Beyond the nanoscale, protein-polymer conjugate could also be used as building blocks for constructing more complicated structures such as microcapsules through hydrophobic interactions. By performing  pickering emulsion technique  to process BSA–pNIPAm nanoconjugates into hollowed microcapsules consisting of a closely packed monolayer of conjugated protein–polymer building blocks (named proteinosomes). [ 34 ]  These proteinosomes exhibit protocellular properties such as guest molecule encapsulation, selective permeability, controllable mobilization, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis. [ 35 ]


Based on the above-mentioned method, a multi responsive microcapsule has been synthesized by incorporating photoswitchable spiropyran units and the thermoresponsive monomer N-isopropylacrylamide into the membrane. [ 36 ]  Stimuli responsive membrane exhibited advantages in the capture and release of different-molecular-weight products by opening and closing the photoresponsive spiropyran ligands, under body temperature, room temperature, UV, redox.


Another effective way to modulate the permeability of microcapsules was based on a  self-sacrificing  strategy. By selectively using lysozyme and BSA as building blocks as well as  self-sacrificing  components, the corresponding pores could be generated in the membrane, and then the permeability of the generated microcapsules could be increased from10 kDa to 22 kDa and then to 71 kDa. By loading FITC-Lys (14 kDa), RBITCdextran (70 kDa) and DNA (90 kDa) into the microcapsules, a programmed release of the encapsulants from low molecular weight to high molecular weight was realized. [ 37 ]


Using similar strategy, pH-sensitive protein-polymer microcapsules were developed. Both doxycycline (DOX) and folic acid were incorporated onto the surface of protein covalently. The very low toxicity of polymer-protein nanoconjugates effectively avoided the high toxicity of DOX, which is expected to not only reduce toxic side effects, but also improve anticancer efficiency in vitro examinations. [ 38 ]


Protein nanocages are natural nanocarriers composed of protein subunits with a porous structure. They benefit from monodispersity, intrinsic high stability for protection of internalized drugs from enzymatic degradation and controllable assembly for cargo loading and release.


However, their application might be blocked by immunogenicity, broad biodistribution and significant function and property variations. The incorporation of polymer chains by performing in situ ATRP on the outer surface of or inside the protein nanocages can be an effective way to mitigate those drawbacks. For example, increased loading density of cargo molecules and enhanced stability of the cage assembly can be obtained via internal ATRP inside the cavity of the virus capsid. [ 39 ]


Beyond virus type particles, large multimeric proteins such as the iron storage protein ferritin have emerged as attractive tools to be used as well-defined nano-containers. Using a grafting from strategy, polymers can be introduced to ferritin in a highly regular fashion for precise spatial control. [ 40 ]  These polymer–ferritin constructs exhibited protease resistance, enabling longer retention time within the bloodstream while reducing possible antibody interactions.


Polymer-Protein nanoparticles not only contain the traditional properties of nanoparticles, but also have their own unique properties based on the properties of specific proteins. Because they are proteinaceous, they have high biocompatibility,  biodegradability  and biofunctionality. [ 35 ]   Protein-polymer bioconjugates which is the building block of Polymer-Protein hybrids exhibit a unique array of properties such as:  light-switching effects, [ 41 ] [ 42 ]  acoustic signal capture, thermal energy transfer, and magnetic signal response. [ 43 ] [ 44 ] [ 45 ] [ 6 ] [ 46 ]


Generally, Polymer-Protein hybrids can be synthesized by interfacial self-assembly of protein–polymer conjugates in emulsions. [ 47 ]


Grafting to approach which is the most common and straightforward methodology refers to directly attaching the synthetic polymers to the target protein. This technique can be engineered for site-specific or random conjugation and, when compared to other conjugation methods, provides simple and thorough characterization of polymer before conjugation. And when using this method, the protein remains unaffected by polymerization methods.


As shown in the figure, a protein is firstly conjugated with the initiator and the polymer chain then grows from the protein core in a controlled manner via living polymerization. Likewise, to the earlier discussed methods, grafting from approach can be designed for site-specific or random attachment.


Not like the grafting from and grafting to approach which can conjugate several polymers onto one protein core, the grafting through approach enables several proteins to connect to one polymer chain due to the multivalent nature of protein.


Thermoresponsive conjugates have been exploited for the subsequent separation of proteins from a complex mixture. This method has been utilized to purify polyclonal antibodies in serum samples. This method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies. [ 48 ]


Thermoresponsive conjugates can also be exploited to mediate bioactivity. One of the utilities of the method is demonstrated temperature control of biotin binding and release. Biotin binding was observed below the LCST, while above the LCST the conjugates aggregated, and the biotin binding affinity was reduced by ~20%. By changing the temperature, the recovery of the biotinylated molecules can be achieved. [ 49 ]


The absorption of proteins for particles in physiological fluids can greatly affect the subsequent medical performance of particles in vivo. Nonspecific protein adsorption can be controlled in vivo by modifying the nanoparticle surface with a non-toxic, biocompatible protein possessing tolerable antigenic properties such as albumin. [ 50 ]


The high recognition ability of proteins can enable high delivery efficiency. Protein-polymer particles have potential to deliver drugs to specific regions of the body using the inherent biorecognition property at the protein interface. [ 51 ]  Additionally, in some cases the presentation of specific proteins on nanoparticle surfaces can be useful for aiding passage through impermeable biological barriers. [ 52 ]


Enzyme-catalyzed reactions can be performed at higher temperatures using enzyme-immobilized nanoparticles, in which the presence of multiple proteins at the nanoparticle surface facilitates the retention of water molecules limiting the denaturation of the attached proteins. After modification with poly(amide), protein activity could remain unchanged over 500 min at 50 °C, while the half-life time of the native lipase at 50 °C is only 30 min in aqueous solution. [ 53 ]  Immobilized enzymes on nanoparticles can significantly improve the efficiency of enzyme reactions by increasing tolerance to a wider range of experimental conditions without significantly reducing biological activity. Besides, polymer-protein particles are reported to control the activity of proteins [ 54 ]  and compartmentalize different enzymes to perform multi-step reactions. [ 55 ]


By immobilizing proteins to polymer nanoparticles or polymer/inorganic hybrid nanoparticles (such as polymer-stabilized iron oxide nanoparticles), proteins or their affinity ligands can be separated from complex solutions by applying magnetic fields or centrifugation. Lipase attached to iron oxide nanoparticles maintained 85% biological activity after 30 reaction and separation cycles. [ 56 ]


As the appropriate target is combined with magnetic nanoparticles, the selected target can be magnetically separated directly from natural biological fluids, [ 57 ]  which offers a fast, gentle, extensible, and easy to automate separation technique. The simplicity of magnetic separation has been applied in a number of disciplines, including mineral processing wastewater treatment, molecular biology, cell sorting, and clinical diagnostics. [ 58 ] [ 59 ]


