Current Pharmaceutical Biotechnology - Volume 9, Issue 4, 2008

In 1934, the polysaccharide hyaluronan [hyaluronic acid or HA] was extracted from bovine eyes by Karl Meyer. As of early 2008, >22,000 research papers on HA-related science, >1,500 US patents with HA claims, and a ∼$1.5 billion/year market (“Global Markets for Hyaluronic Acid” Millenium Research 2006) definitively prove the value of HA. This sugar polymer is in the same chemical family as heparin, an anticoagulant and an antithrombotic that is the most widely used drug in hospitals. This series of articles describe aspects, both historical and emerging, of HA in the world of pharmacology and biotechnology. The sheer abundance of HA in mammalian tissues in certain structures such as the vitreus of eye, the synovial fluid of joints and the dermis of skin spawned the initial idea that HA was a space filler with superb viscoelastic and hydrating properties. The simple repeating unit structure of HA composed of two monosaccharides, (-beta-4-GlcA-1-beta-3-GlcNAc-1)n, also masked its potential for biological complexity; this prejudice is reminiscent of the ‘boring’ DNA molecule composed of just 4 simple bases! HA is also important for boosting infection by certain microbes; these pathogens coat themselves with a molecule that is identical to vertebrate HA to avoid host defenses. The complex machinery for the synthesis (3 genes) and the degradation (perhaps 7 genes) of HA in mammals as well as its effects on the behavior and the proliferation of a variety of cells suggest that in many circumstances, HA and its metabolic products contain information. In the most current hypotheses, the molecular weight (MW), the amount, and the location of the HA molecule effects its ability to trigger various signals that appear to cue development as well as alter inflammation, cell mobility and adhesion in both health and disease. For example, very large mass (2- 8x106 Da or n=∼103-4) and very small mass (∼103 Da or n=∼2-20) HA molecules can have opposite effects on cells with respect to angiogenesis and to cancer cell growth and metastasis (B. Toole et. al). Thus the recent availability of pure, defined narrow size distribution HA preparations (P. DeAngelis) bode well for drug discovery and development. Purified HA from a variety of sources is used in a variety of medical applications. HA was initially isolated from rooster (chicken) comb and used as medical devices for eye surgeries (E. Balazs) and arthritic pain. Extraction of native and now recombinant bacteria (S. Brown & P. Pummill) is now a significant source of less expensive HA. In addition to the native polymeric form of HA, a variety of chemical analogs have been made to alter its physical properties (G. Prestwich & J. Kuo). Cross-linking or esterification have been used to convert HA from its normal extremely viscous liquid state to more solid forms (e.g., gels, fabrics, etc) with longer biological half-life that retain shape or maintain volume. In the near future, more tissue engineering applications and drug delivery systems (T. Brown) for HA should be in clinical trials. Overall, it is a safe wager that the scope of HA applications will expand with the availability of HA formulations with improved performance and as more HA-related biology becomes elucidated.

Hyaluronan solutions known as ophthalmic viscoelastic devices (OVDs) are used in surgical procedures within the eye and on the surface of the eye to prevent dryness and to facilitate wound healing. HA and a variety of derivatives facilitate procedures including vitreoretinal surgery, anterior segment surgery, glaucoma surgery, and corneal transplantation.

Presently, the two main commercial sources of hyaluronic acid (HA) are rooster combs and streptococci. Harvesting from rooster combs is complex and costly. Streptococci are difficult to genetically manipulate and require complex media for growth. Both sources have potential problems with unwanted by-products, such as allergens and toxins. These problems can be solved by producing the HA with safe bacilli that are expressing a recombinant HA synthase (HAS).

Hyaluronan (HA) is a very useful polymer, but its properties sometimes need to be altered or enhanced by chemical modification for biomedical applications. A wide variety of HA derivatives are currently used for eye surgery, joint viscoelastic supplementation, and anti-adhesion films. The future promises to deliver new classes of HA-based reagents as well as new polymers that can be used in situ with living cells or within the body.

In many cases, the cellular response to hyaluronan (HA) depends on the molecular weight (MW) of the polymer chain. Most HA preparations from Nature or its derivatives possess wide size distributions called polydisperse. New chemoenzymatic synthesis technology allows the production of very narrow size distribution polymers called monodisperse. The use of stoichiometrically controlled and synchronized polymerization reactions allows an assortment of new HA reagents in the range of 10 kDa to 2,500 kDa for answering HA biology questions or potentially treating disease.

Hyaluronan (HA) polysaccharide has differential effects on cells depending on polymer size. One of the more exciting findings is that small chains or oligosaccharides of HA (6-18 sugar units), but not large polymers, will kill many types of cancer cells by triggering apoptosis while leaving normal cells unaffected. Even chemoresistant cells become drug-sensitive when co-treated with HA oligosaccharides. Overall, these observations form the basis for new anticancer therapeutics.

