- Atkinson JM, Siller CS, Gill JH (2008) Tumour endoproteases: the cutting edge of cancer drug delivery? J Pharmacol 153: 1344-1352.
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