Microcapsules termed protocells prepared by polymer-protein hybrids are the hotspot of the research area recently, enabling various functions such as bioreactors, [ 45 ]  cascade system [ 60 ]  and multiresponsive membranes, etc. [ 37 ]


Polypharmacology  is the design or use of pharmaceutical agents that act on multiple targets or disease pathways. [ 1 ]


Despite scientific advancements and an increase of global  R&D  spending, drugs are frequently withdrawn from markets. This is primarily due to their  side effects  or toxicities. Drug molecules often interact with multiple targets and the unintended  drug-target  interactions can cause side effects. Polypharmacology remains to be one of the major challenges in drug development, and it opens novel avenues to rationally design the next generation of more effective but less toxic therapeutic agents. [ 2 ]  Polypharmacology suggests that more effective drugs can be developed by specifically modulating multiple targets. [ 3 ] [ 4 ]


It is generally thought that complex diseases such as cancer and central nervous system diseases may require complex therapeutic approaches. In this respect, a drug that "hits" multiple sensitive nodes belonging to a network of interacting targets offers the potential for higher efficacy and may limit drawbacks generally arising from the use of a single-target drug or a combination of multiple drugs. [ 5 ]  In contrast,  chemical biology  continues to be a reductionist discipline, still regarding chemical probes as highly selective small molecules that enable the modulation and study of one specific target. Chemical biology cannot continue to overlook the existence of polypharmacology text [ according to whom? ]  and its urge to become a more holistic discipline that looks at the use of tool compounds from a systems perspective. [ 6 ]  The use of  chemoproteomics  offers strategies to develop a more holistic understanding of the proteome-wide range of targets a drug interacts with. [ 7 ]


The primordial idea of polypharmacology was first proposed in 2004 by  Bryan Roth . [ 8 ]  He reasoned that most common central nervous system disorders are polygenic in origin, and attempts to develop more effective treatments for diseases such as schizophrenia and depression by discovering drugs selective for single molecular targets ('magic bullets') have been largely unsuccessful. He therefore proposed a proof of concept that designing selectively non-selective drugs (that is, 'magic shotguns') that interact with several molecular targets will lead to new and more effective medications for a variety of central nervous system disorders.


A similar concept was independently proposed in the year of 2006 by Professor Zhiguo Wang [ 9 ]  who used the term 'single agent–multiple targets' (SAMT) to describe the same principle as 'magic shotguns' and his research team provided the first experimental evidence for the feasibility, effectiveness and advantages of SAMT, specifically the 'complex decoy oligodeoxynucleotides technology cdODN' attacking multiple target transcription factors, in the treatment of xenograft breast cancer in mice. Subsequently, Wang's team extended the SAMT to designing single agent that can act on multiple miRNAs targeting cancer cells and cardiac pacemaker channel genes and calcium channel genes as a new therapeutic approach. [ 10 ] [ 11 ] [ 12 ]  Wang has published two monographs on polypharmacology:<Polypharmacology: Principles and Methodologies> [ 13 ]  and <Anti-Aging Polypharmacology> Wang's work is now categorized as 'Epigenetic Polypharmacology' or 'Targeted Polypharmacology', a branch of Polypharmacology. [ 14 ]  In 2008, Professor Keven Shokat and his colleagues described a single compound that blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases and termed it 'multitargeted drug' along with the concept of 'Polypharmacology'. [ 15 ]  Since then, Polypharmacology has become a new branch of Pharmacology discipline and research field as well as one of the new direction and strategies for drug development. [ 16 ]


The  Sheild Professorship of Pharmacology  is the senior  professorship  in  pharmacology  at the  University of Cambridge . It is named in honour of  Marmaduke Sheild . [ 1 ]


The position was originally established on 7 June 1946 as a personal chair for the tenure of  Ernest Basil Verney . On 11 March 1961 the professorship was re-established on a permanent basis. [ 2 ]


A  proteolysis targeting chimera  ( PROTAC ) [ 2 ]  is a molecule that can remove specific unwanted proteins. Rather than acting as a conventional  enzyme inhibitor , a PROTAC works by inducing selective  intracellular   proteolysis . A heterobifunctional molecule with two active domains and a linker, PROTACs consist of two covalently linked protein-binding molecules: one capable of engaging an  E3 ubiquitin ligase , and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in  ubiquitination  and subsequent degradation of the target protein via the  proteasome . Because PROTACs need only to bind their targets with high selectivity (rather than inhibit the target protein's enzymatic activity), there are currently many efforts to retool previously ineffective inhibitor molecules as PROTACs for next-generation drugs. [ 3 ] [ 4 ]


Initially described by Kathleen Sakamoto,  Craig Crews  and Ray Deshaies in 2001, [ 5 ]  the PROTAC technology has been applied by a number of drug discovery labs using various E3 ligases, [ 6 ]  including  pVHL , [ 7 ] [ 8 ] [ 9 ]   CRBN , [ 10 ] [ 11 ]   Mdm2 , [ 12 ]   beta-TrCP1 , [ 5 ]   DCAF11 , [ 13 ] [ 14 ]   DCAF15 , [ 15 ]  DCAF16, [ 15 ]   RNF114 , [ 15 ]  and  c-IAP1 . [ 16 ]   Yale University  licensed the PROTAC technology to Arvinas in 2013–14. [ 17 ] [ 18 ]


In 2019, Arvinas put two PROTACs into clinical trials:  bavdegalutamide  (ARV-110), an  androgen receptor  degrader, and  vepdegestrant  (ARV-471), an  estrogen receptor  degrader. [ 19 ] [ 20 ]  In 2021, Arvinas put a second androgen receptor PROTAC,  Luxdegalutamide  (ARV-766), into the clinic. [ 21 ]


PROTACs achieve degradation through "hijacking" the cell's ubiquitin–proteasome system (UPS) by bringing together the target protein and an E3 ligase. [ 22 ]


First, the E1 activates and conjugates the ubiquitin to the E2. [ 15 ]  The E2 then forms a complex with the E3 ligase. The E3 ligase targets proteins and covalently attaches the ubiquitin to the protein of interest. [ 22 ]  Eventually, after a ubiquitin chain is formed, the protein is recognized and degraded by the  26S proteasome . [ 19 ]  PROTACs take advantage of this cellular system by putting the protein of interest in close proximity to the E3 ligase to catalyze degradation. [ 19 ]


Unlike traditional inhibitors, PROTACs have a  catalytic mechanism , with the PROTAC itself being recycled after the target protein is degraded. [ 19 ]


The protein targeting warhead, E3 ligase, and linker must all be considered for PROTAC development. Formation of a  ternary complex  between the protein of interest, PROTAC, and E3 ligase may be evaluated to characterize PROTAC activity because it often leads to ubiquitination and subsequent degradation of the targeted protein. [ 15 ]  A  hook effect  is commonly observed with high concentrations of PROTACs due to the bifunctional nature of the degrader. [ 15 ]


Currently,  pVHL  and  CRBN  have been used in preclinical trials as E3 ligases. [ 15 ]  However, there still remains hundreds of E3 ligases to be explored, with some giving the opportunity for cell specificity.