Despite advances in chemotherapeutic regimens, the treatment of metastatic cancer remains a challenge. A key problem with chemotherapy drugs is nonspecific drug distribution, resulting in low tumor concentrations and systemic toxicity. The holy grail of clinical cancer research has been to establish more specific ways of directing therapeutics to tumors, whether through more targeted anti-cancer agents or via the method of delivery. Many tumor cells show upregulated expression of receptors for the polysaccharide hyaluronan (HA), resulting in HA having a high affinity for tumors. This observation has led to the preclinical development of HA-cytotoxin bioconjugates that utilize HA as the tumor recognition moiety. The primary challenges have been organ-directed toxicity and limited efficacy. An alternative, simpler strategy has been to use the large volumetric domain of HA to entrain small chemotherapeutic drugs within the HA matrix. The resultant HA/drug formulation accumulates in the microvascular of the tumor, forming a microembolism that increases drug retention at the tumor site and allows for active tumor uptake through HA receptors. Clinical trials of HA formulations of three anti-cancer drugs have been undertaken and have demonstrated that such formulations are safe and efficacious. Within these formulations we postulate that HA is acting as a novel excipient, capable of improving the therapeutic index of the active anti-cancer agent.

Biofilm formation occurs spontaneously on both inert and living systems and is an important bacterial survival strategy. In humans bioflms are responsible for many pathologies, most of them associated with the use of medical devices. A major problem of biofilms is their inherent tolerance to host defences and antibiotic therapies; there is therefore an urgent need to develop alternative ways to prevent and control biofilm-associated clinical infections. Several in vitro experiments have shown that phages are able to infect biofilm cells and that those phages inducing the production of depolymerases have an advantage since they can penetrate the inner layers of the biofilm by degrading components of the biofilm exopolymeric matrix. In practice clinically relevant biofilms and especially those associated with the use of medical devices can possibly be controlled for example by the topic application or the impregnation of the surface of the device with a phage solution. Another interesting approach has been the use of a phage encoding a phage polysaccharide lyase to treat Pseudomonas aeruginosa biofilms in cystic fibrosis patients by aerosol administration. All these strategies require prior identification of the phage and/or polysaccharide depolymerase capable of infecting the bacterial cells and degrading the polysaccharide within the biofilm, respectively. The biofilm organisms must therefore be isolated and screened against a bank of phages. This procedure is essential and raises important biotechnological challenges: the existence of a bank of phages well characterised (physiologically and genetically) whose efficacy in vivo has been tested and pharmacokinetics studied; the existence of economical and safe production protocols and purification methods (e.g. the presence of endotoxins in a phage preparation may compromise phage therapy). It is however important to consider the fact that the chances of getting a specific phage with a high lytic capability and preferential expressing a relevant exopolymer degrading enzyme is likely to be low. Genetically engineered phages can play an important role in this process. Phages can be genetically manipulated to alter their host range and to induce the production of depolymerases. It is therefore important to reinforce the application of synthetic biology to engineer phages able to efficiently degrade medical biofilms. It is also important to develop efficient methods of phage delivery and to study “in vivo” the phage performance against biofilms. It is still not clear how effective the biofilm can be in protecting the phages against the immune system. Efficient and economic phage production and purification protocols need also to be addressed before one can hope to use phage treatment to prevent or control infectious biofilms.

The concept of probiotics now has been around for more than a century, with its consumption increasing exponentially; owing to exciting scientific and clinical findings, limiting side effects of existing pharmaceutical agents and increased consumer demand for natural products. But, the evidence for their safety and efficacy has largely been anecdotal, lacking an integrated scientific basis. Clinical studies conducted with probiotics were of inadequate design and resulted in unreliable data. That is the reason why despite having innumerable potential therapeutic uses probiotics are not being universally accepted. The purpose of present article is to amalgamate various branches of research which would help in development of “better”, “commercial” and “pharmaceutical” probiotic products with defined strength, mechanism of action and indication. Probiotics have been classified into oral and vaginal in accordance to their route of administration, describing the health benefits. The article summarizes the research on significance of strain selection, interactions with coadministered agents and appropriate clinical studies uncovering the safety issues. There is a special emphasis on pharmaceutical issues including probiotic delivery systems, technological challenges during formulation, regulatory concerns, quality control and market potential. Developments in the techniques for in vitro evaluation have also been discussed.

