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Ongoing pharmaceutical endeavor to develop orally administrable drugs including oligonucleotide therapeutics for inflammation, cancer and other disorders

One of the key issues discussed at the San Antonio Breast Cancer Symposium concerns drug delivery.  The notion of administering drugs orally seems unconventional given that most cancer chemotherapies are given at an infusion center located in a hospital.  To follow specific dosing schedule, chemotherapy drugs have generally been administered intravenously.  For cytotoxic drugs, unintentional deviation from a particular dosing schedule may limit their therapeutic efficacy as well as increase adverse side effects.  Nevertheless, the option to administer orally may prove to be critical for those who cannot travel easily to receive cancer treatment.

 The anticancer drug Taxol has been used extensively to treat breast or ovarian cancer (also lung cancer, esophageal cancer, head and neck cancer, pancreatic cancer).  Taxols' anticancer property derives from its ability to interfere with 'dynamic instability', the mechanism through which microtubule polymers are maintained in vivo, thus interfering with chromosome segregation during the mitosis.  Taxol was originally isolated from the bark of pacific yew plants; however, the processing of the bark (provides immunity) leads to the death of the plants (38,000 trees yield 25 kilogram Taxol annually).  Yet the demand for Taxol continues to rise drastically (~250 kg annually as of 2012) for treating cancer and other diseases.

 This had led to the semisynthesis of Taxol from 10-deacetylbaccatin isolated from the needles of European yew (Taxus baccata), which leaves the plants viable.  Though the total organic synthesis (40 steps) of Taxol was achieved chemically (Nicolaou et al., 1994), it did not reach commercial application due to a low yield.  More recently, Bristol-Meyer Squibb Pharmaceutical has been relying on a plant cell line (Taxus) to harvest Taxol (by purifying Taxol secreted into growth medium via chromatography and crystallization).  For this, Phyton Biotech Inc. (Germany), which operates the largest GMP plant cell fermentation facility, supplied the Taxol-containing medium.

 For clinical application, Taxol (due to its poor solubility in aqueous media) is dissolved in Cremophor EL (polyoxyethylated castor oil in 1 : 1 mixture with dehydrated ethanol), which causes significant side effects (i.e. hypotension, brochospasm, hypersensitivity) (Surapaneni, et al., 2012).  To avoid using organic solvents for dissolution, 'Abraxane' was developed, which consists of Taxol encased in a nanoparticle comprised of albumin (Wilson et al., 2012).  The intravenously administered Abraxane exits blood vessels via binding to albumin receptor present on endothelial cells, followed by its putative binding to SPARC (Secreted Protein Acidic Rich in Cysteine) overexpressed in various solid tumors.  However, to prevent clogging of capillaries, other types of experimental nanoparticles are being developed using ingredients such as PLGA [poly(lactic-co-glycolic acid)] (Surapaneni, et al., 2012).
     
                                                                                        


The latest innovation concerns the attempt to administer Taxol orally.  Normally, intestinal accumulation of toxic products is prevented by p-glycoprotein (a transporter expressed by the intestinal cells), which pumps them out of the cells.  Unfortunately, p-glycoprotein also prevents the uptake of cytotoxic drugs through effluxing, causing multi-drug resistance.  To allow intestinal absorption, patients with metastatic breast cancer were treated with Encequidar, an inhibitor of p-glycoprotein, along with Taxol in a Phase 3 clinical trial.  Though the study yielded comparable results as intravenously administered Taxol, it led to lower white blood cell counts (neutropenia), increasing the risk of infection (
https://www.abstractsonline.com/pp8/#!/7946/presentation/2050 ).  Also, the question remained as to whether the patients alone could follow complex dosing schedule.