Compared to traditional inhibitors, PROTACs display multiple benefits that make them desirable drug candidates. Due to their catalytic mechanism, PROTACs can be administered at lower doses compared to their inhibitor analogues, [ 20 ]  though care needs to be taken in achieving oral bioavailability if administered by that route. [ 23 ]  Some PROTACs have been shown to be more selective than their inhibitor analogues, reducing off-target effects. [ 20 ]  PROTACs have the ability to target previously undruggable proteins, as they do not need to target catalytic pockets. [ 20 ]  This also helps prevent mutation-driven  drug resistance  often found with enzymatic inhibitors.


Pullulan bioconjugates  are systems that use  pullulan  as a scaffold to attach biological materials to, such as drugs. These systems can be used to enhance the delivery of drugs to specific environments or the mechanism of delivery. These systems can be used in order to deliver drugs in response to stimuli, create a more controlled and sustained release, and provide a more targeted delivery of certain drugs.


Pullulan is generated by the microbial  A. pullulans  through the processing mainly of glucose, but can also be produced from  maltose ,  fructose ,  galactose ,  sucrose , and  mannose . [ 1 ]  In a commercial setting, pullulan is obtained from a strain of  A. pullulans  that is non-toxic, non-pathogenic, and unmodified genetically that is given a liquid form of starch in a set environment. [ 1 ]  The pullulan produced can be modified by different conditions such as the nutrients provided, temperature, pH, oxygen content, and other supplements. The microbial needs to be provided with a source of carbon and nitrogen in order to produce pullulan and the ratio of carbon to nitrogen needs to be precise in order to maximize pullulan production. Higher levels of nitrogen than carbon are required as excess carbon can decrease the efficiency of the enzymes and excess nitrogen can increase the production of  biomass , but does not affect the pullulan production. [ 1 ]  Oxygen is also important for the proliferation of the  A. pullulans  cells and the production of pullulan. [ 1 ]  Further supplements can be used in order to increase the level of pullulan production, such as olive oil and tween 80. [ 1 ]


While the manufacturing conditions of pullulan can be altered in order to increase yield, chemical modifications of pullulan can also be used to alter the properties of the pullulan. The unmodified structure of pullulan contains nine  hydroxyl  groups attached to the backbone of the molecule, and these hydroxyl groups can be replaced with other functional groups. [ 1 ]  Some examples of processes that can modify the functional groups of pullulan include  sulfation ,  esterification ,  oxidation ,  etherification ,  copolymerization , amidification, and others. [ 1 ]  Pullulan can be given a negative charge through creating an ester linkage that attaches a carboxylate group to the hydroxyl, which yields a carboxymethyl pullulan. [ 1 ]  Pullulan is hydrophilic and can be modified to have hydrophobic functionality by adding a cholesterol group. [ 1 ]  The main benefit of the added hydrophobic functionality is that it makes it so the pullulan can form self assembling  micelles. [ 1 ]  Another notable modification to pullulan is the  acetylation  of pullulan in order to create pullulan acetate (PA), which also has a hydrophobic functionality. [ 2 ]  PA has the benefit of forming self-assembled  nanoparticles , which can simplify manufacturing of certain pullulan bioconjugates. [ 2 ]  Pullulan and pullulan derivatives can also be folated in order to improve cancer cell targeting as the nanoparticle can be endocytosed into the cancer cells through folate-mediated  endocytosis . [ 3 ]


Pullulan bioconjugate systems can be formed to respond to many different stimuli to enhance the release of the drug to the target tissue. These stimuli include pH, temperature, photothermal, electrical, ultrasonic, magnetic, and enzymatic. [ 1 ]  The pH is often used to target tumor tissues, as the extracellular pH of tumors is more acidic than the normal cells. [ 3 ]


A pullulan and polydopamine hydrogel loaded with crystal violet demonstrated pH responsive behavior due to the  protonation  of the  polydopamine , which increased the release of the  crystal violet  in the acidic environment. [ 4 ]  The study showed that at a pH of a normal cell's extracellular environment, 7.4, about 60% of the crystal violet was released compared to the 87% release when in a pH of 5.0. [ 4 ]  The use of pH responsive systems for the treatment of cancer may aid in the ability to overcome resistance of the drug as well as prevent excess damage to healthy tissue. [ 5 ]


Another pH responsive pullulan system was formed with pullulan and  doxorubicin  where the doxorubicin is attached to the pullulan by  hydrazone  bonds. [ 6 ]  The drug release of the doxorubicin was tested at two pHs, 7.4 and 5, where the hydrazine is stable at 7.4 and cleaves in acidic environments. [ 6 ]  The results from this study supported the results from the pullulan and polydopamine study, as doxorubicin was released faster in the acidic environment than the pH that reflected a normal cell's extracellular environment. [ 6 ]


Temperature can also be used as a trigger to control the drug release from pullulan systems. Thermal responsive pullulan systems can be used in conjunction with thermal generating treatments for cancer in order to improve the treatment. [ 1 ]  Nanoparticles composed of periodate oxidized carboxymethyl pullulan crosslinked with two Jeffamines were synthesized and demonstrated that the nanoparticle size could be decreased with increased temperature. [ 7 ]  The nanoparticles decrease in size with increasing temperature due to the increased temperature promoting the hydrophobic interactions of the structure. [ 7 ]  Altering the temperature can induce heating or cooling dynamics that are reversible, which allows for unique properties in terms of drug release. [ 3 ]  Pullulan can be altered with  photosensitizers  in order to provide a controlled thermal reaction in a target area. [ 1 ]   Spiropyrane  can be added to pullulan in order to act as a photosensitizer. [ 3 ]


Electrical stimuli can be used to alter the delivery of drugs through pullulan constructs. A copolymer polyacrylamide-graft-pullulan was synthesized and used for transdermal delivery of rivastigmine tartarate. [ 8 ]  In this study, the use of electric stimuli demonstrated the ability to increase the diffusion rate and in a way acted as a controllable switch to control diffusion rate. [ 8 ]  Pullulan systems can be used to enhance ultrasound imaging, as pullulan-graft-poly(carboxybetaine methacrylate) demonstrated the ability to generate carbon dioxide in response to ultrasound, which enhanced the contrast. [ 1 ]   Superparamagnetic iron oxide nanoparticles (SPIONs)  have been generated which have magnetic properties, which showed to improve uptake and also decrease the cytotoxicity. [ 1 ]  Enzymes can also be used to trigger drug release mechanisms, such as how esterase has been used to cleave photosensitizers from pullulan in order to increase the photodynamic reaction. [ 1 ]


As demonstrated in the last example, these stimuli response mechanisms do not have to be independent. They can be used in combinations in order to improve the efficacy of the drug delivery.


