scholarly journals Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 625
Author(s):  
Mohammed S. Aldughaim ◽  
Fatimah Alsaffar ◽  
Michael D. Barker
Keyword(s):  
Fold Increase ◽  
Repeated Administration ◽  
Cytotoxic Drugs ◽  
Peptide Sequence ◽  
Tumour Site ◽  
Enzyme Prodrug Therapy ◽  
Nickel Affinity Chromatography ◽  
Glutamic Acid Residue ◽  
Carboxypeptidase G2 ◽  
Effective Delivery

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

scholarly journals Cell-Penetrating Peptides as a Tool for the Cellular Uptake of a Genetically Modified Nitroreductase for use in Directed Enzyme Prodrug Therapy

2019 ◽  
Vol 10 (4) ◽  
pp. 45 ◽  
Author(s):  
Anderson ◽  
Hobbs ◽  
Gwenin ◽  
Ball ◽  
Bennie ◽  
...  
Keyword(s):  
Magnetic Nanoparticle ◽  
Genetically Modified ◽  
Cell Penetrating Peptides ◽  
Target Cells ◽  
Tumour Site ◽  
Enzyme Prodrug Therapy ◽  
Delivery Vector ◽  
Cell Penetrating ◽  
Subsequent Activation ◽  
Potential Use

Directed enzyme prodrug therapy (DEPT) involves the delivery of a prodrug-activating enzyme to a solid tumour site, followed by the subsequent activation of an administered prodrug. One of the most studied enzyme–prodrug combinations is the nitroreductase from Escherichia coli (NfnB) with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitro-benzamide]. One of the major issues faced by DEPT is the ability to successfully internalize the enzyme into the target cells. NfnB has previously been genetically modified to contain cysteine residues (NfnB-Cys) which bind to gold nanoparticles for a novel DEPT therapy called magnetic nanoparticle directed enzyme prodrug therapy (MNDEPT). One cellular internalisation method is the use of cell-penetrating peptides (CPPs), which aid cellular internalization of cargo. Here the cell-penetrating peptides: HR9 and Pep-1 were tested for their ability to conjugate with NfnB-Cys. The conjugates were further tested for their potential use in MNDEPT, as well as conjugating with the delivery vector intended for use in MNDEPT and tested for the vectors capability to penetrate into cells.

scholarly journals Effect of Electro-Acupuncture at ST36 and SP6 on the cAMP -CREB Pathway and mRNA Expression Profile in the Brainstem of Morphine Tolerant Mice

2021 ◽  
Vol 15 ◽  
Author(s):  
Qisheng Wang ◽  
Fenfen Qin ◽  
Hui Wang ◽  
Huanya Yang ◽  
Qingyang Liu ◽  
...  
Keyword(s):  
Antinociceptive Effect ◽  
Cyclic Adenosine Monophosphate ◽  
Fold Increase ◽  
Adenosine Monophosphate ◽  
Repeated Administration ◽  
Underlying Mechanism ◽  
Camp Response Element Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tail Flick ◽  
Hot Plate Test