Jatropha curcas is a stress - resistant perennial plant growing on marginal soils. This plant is widespread throughout arid and semiarid tropical regions of the world and has been used as a traditional folk medicine in many countries. J.curcas is a source of several secondary metabolites of medicinal importance. The leaf, fruits, latex and bark contain glycosides, tannins, phytosterols, flavonoids and steroidal sapogenins that exhibit wide ranging medicinal properties. The plant products exhibit anti-bacterial and anti-fungal activities. The paper highlights the ability of various metabolites present in the plant to act as therapeutic agents and plant protectants. The plant is designated as an energy plant and use of J.curcas oil as biodiesel is a promising and commercially viable alternative to diesel oil. The seeds of the plant are not only a source of biodiesel but also contain several metabolites of pharmaceutical importance. Commercial exploitation for biopharmaceuticals and bio-energy production are some of the prospective future potential of this plant. Further reclamation of wastelands and dry lands is also possible with J.curcas cultivation.

Silkworm pupae protein is a good source of high quality protein. The hydrolyzates of silkworm pupae protein catalyzed by neutrase, pepsin, acidic protease (Asperqiius usamii NO. 537), flavourzyme, alcalase, and trypsin with inhibitory activity on angiotensin I-converting enzyme (ACE) were identified by HPLC. The hydrolyzates catalyzed by acidic protease exerted the highest inhibitory activity on ACE. The hydrolyzing conditions were optimized by one-factor, factional factorial (FFD), and center composite (CCD) design methods, and response surface methodology (RSM). Statistical analyses showed that regression of the second-order model equation is suitable to describe ACE inhibitory bioactivity. The predicted inhibitory activity of hydrolyzates on ACE was 73.5 % at a concentration of 2.0 mg/ml. Optimized RSM technique decreased IC50 of hydrolyzates inhibiting ACE to 1.4 mg/mL from 2.5mg/ml. The molecular weight of the components of the hydrolyzates with inhibitory activity on ACE varied from less than 500 to about 1000 Da by ultra-filter analysis. These studies suggest that hydrolyzates of silkworm protein contain ACE inhibitory activity that could form a potential source of ACE inhibitor drugs.

Jatropha curcas is a drought resistant, perennial plant that grows even in the marginal and poor soil. Raising Jatropha is easy. It keeps producing seeds for many years. In the recent years, Jatropha has become famous primarily for the production of biodiesel; besides this it has several medicinal applications, too. Most parts of this plant are used for the treatment of various human and veterinary ailments. The white latex serves as a disinfectant in mouth infections in children. The latex of Jatropha contains alkaloids including Jatrophine, Jatropham and curcain with anti-cancerous properties. It is also used externally against skin diseases, piles and sores among the domestic livestock. The leaves contain apigenin, vitexin and isovitexin etc. which along with other factors enable them to be used against malaria, rheumatic and muscular pains. Antibiotic activity of Jatropha has been observed against organisms including Staphylococcus aureus and Escherichia coli. There are some chemical compounds including curcin (an alkaloid) in its seeds that make it unfit for common human consumption. The roots are known to contain an antidote against snake venom. The root extract also helps to check bleeding from gums. The soap prepared from Jatropha oil is efficient against buttons. Many of these traditional medicinal properties of Jatropha curcas need to be investigated in depth for the marketable therapeutic products vis-à-vis the toxicological effects thereof. This mini review aims at providing brief biological significance of this plant along with its up-to-date therapeutic applications and risk factors.

Caffeine and other methylxanthines produce multiple physiologic effects throughout the human body, many of these effects could potentially modulate the activity of anticancer therapy. Caffeine may directly interfere with drug transport to tumor cells by formation of mixed stacking complexes with polyaromatic drugs. If formed in cells, these complexes may also prevent of intercalating drugs from DNA binding and, in this way, lower their antitumor activity. Since many of potent carcinogens are polyaromatic compounds, formation of stacking complexes with carcinogens could be associated with anti-genotoxic activity of caffeine and its use in cancer chemoprevention. Caffeine has also been reported to inhibit ATM and ATR kinases which leads to the disruption of multiple DNA damage-responsive cell cycle checkpoints and greatly sensitizes tumor cells to antitumor agents which induce genotoxic stress. Caffeine may inhibit repair of DNA lesions through a direct intereference with DNA-PK activity and other repair enzymes. A number of in vitro and in vivo studies demonstrated that caffeine modulates both innate and adaptive immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase. Finally, another group of effects induced by caffeine is mediated through its inhibitory action on adenosine receptors. This may modulate the stability of HIF1 alpha as well as VEGF and interleukin-8 expression in tumor cells, which could have a direct impact on neovascularization of human tumors. In this review, we present different molecular mechanisms by which caffeine and other methylxanthines may directly or indirectly modulate the effect of antitumor treatment in tumor cells and in cancer patients.