When pullulan is modified with a hydrophobic functionality, such as cholesterol, the pullulan derivative forms self-assembled vesicles that can encapsulate a hydrophobic drug. [ 1 ]  With the hydrophobic functional group, the pullulan derivative is an amphiphilic molecule, which when in an aqueous environment forms a  micelle . This micelle has a hydrophilic exterior with the pullulan backbone and a hydrophobic core due to the functional group added to the pullulan. [ 3 ]  The nanoparticles formed are spherical, have an average size of 20-30 nanometers according to dynamic light scattering measurements, and are able to be maintained in physiological conditions. [ 9 ]  Cholesteryl-pullulan (CHP) is an example of a pullulan derivative that is capable of forming self-assembled mechanisms and has been used to anticancer drugs. [ 5 ]  The size of the self-assembled nanoparticle can be adjusted by changing the amount of cholesterol attached to the pullulan. The higher the number of cholesterol substitutions, the smaller the nanoparticle created. [ 3 ]   PA and folated PA (FPA) have been created and form self-assembled nanoparticles, which have been used to deliver  epirubicin . [ 2 ]  Pullulan derivatives have been combined with gold to form self-assembled nanoparticles that were capable of loading doxorubicin. [ 10 ]  Pullulan-dexamethasone bioconjugates have been created which also exhibit self-assembling nanoparticles that have an approximate size of 400 nanometers and have shown to extend the release of the  dexamethasone . [ 11 ]


Pullulan is used as a bioconjugate platform in order to enhance the delivery of  chemotherapeutics . Pullulan derivatives can be created in order to specifically target cancer cells. In terms of cancer therapeutics, pullulan can be used to encapsulate hydrophobic cancer therapeutics through self assembled micelles, can be linked to drugs in the form of a bioconjugate, and can be utilized for its pH responsive nature. [ 12 ]  Cancer drugs that have been used with pullulan include doxorubicin,  paclitaxel , epirubicin,  mitoxantrone , and 10-hydroxycamptothecin. [ 12 ]


Pullulan derivatives can be folated in order to take advantage of the higher density of folate receptors on cancer cells. [ 5 ]  Doxorubicin has been loaded into pullulan micelles and folated micelles for targeted delivery to cancer cells through folate mediated endocytosis. [ 5 ]  The use of folated pullulan nanoparticles shows lower toxicity and higher levels of drug accumulation within the cancer cells. [ 5 ]  The pH sensitivity of pullulan also makes pullulan a good candidate for chemotherapeutic delivery, as the pullulan can be altered by the acidic environment of the tumor to provide targeted release. [ 5 ]


Pullulan nanoparticles have also been used to deliver paclitaxel and proved to be stable under different environmental conditions. [ 5 ]   Curcumin  pullulan derivatives have a great effect in targeting  hepatocarcinoma  cells, as the pullulan increases the ability of curcumin to solubilize, and therefore allows for the cells to properly uptake the curcumin. [ 5 ]  Pullulan micelles can also be used to deliver genes, such as  p53 , in order to suppress tumor development. [ 5 ]  The pullulan protects the RNA or DNA from degradation from enzymes within the body, which enables the ability of gene therapy for treatment of cancer. [ 5 ]  The addition of ascorbic acid to pullulan bioconjugates has demonstrated antimetastic properties, which can improve cation modified pullulan derivatives. [ 5 ]  There are many factors that make pullulan a suitable drug delivery platform for cancer therapeutics. Some of these factors include the chemical modifications, the pH responsiveness, as well as the ability for the pullulan to form self-assembled micelles that can protect the therapeutics from the immune system. [ 5 ]


In vitro research has been conducted that synthesized pullulan acetate nanoparticles altered with folate and then loaded with epirubicin. [ 2 ]   This study showed that the use of folate modification to pullulan increased the cytotoxicity of the drug as well as released the drug at a faster rate than unfolated pullulan acetate. [ 2 ]  Another pullulan folated system was researched, where pullulan gold nanoparticles were folated and encapsulated doxorubicin. [ 10 ]  The pullulan gold nanoparticle provided pH controlled release of the doxorubicin and demonstrated lower toxicity to non cancer cells than doxorubicin without a carrier platform. [ 10 ]  CHP systems have been developed to deliver protein vaccines and have shown success in generating different degrees of immune responses mostly with  CD4 T  cells. [ 3 ]  Biotinylated pullulan acetate (BPA) have been created as they have vitamin H functionality, which helps increase the level of interaction with cancer cells. [ 13 ]  The drawback with vitamin H is that increasing the vitamin H increases the interaction of the nanoparticles with cancer cells, but also lowers the concentration of the drug in the nanoparticle due to the altered hydrophobicity. [ 13 ]  Modifications to pullulan can be made to enhance the controlled release of drugs, such as pullulan-g-poly(L-lactide) due to the water insoluble nature of the polymeric component. [ 13 ]   Doxorubicin has been conjugated to pullulan through hydrazone bonds, but was shown to have lower cytotoxic activity than doxorubicin without a delivery platform. [ 6 ]


The ocular space is a difficult area to deliver drugs into and therefore special drug delivery considerations need to be taken into account.  Intravitreal injections  are a common method of delivery drugs to the eye. Pullulan systems can be utilized in intravitreal injections in order to develop drugs that are long lasting and therefore require less frequent injections. [ 11 ]  One study looked at different chemical linkers to pullulan to test efficacy of said linkers in extending the release of  rhodamine B  (RhB). [ 9 ]  This study used ether (Pull-Et-RhB), hydrazone (Pull-Hy-RhB), and ester (Pull-Es-RhB) linkers to generate pullulan bioconjugates. [ 9 ]  Ex vivo modeling of the drug release indicated that the drug diffuses slower in the vitreous humor than in water. [ 9 ]  The ether bond was stable at differing pH, while the hydrazone and ester bond released the drug faster in more acidic pH, that reflected the pH of endosomes. [ 9 ]  The Pull-Hy-RhB demonstrated that this drug delivery system was capable of delivering the drug to the retina through testing of the blood in the vessels of the retina. [ 9 ]


Further studies have investigated the creation and efficacy of pullulan-dexamethasone bioconjugates for intravitreal injections. [ 14 ]  The study synthesized self-assembling pullulan nanoparticles with dexamethasone attached through hydrazone bonds. [ 11 ]   This study reiterated that the drug release was fast in acidic pH that mimicked the pH of  lysosomes. [ 11 ]  The variation in drug release was that at the pH of the vitreous humor the drug took two weeks to release half of the drug, while took only two days, when in a lysosomal pH. [ 11 ]   Pharmacokinetic  analysis was performed on this bioconjugate system and revealed that dexamethasone was released in the vitreous humor and that it remained for sixteen days and that a substantial amount of the bioconjugate left the vitreous humor intact. [ 14 ]  Overall the studies regarding pullulan bioconjugates for the application in intravitreal injections demonstrate that pullulan can provide sustained release as well as allow the drug to reach the retina.