Undoubtedly, opioid drugs have been the most popular treatment for refractory pain since found, such as morphine. However, tolerance to the analgesic effects caused by repeated use is inevitable, which greatly limits the clinical application of these drugs. Nowadays, it has become the focus of the world that further development of non-opioid-based treatment along with efficient strategies to circumvent opioid tolerance are urgently needed clinically. Fortunately, electro-acupuncture (EA) provides an alternative to pharmaceutic treatment, remaining its potential mechanisms unclear although. This study was aimed to observe the effects of EA on morphine-induced tolerance in mice and discover its underlying mechanism. Tail-flick assay and hot-plate test were conducted to assess the development of tolerance to morphine-induced analgesia effect. As a result of repeated administration scheme (10 mg/kg, twice per day, for 7 days), approximately a two-fold increase was observed in the effective dose of 50% (ED50) of morphine-induced antinociceptive effect. Interestingly, by EA treatment (2/100Hz, 0.5, 1.0, and 1.5 mA, 30 min/day for 7 days) at the acupoints Zusanli (ST36) and Sanyinjiao (SP6), morphine ED50 curves was remarkably leftward shifted on day 8. In addition, the RNA sequencing strategy was used to reveal the potential mechanisms. Due to the well described relevance of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), extracellular regulated protein kinases (ERK), and cAMP response element-binding (CREB) in brainstem (BS) to analgesia tolerance, the cAMP-PKA/ERK-CREB signaling was deeply concerned in this study. Based upon Enzyme-Linked Immunosorbent Assay, the up-regulation of the cAMP level was observed, whereas reversed with EA treatment. Similarly, western blot revealed the phosphorylation levels of PKA, ERK, and CREB were up-regulated in morphine tolerant mice, whereas the EA group showed a significantly reduced expression level instead. This study observed an attenuating effect of the EA at ST36 and SP6 on morphine tolerance in mice, and suggested several potential biological targets by RNA-seq, which include the cAMP-PKA/ERK-CREB signaling pathway, strongly supporting a useful treatment for combatting the opioid epidemic, and opioid-tolerant patients.

scholarly journals Mucoadhesion and Mucopenetration of Cannabidiol (CBD)-Loaded Mesoporous Carrier Systems for Buccal Drug Delivery

2021 ◽  
Vol 89 (3) ◽  
pp. 35
Author(s):  
Ulrike Söpper ◽  
Anja Hoffmann ◽  
Rolf Daniels
Keyword(s):  
Drug Delivery ◽  
Propylene Glycol ◽  
Fold Increase ◽  
Carrier System ◽  
Mucoadhesive Polymers ◽  
Buccal Drug Delivery ◽  
Penetration Ability ◽  
Effective Delivery ◽  
Mucosal Absorption ◽  
Carrier Systems

Transmucosal drug delivery represents a promising noninvasive option when drugs are employed which have a low oral bioavailability like CBD. However, this concept can only be successful as long as the formulation provides sufficient buccal retention and mucosal penetration. In this study, mucoadhesive carrier systems were evaluated consisting of CBD-loaded silica (Aeroperl 300) carriers, mucoadhesive polymers (Hypromellose (HPMC), chitosan and carbomer) and propylene glycol as a penetration enhancer. Mucoadhesive effect, drug release and penetration ability were evaluated for each carrier system. The results show that the addition of HPMC and carbomer substantially improve mucoadhesion compared to pure CBD, with an increase of 16-fold and 20-fold, respectively. However, due to their strong swelling, HPMC and carbomer hinder the penetration of CBD and rely on co-administration of propylene glycol as an enhancer to achieve sufficient mucosal absorption. Chitosan, on the other hand, achieves an 8-fold increase in mucoadhesion and enhances the amount of CBD absorbed by three times compared to pure CBD. Thus, chitosan represents a promising polymer to combine both effects. Considering the results, the development of silica-based buccal drug delivery systems is a promising approach for the effective delivery of CBD.

scholarly journals Application of Gene-Directed Enzyme Prodrug Therapy in Cancer Treatment

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Cindy Yeoh Shin Ly ◽  
Anil Philip Kunnath
Keyword(s):  
Tumor Cells ◽  
Tumor Regression ◽  
Cytosine Deaminase ◽  
Therapeutic Index ◽  
Specific Gene ◽  
Enzyme Prodrug Therapy ◽  
E Coli ◽  
Carboxypeptidase G2 ◽  
High Concentrations ◽  
Simplex Virus