 Increasingly, oligonucleotide based drugs (siRNA, gapmer, miRNA, aptamer) are being recognized as a viable therapeutic modality.  The hurdles once thought insurmountable are slowly being chipped away through chemical modification of oligonucleotides (i.e. nucleobase, internucleotide linkage, sugar moieties) as well as improvements in delivery vector (ex. GlcNAc, cholesterol, PEGylation, peptide) (O'Driscoll et al., 2019).  One of the key barriers for oral delivery of oligonucleotide therapeutics is the presence of gastroinstestinal nucleases (ex. DNase), which has prompted encasing of the oligonucleotides in oil drops such as SEDDS (self-microemulsifying drug delivery system) or nanoparticles like the liposomes to avoid degradation (Hauptstein et al., 2015).  The second barrier is the mucus layer (glycoproteins crosslinked by disulfide bonds; 400 micrometer thick; mesh size 10-200 nanometer; dynamic) making it nearly impossible for bulky plasmid DNA (for gene therapy) to penetrate.  To infiltrate, drug delivery vectors that are mucoinert and less than 100 nanometers may need to be administered along with mucolytic agents (ex. enzymes, sulfhydryl compounds).  Intriguingly, viruses with "slippery" exterior due to a high density of of positive and negative charges are known to permeate through mucus efficiently.  The third barrier is electrostatic repulsion of oligonucleotides (negatively charged) by the anionic charge associated with brush border microvilli (contains charged residues) of the epithelial cells lining the gastrointestinal tract.

Despite the challenge, nearly 20 works have been published regarding the oral delivery of oligonucleotide based drugs (antisense, siRNA) or plasmid DNA (O'Driscoll et al., 2019).    Among their targets include the mRNAs encoding Map4kf4, proinfoammatory cytokines, tumor necrosis factor alpha, VEGF (vascular endothelial growth factor) and survivin.  To suppress systemic inflammation, the investigators at the University of Massachusetts (USA) reported the successful oral delivery of oligonucleotides encased in hollow β1,3-D-glucan particles (purified from baker's yeast)  to suppress TNF and Map4kf4 expression in macrophages found in spleen, liver and lung of mice (Aouadi et al., 2009).  Of relevance to cancer, double gene silencers (shRNA downregulating survivin mRNA; siRNA targeting VEGF mRNA) enclosed in a nanoparticle composed of GTC (galactose modified trimethyl chitosan-cysteine) were delivered orally, which suppressed tumor growth in a mouse model (Han et al., 2014).   Another group utilized glycol chitosan-taurocholic acid conjugate to protect gold-siRNA (inhibit Akt2) conjugate from gastrointestinal degradation, which inhibited colorectal liver metastasis in a murine model after oral delivery (Kang et al., 2017).

 The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as to conjugate to peptides or antibodies.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

 

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/covid-19.aspx

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/tew/fda-approves-3rd-sirna-therapeutic-oxlumo-to-treat-the-metabolic-disease-primary-hyperoxaluria-type-1-and-the-structural-significance-of-2-nucleotide-3-overhang.aspx

 

 References

Aouadi M, Tesz GJ, et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.  Nature.  458:1180-4 (2009).  PMID: 19407801

Han L, Tang C, et al. Oral delivery of shRNA and siRNA via multifunctional polymeric nanoparticles for synergistic cancer therapy.  Biomaterials. 35:4589-600 (2014).  PMID: 24613049

Hauptstein S, Prüfert F, et al.  Self-nanoemulsifying drug delivery systems as novel approach for pDNA drug delivery.  Int J Pharm.  487:25-31 (2015).  PMID: 25839413

Kang SH, Revuri V, et al. Oral siRNA Delivery to Treat Colorectal Liver Metastases.  ACS Nano.  11:10417-10429 (2017).  PMID: 28902489

Nicolaou KC, Yang Z, et al.  Total synthesis of taxol. Nature.  367:630-4 (1994).  PMID: 7906395

O'Driscoll CM, Bernkop-Schnürch A, et al.   Oral delivery of non-viral nucleic acid-based therapeutics - do we have the guts for this?  Eur J Pharm Sci.  133:190-204 (2019).  PMID: 30946964

Surapaneni MS, Das SK, et al. Designing Paclitaxel drug delivery systems aimed at improved patient outcomes: current status and challenges.  ISRN Pharmacol.  2012:623139 (2012).  PMID: 22934190

Wilson SA, Roberts SC.  Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol J.  10:249-68 (2012).   PMID: 22059985