Pullulan has many other applications. Pullulan can be used as a scaffold material for stem cells, such as  mesenchymal stem cells . [ 1 ]  Pullulan can be conjugated with photosensitive molecules in order to be used with photodynamic therapy. [ 1 ]  Pullulan can be modified to be a contrast agent for MRI in multiple ways such as oxidation, iron-oxide conjugates, and cation conjugates. [ 1 ]  Pullulan has been thiolated in order to generate mucoadhesive properties. [ 15 ]  This mucoadhesive system has been further modified by polyaminating pullulan to provide sustained drug release. [ 15 ]  A study developed a transdermal pullulan system that is capable of delivering rivastigmine tartarate in response to external electrical stimuli. [ 8 ]  Pullulan systems can be loaded with a plethora of different drugs including anti-inflammatory, antilipidemic, and antiglycemic drugs. [ 12 ]  Pullulan systems can be used to treat heart conditions through the delivery of  beta blockers  and inhibitors of  angiotensin-converting enzyme . [ 12 ]  Pullulan can also be utilized in regards to bone disease as they can be used to deliver  bisphosphonates  and can help to image bone regeneration through MRI. [ 12 ]


Quantitative systems pharmacology  (QSP) is a discipline within biomedical research that uses mathematical computer models to characterize biological systems, disease processes and drug pharmacology. [ 1 ] [ 2 ]  QSP can be viewed as a sub-discipline of  pharmacometrics  that focuses on modeling the mechanisms of drug  pharmacokinetics  (PK),  pharmacodynamics  (PD), and disease processes using a  systems pharmacology  point of view.  QSP models are typically defined by systems of  ordinary differential equations  (ODE) that depict the dynamical properties of the interaction between the drug and the biological system.


QSP can be used to generate biological/pharmacological hypotheses  in silico  to aid in the design of  in vitro  or  in vivo  non-clinical and clinical experiments.  This can help to guide biomedical experiments so that they yield more meaningful data.  QSP is increasingly being used for this purpose in pharmaceutical research & development to help guide the discovery and development of new therapies. [ 3 ] [ 4 ]  QSP has been used by the FDA in a clinical pharmacology review. [ 5 ]


QSP emerged as a discipline through two workshops held at the National Institutes of Health (NIH) in 2008 and 2010, with the goal of merging of systems biology and pharmacology. The workshops outlined a need for a mathematical discipline to aid in translational medicine. QSP proposed integrating concepts, methods, and investigators from computational biology, systems biology, and biological engineering into pharmacology.  [ 2 ]


A review of the history and future of QSP identified areas where it has advanced understanding of drug mechanisms, supported preclinical to clinical translation, and in general aided in drug development. The FDA has included QSP as a component of the Model-Informed Drug Development Program.  [ 6 ]   [ 7 ]


Receptor theory  is the application of receptor models to explain drug behavior. [ 1 ]  Pharmacological receptor models preceded accurate knowledge of  receptors  by many years. [ 2 ]   John Newport Langley  and  Paul Ehrlich  introduced the concept that receptors can mediate drug action at the beginning of the 20th century.  Alfred Joseph Clark  was the first to quantify drug-induced biological responses (specifically, f-mediated receptor activation). So far, nearly all of the quantitative theoretical modelling of receptor function has centred on  ligand-gated ion channels  and  G protein-coupled receptors . [ 3 ]


In 1901, Langley challenged the  dominant hypothesis  that drugs act at nerve endings by demonstrating that nicotine acted at sympathetic ganglia even after the degeneration of the severed preganglionic nerve endings. [ 4 ]  In 1905 he introduced the concept of a receptive substance on the surface of skeletal muscle that mediated the action of a drug. Langley postulated that these receptive substances were different in different species (citing the fact that nicotine-induced muscle paralysis in mammals was absent in crayfish). [ 5 ]  Around the same time,  Ehrlich  was trying to understand the basis of selectivity of agents. [ 6 ]  He theorized that selectivity was the basis of a preferential distribution of lead and dyes in different body tissues. However, he later modified the theory in order to explain immune reactions and the selectivity of the immune response. [ 6 ]  Thinking that selectivity was derived from interaction with the tissues themselves, Ehrlich envisaged molecules extending from cells that the body could use to distinguish and mount an immune response to foreign objects. However, it was only after Ahlquist demonstrated the differential effects of adrenaline on two distinct receptor populations, that the theory of receptor-mediated drug interactions gained acceptance. [ 7 ] [ 8 ]


The receptor occupancy model, which describes agonist and competitive antagonists, was built on the work of Langley,  Hill , and Clark. The occupancy model was the first model put forward by Clark to explain the activity of drugs at receptors and quantified the relationship between drug concentration and observed effect. It is based on mass-action kinetics and attempts to link the action of a drug to the proportion of receptors occupied by that drug at equilibrium. [ 9 ] [ 10 ]   In particular, the magnitude of the response is directly proportional to the amount of drug bound, and the maximum response would be elicited once all receptors were occupied at equilibrium. He applied mathematical approaches used in enzyme kinetics systematically to the effects of chemicals on tissues. [ 2 ]
He showed that for many drugs, the relationship between drug concentration and biological effect corresponded to a hyperbolic curve, similar to that representing the adsorption of a gas onto a metal surface [ 11 ]  and fitted the  Hill–Langmuir equation . [ 3 ]  Clark, together with  Gaddum , was the first to introduce the  log concentration–effect curve  and described the now-familiar 'parallel shift' of the log concentration–effect curve produced by a competitive antagonist. [ 3 ]  Attempts to separate the binding phenomenon and activation phenomenon were made by  Ariëns  in 1954 and by Stephenson in 1956 to account for the intrinsic activity (efficacy) of a drug (that is, its ability to induce an effect after binding). [ 9 ] [ 12 ] [ 13 ]  Classic occupational models of receptor activation failed to provide evidence to directly support the idea that receptor occupancy follows a Langmuir curve as the model assumed leading to the development of alternative models to explain drug behaviour. [ 12 ]


The development of the classic theory of drug antagonism by Gaddum, Schild and Arunlakshana built on the work of Langley,  Hill  and Clark. [ 12 ]   Gaddum  described a model for the competitive binding of two ligands to the same receptor in short communication to  The Physiological Society  in 1937. The description referred only to binding, it was not immediately useful for the analysis of experimental measurements of the effects of antagonists on the response to agonists. [ 14 ]  It was Heinz Otto Schild who made measurement of the equilibrium constant for the binding of an antagonist possible.  He developed the  Schild equation  to determine a dose ratio, a measure of the potency of a drug. In Schild regression, the change in the dose ratio, the ratio of the EC 50  of an agonist alone compared to the EC 50  in the presence of a competitive antagonist as determined on a dose response curve used to determine the affinity of an antagonist for its receptor.