Gene-directed enzyme prodrug therapy (GDEPT) is an advanced cancer therapy that has potential use against localized and metastasized cancer. This strategy aims to improve the limitations of chemotherapy and existing cancer treatments by specific gene delivery, which allows the conversion of systemically administered nontoxic prodrugs to active chemotherapeutic drugs inside the target tumor cells, thereby resulting in a significant therapeutic index by introducing high concentrations of cytotoxic compounds to the tumor cells while limiting the systemic toxicity. The main attraction of GDEPT is by expanding the toxicity to adjacent non-expressing target cancer cells through local and distal bystander effects, leading to tumor regression. This review focused on the application of the six main GDEPT systems for treating cancer, including herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV), cytosine deaminase (CD) from bacteria or yeast with 5-fluorocytosine (5-FC), E. coli nitroreductase (NfsB) with 5-(aziridin-1-yl)-2,4- initrobenzamide (CB1954), hepatic cytochrome P4l50 (CYP450) with cyclophosphamide (CPA), purine nucleoside phosphorylase (PNP) from E. coli with 6-methylpurine deoxyriboside (MEP), and bacterial carboxypeptidase G2 (CPG2) with 4-[(2-chloroethyl)(2-mesloxyethyl)amino] benzoyl-L-glutamic acid (CMDA). In each system, the mechanism of action, clinical trials for the past decades, limitations, and areas that need improvement are discussed.

A Novel Epidural Catheter Fixation Device

2017 ◽  
Author(s):  
Daniel P. Cavanagh ◽  
Asena Abay ◽  
Jessica M. Brito ◽  
Jasmine R. Joyner ◽  
Jordyn N. Nally ◽  
...  
Keyword(s):  
Pain Relief ◽  
State Level ◽  
Repeated Administration ◽  
The Body ◽  
Secondary Migration ◽  
Epidural Injections ◽  
Vaginal Deliveries ◽  
Effective Delivery ◽  
Epidural Catheters ◽  
Healthcare Expenses

Epidurals are a method of long-term pain relief administered by injecting and continuously delivering an anesthetic via catheter in the spine. This method of pain relief is often used for patients in the Obstetrics/Gynecology unit as well as those in pre- and post-operational care. For almost 2 million singleton vaginal deliveries across 27 states in 2008 (representing 65% of all US singleton vaginal births in 2008), 61% of patients received some form of an epidural or spinal injection [1]. Additionally, this number has been increasing. For the 18 states for which 2006 and 2008 data are available, the average of the state-level increases in epidural/spinal injections is approximately 4.2% revealing an overall increase in these injections. Just between 2000 and 2010, the use of epidural injections increased by 160% [2]. Commonly, epidural catheters are inserted into the patient’s back in the appropriate location and then secured to the body with an adhesive medical dressing. Movement and subsequent dislocation of the catheter beneath the adhesive medical dressing can result in inefficient anesthetic delivery, increased patient discomfort, and repeated administration of the epidural. Secondary migration of epidural catheters is a problem responsible for failure in approximately 6.8% of epidurals administered [3]. Requiring an anesthesiologist to repeat the procedure is also an increased cost. A solution to secondary migration of epidural catheters would ensure effective delivery of the anesthetic to the patient, reduce the need for a repeated procedure, and prevent unwanted additional healthcare expenses.

Novel Inhibitors of Carboxypeptidase G2(CPG2):  Potential Use in Antibody-Directed Enzyme Prodrug Therapy

1999 ◽  
Vol 42 (6) ◽  
pp. 951-956 ◽  
Author(s):  
Tariq H. Khan ◽  
Ebun A. Eno-Amooquaye ◽  
Frances Searle ◽  
Pat J. Browne ◽  
Helen M. I. Osborn ◽  
...  
Keyword(s):  
Enzyme Prodrug Therapy ◽  
Carboxypeptidase G2 ◽  
Potential Use

scholarly journals Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

2000 ◽  
Vol 16 (1-2) ◽  
pp. 53-62 ◽  
Author(s):  
K. A. Chester ◽  
J. Bhatia ◽  
G. Boxer ◽  
S. P. Cooke ◽  
A. A. Flynn ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Gamma Camera ◽  
Single Chain ◽  
Enzyme Prodrug Therapy ◽  
Radioimmunoguided Surgery ◽  
Patients With Cancer ◽  
Carboxypeptidase G2 ◽  
Single Chain Fv ◽  
Surgery Trial ◽  
Colorectal Adenocarcinomas

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using123I-MFE-23 and a radioimmunoguided surgery trial using125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

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