The flaw in Clark's receptor-occupancy model was that it was insufficient to explain the concept of a  partial agonist . This led to the development of agonist models of drug action by Ariens in 1954 and by Stephenson in 1956 to account for the intrinsic activity (efficacy) of a drug (that is, its ability to induce an effect after binding). [ 12 ] [ 13 ]


The two-state model is a simple linear model to describe the interaction between a ligand and its receptor, but also the active receptor (R * ). [ 15 ]  The model uses an equilibrium dissociation constant to describe the interaction between ligand and receptor.  It proposes that ligand binding results in a change in receptor state from an inactive to an active state based on the  receptor's conformation . A receptor in its active state will ultimately elicit its biological response. It was first described by Black and Leff in 1983 as an alternative model of receptor activation. [ 16 ]  Similar to the receptor occupancy model, the theory originated from earlier work by del Castillo & Katz on observations relating to ligand-gated ion channels. [ 3 ]  In this model,  agonists  and  inverse agonists  are thought to have selective binding affinity for the pre-existing resting and active states [ 3 ] [ 17 ]  or can induce a conformational change to a different receptor state.  Whereas  antagonists  have no preference in their affinity for a receptor state. [ 18 ] [ 19 ]  The fact that receptor conformation (state) would affect binding affinity of a ligand was used to explain a mechanism of partial agonism of receptors by del Castillo & Katz in 1957 was based on their work on the action of acetylcholine at the motor endplate [ 3 ]  build on similar work by Wyman & Allen in 1951 on conformational-induced changes in hemoglobin's oxygen binding affinity occurring as a result of oxygen binding. [ 20 ]  The del Castillo-Katz mechanism divorces the binding step (that can be made by agonists as well as antagonists) from the receptor activation step (that can be only exerted by agonists), describing them as two independent events. [ 20 ]


The original Ternary complex model was used to describe ligand, receptor, and G-protein interactions.  It uses equilibrium dissociation constants for the interactions between the receptor and each ligand (K a  for ligand A; K b  for ligand B), as well as a cooperativity factor (α) that denotes the mutual effect of the two ligands on each other's affinity for the receptor. An α > 1.0 refers to positive allosteric modulation, an α < 1.0 refers to negative allosteric modulation, and an α = 1.0 means that binding of either ligand to the receptor does not alter the affinity of the other ligand for the receptor (i.e., a neutral modulator). [ 15 ]  Further, the α parameter can be added as a subtle but highly useful extension to the ATCM in order to include effects of an allosteric modulator on the efficacy (as distinct from the affinity) of another ligand that binds the receptor, such as the orthosteric agonist. Some ligands can reduce the efficacy but increase the affinity of the orthosteric agonist for the receptor. [ 15 ]


Although it is a simple assumption that the proportional amount of an active receptor state should correlate with the biological response, the experimental evidence for receptor overexpression and spare receptors suggests that the calculation of the net change in the active receptor state is a much better measure for response than is the fractional or proportional change. This is demonstrated by the effects of agonist/ antagonist combinations on the desensitization of receptors. This is also demonstrated by receptors that are activated by overexpression, since this requires a change between R and R* that is difficult to understand in terms of a proportional rather than a net change, and for the molecular model that fits with the mathematical model. [ 21 ] [ 22 ] [ 23 ]


The classic  Monod–Wyman–Changeux model  (MWC) for cooperativity is generally published in an irreversible form. That is, there are no product terms in the rate equation which can be problematic for those wishing to build metabolic models since there are no product inhibition terms. [ 1 ]  However, a series of publications by Popova and Sel'kov [ 2 ]  derived the MWC rate equation for the reversible, multi-substrate, multi-product reaction.


The same problem applies to the classic  Hill equation  which is almost always shown in an irreversible form. Hofmeyr and Cornish-Bowden first published the reversible form of the Hill equation. [ 1 ]  The equation  has since been discussed elsewhere [ 3 ] [ 4 ]  and the model has also been used in a number of kinetic models such as a model of Phosphofructokinase and Glycolytic Oscillations in the Pancreatic β-cells [ 5 ]  or a model of a glucose-xylose co-utilizing S. cerevisiae strain. [ 6 ]  The model has also been discussed in modern enzyme kinetics textbooks. [ 7 ] [ 8 ]


Consider the simpler case where there are two binding sites. See the scheme shown below. Each site is assumed to bind either molecule of substrate S or product P. The catalytic reaction is shown by the two reactions at the base of the scheme triangle, that is S to P and P to S. The model assumes the binding steps are always at equilibrium. The reaction rate is given by:


v
 =

 k

 1

 (

 E
 S
 +
 2
 E

 S

 2

 +
 E
 S
 P

 )

 −

 k

 2

 (

 E
 P
 +
 2
 E

 P

 2

 +
 E
 S
 P

 )

 {\displaystyle v=k_{1}\left(ES+2ES_{2}+ESP\right)-k_{2}\left(EP+2EP_{2}+ESP\right)}


Invoking the  rapid-equilibrium assumption  we can write the various complexes in terms of equilibrium constants to give:


v
 =

 V

 f

 σ
 (
 1
 −
 ρ
 )
 (
 σ
 +
 π
 )

 1
 +
 (
 σ
 +
 π

 )

 2

 {\displaystyle v={\frac {V_{f}\sigma (1-\rho )(\sigma +\pi )}{1+(\sigma +\pi )^{2}}}}


where

 ρ
 =
 Γ

 /

 K

 e
 q

 {\displaystyle \rho =\Gamma /K_{eq}}

 . The

 σ

 {\displaystyle \sigma }

  and

 π

 {\displaystyle \pi }

  terms are the ratio of substrate and product to their respective half-saturation constants, namely

 σ
 =
 S

 /

 S

 0.5

 {\displaystyle \sigma =S/S_{0.5}}

  and

 π
 =
 P

 /

 P

 0.5

 {\displaystyle \pi =P/P_{0.5}}

  and


Using the author's own notation, if an enzyme has

 h

 {\displaystyle h}

  sites that can bind ligand, the form, in the general case, can be shown to be:


v
 =

 V

 f

 σ
 (
 1
 −
 ρ
 )
 (
 σ
 +
 π

 )

 h
 −
 1

 1
 +
 (
 σ
 +
 π

 )

 h

 {\displaystyle v={\frac {V_{f}\sigma (1-\rho )(\sigma +\pi )^{h-1}}{1+(\sigma +\pi )^{h}}}}


The non-cooperative reversible Michaelis-Menten equation can be seen to emerge when we set the Hill coefficient to one.


If the enzyme is irreversible the equation turns into the simple Michaelis-Menten equation that is irreversible. When setting the equilibrium constant to infinity, the equation can be seen to revert to the simpler case where the product inhibits the reverse step.


A comparison has been made between the MWC and reversible Hill equation. [ 9 ]


A modification of the reversible Hill equation was published by Westermark et al [ 10 ]  where modifiers affected the catalytic properties instead.  This variant was shown to provide a much better fit for describing the kinetics of muscle  phosphofructokinase .


Ro05-4082  ( N-methylclonazepam ,  ID-690 ) is a  benzodiazepine  derivative developed in the 1970s. It has  sedative  and  hypnotic  properties, and has around the same potency as  clonazepam  itself. [ 1 ]  It was never introduced into clinical use. It is a  structural isomer  of  meclonazepam  (3-methylclonazepam), and similarly has been sold as a  designer drug , first being identified in Sweden in 2017. [ 2 ]


This  sedative -related article is a  stub . You can help Wikipedia by  expanding it .


Safety pharmacology  is a branch of  pharmacology  specialising in detecting and investigating potential undesirable  pharmacodynamic  effects of  new chemical entities  (NCEs) on physiological functions in relation to exposure in the therapeutic range and above. [ 1 ]


Primary organ systems (so-called core battery systems) are:


Secondary organ systems of interest are:


Safety pharmacology studies are required to be completed prior to human exposure (i.e., Phase I clinical trials), and regulatory guidance is provided in ICH S7A and other documents. [ 2 ] [ 3 ]


The aims of nonclinical safety pharmacology evaluations are three-fold:


The following key issues have to be considered within safety pharmacology: [ 4 ]


The first appearance of the term ‘safety pharmacology’ in the published literature dates back to 1980. [ 5 ]  The term was certainly in common usage in the 1980s within the pharmaceutical industry to describe nonclinical pharmacological evaluation of unintended effects of candidate drugs for regulatory submissions. [ 6 ]  Back then, it was part of a wider ‘general pharmacology’ assessment, which addressed actions of a drug candidate beyond the therapeutically intended effects. The only detailed guidelines indicating the requirements from drug regulatory authorities for general pharmacology studies were from the  Ministry of Health, Labour, and Welfare . Nowadays, the term ‘general pharmacology’ is no longer used, and the ICH S7A guidelines [ 7 ]  distinguish between primary pharmacodynamics (“studies on the mode of action and/or effects of a substance in relation to its desired therapeutic target”), secondary pharmacodynamics (“studies on the mode of action and/or effects of a substance not related to its desired therapeutic target”) and safety pharmacology (“studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above.”).
A major stimulus to the discipline of safety pharmacology was the release in 1996 of a draft ‘Points to Consider’ document on QT prolongation by the European Medicines Agency's Committee for Proprietary Medicinal Products (CPMP), issued in final form the following year. [ 8 ]  This initiative had been prompted by growing concern of sudden death caused by drug-induced torsade de pointes, a potentially lethal cardiac tachyarrhythmia. Later, in 2005,  this concern was addressed by issue of the ICH S7B guidelines. [ 9 ]


Preclinical safety pharmacology integrates  in silico ,  in vitro ,  and  in vivo  approaches. [ 10 ]   In vitro  safety pharmacology studies are focused on early hazard identification and subsequent compound profiling in order to guide preclinical  in vivo   safety and toxicity studies. Early compound profiling can flag for receptor-, enzyme-, transporter-, and ion channel-related liabilities of NCEs (e.g., inhibition of the human ether-a-go-go related gene protein ( hERG )). Classically,  in vivo  investigations comprise the use of young adult conscious animals.


Safety pharmacology studies have to be designed for defining the  dose-response relationship  of the adverse effect observed. Justification should be provided for the selection of the particular animal model or test system. The time course (e.g., onset and duration of response) of the adverse effect is investigated through selected time points for the measurements based on pharmacodynamic and pharmacokinetic considerations. Generally, the doses eliciting the adverse effect have to be compared to the doses eliciting the primary pharmacodynamic effect in the test species or the proposed therapeutic effect in humans.


The primary reference document for safety pharmacology is ICH S7A, followed by many key regulatory documents which either focus on or mention safety pharmacology:


A  subunit vaccine  is a  vaccine  that contains purified parts of the  pathogen  that are  antigenic , or necessary to elicit a protective  immune response . [ 1 ] [ 2 ]  Subunit vaccine can be made from dissembled viral particles in cell culture or  recombinant DNA  expression, [ 3 ]  in which case it is a  recombinant subunit vaccine .


A "subunit" vaccine doesn't contain the whole pathogen, unlike  live attenuated  or  inactivated vaccine , but contains only the antigenic parts such as  proteins ,  polysaccharides [ 1 ] [ 2 ]  or  peptides . [ 4 ]  Because the vaccine doesn't contain "live" components of the pathogen, there is no risk of introducing the disease, and is safer and more stable than vaccines containing whole pathogens. [ 1 ]
Other advantages include being well-established technology and being suitable for  immunocompromised  individuals. [ 2 ]  Disadvantages include being relatively complex to manufacture compared to some vaccines, possibly requiring  adjuvants  and  booster shots , and requiring time to examine which antigenic combinations may work best. [ 2 ]


The first recombinant subunit vaccine was produced in the mid-1980s to protect people from  Hepatitis B . Other recombinant subunit vaccines licensed include  Engerix-B  ( hepatitis B ),  Gardasil 9 [ 5 ]  ( Human Papillomavirus ),  Flublok [ 6 ]  ( influenza ),  Shingrix [ 7 ]  ( Herpes zoster ) and  Nuvaxovid [ 8 ]  ( Coronavirus disease 2019 ).


After  injection , antigens trigger the production of antigen-specific  antibodies , which are responsible for recognising and neutralising foreign substances. Basic components of recombinant subunit vaccines include recombinant subunits, adjuvants and carriers. Additionally, recombinant subunit vaccines are popular candidates for the development of  vaccines  against  infectious diseases  (e.g.  tuberculosis , [ 9 ]   dengue [ 10 ] ).


Recombinant subunit vaccines are considered to be safe for injection. The chances of  adverse effects  vary depending on the specific type of  vaccine  being administered. Minor side effects include injection site pain, fever, and  fatigue , and serious  adverse effects  consist of  anaphylaxis  and potentially fatal  allergic reaction . The  contraindications  are also vaccine-specific; they are generally not recommended for people with the previous history of  anaphylaxis  to any component of the vaccines. Advice from medical professionals should be sought before receiving any vaccination.


The first certified subunit vaccine by clinical trials on humans is the hepatitis B vaccine, containing the surface antigens of the hepatitis B virus itself from infected patients and adjusted by newly developed technology aiming to enhance the vaccine safety and eliminate possible contamination through individuals plasma. [ 11 ]


Subunit vaccines contain fragments of the pathogen, such as protein or polysaccharide, whose combinations are carefully selected to induce a strong and effective immune response. Because the immune system interacts with the pathogen in a limited way, the risk of  side effects  is minimal. [ 2 ]
An effective vaccine would elicit the immune response to the antigens and form  immunological memory  that allows quick recognition of the pathogens and quick response to future infections. [ 1 ]


A drawback is that the specific antigens used in a subunit vaccine may lack  pathogen-associated molecular patterns  which are common to a class of pathogen. These  molecular structures  may be used by  immune cells  for danger recognition, so without them, the immune response may be weaker.  Another drawback is that the antigens do not infect  cells , so the immune response to the subunit vaccines may only be  antibody-mediated , not  cell-mediated , and as a result, is weaker than those elicited by other types of vaccines.
To increase immune response,  adjuvants  may be used with the subunit vaccines, or booster doses may be required. [ 2 ]


A  protein subunit  is a  polypeptide chain  or  protein  molecule that assembles (or " coassembles ") with other protein molecules to form a  protein complex . [ 12 ] [ 13 ] [ 14 ]  Large assemblies of proteins such as  viruses  often use a small number of types of protein subunits as building blocks. [ 15 ]   A key step in creating a recombinant protein vaccine is the identification and isolation of a protein subunit from the pathogen which is likely to trigger a strong and effective immune response, without including the parts of the virus or bacterium that enable the pathogen to reproduce.  Parts of the protein shell or  capsid  of a virus are often suitable.  The goal is for the protein subunit to prime the immune system response by mimicking the appearance but not the action of the pathogen. [ 16 ]  Another protein-based approach involves self‐assembly of multiple protein subunits into a  virus-like particle  (VLP) or nanoparticle. The purpose of increasing the vaccine's surface similarity to a whole virus particle (but not its ability to spread) is to trigger a stronger immune response. [ 17 ] [ 16 ] [ 18 ]


Protein subunit vaccines are generally made through  protein production , manipulating the  gene expression  of an organism so that it  expresses  large amounts of a  recombinant gene . [ 16 ] [ 19 ]  A variety of approaches can be used for development depending on the vaccine involved. [ 17 ]   Yeast ,  baculovirus , or  mammalian  cell cultures can be used to produce large amounts of proteins in vitro. [ 16 ] [ 19 ] [ 20 ]


Protein-based vaccines are being used for  hepatitis B  and for  human papillomavirus  (HPV). [ 17 ] [ 16 ]   The approach is being used to try to develop vaccines for difficult-to-vaccinate-against viruses such as  ebolavirus  and  HIV . [ 21 ]  Protein-based vaccines for COVID-19 tend to target either its spike protein or its receptor binding domain. [ 17 ]  As of 2021, the most researched vaccine platform for COVID-19 worldwide was reported to be recombinant protein subunit vaccines. [ 16 ] [ 22 ]


Vi capsular polysaccharide vaccine  (ViCPS) against  typhoid  caused by the Typhi serotype of  Salmonella enterica . [ 23 ]  Instead of being a protein, the Vi antigen is a  bacterial capsule  polysacchide, made up of a long sugar chain linked to a lipid. [ 24 ]  Capsular vaccines like ViCPS tend to be weak at eliciting immune responses in children. Making a  conjugate vaccine  by linking the polysacchide with a  toxoid  increases the efficacy. [ 25 ]


A  conjugate vaccine  is a type of  vaccine  which combines a weak  antigen  with a strong antigen as a carrier so that the  immune system  has a stronger response to the weak antigen. [ 26 ]


A  peptide-based subunit vaccine  employs a  peptide  instead of a full  protein . [ 27 ]  Peptide-based subunit vaccine mostly used due to many reasons,such as, it is easy and affordable for massive production. Adding to that, its greatest stability, purity and exposed composition. [ 28 ]  Three steps occur leading to creation of peptide subunit vaccine; [ 29 ]


When compared with conventional  attenuated vaccines  and  inactivated vaccines , recombinant subunit vaccines have the following special characteristics:


However, there are also some drawbacks regarding recombinant subunit vaccines:


Vaccination  is a potent way to protect individuals against  infectious diseases . [ 35 ]


Active immunity  can be acquired artificially by  vaccination  as a result of the body's own defense mechanism being triggered by the exposure of a small, controlled amount of  pathogenic  substances to produce its own antibodies and memory cells without being infected by the real pathogen. [ 36 ]


The processes involved in primary immune response are as follows:


Under specific circumstances, low doses of  vaccines  are given initially, followed by additional doses named  booster doses . Boosters can effectively maintain the level of  memory cells  in the human body, hence extending a person's  immunity . [ 33 ] [ 34 ] [ 43 ]


The manufacturing process of recombinant subunit  vaccines  are as follows: [ citation needed ]


Candidate subunits will be selected primarily by their  immunogenicity . [ 44 ]  To be  immunogenic , they should be of foreign nature and of sufficient complexity for the reaction between different components of the  immune system  and the candidates to occur. [ 45 ]  Candidates are also selected based on size, nature of function (e.g.  signalling ) and cellular location (e.g.  transmembrane ). [ 44 ]


Upon identifying the target subunit and its encoding  gene , the  gene  will be isolated and transferred to a second, non-pathogenic organism, and cultured for  mass production . [ 46 ]  The process is also known as  heterologous expression . [ citation needed ]


A suitable  expression system  is selected based on the requirement of  post-translational modifications , costs, ease of product extraction and production efficiency. Commonly used systems for both licensed and developing recombinant subunit  vaccines  include  bacteria ,  yeast ,  mammalian  cells,  insect  cells. [ 47 ]


Bacterial cells  are widely used for  cloning processes ,  genetic modification  and small-scale productions. [ 48 ]   Escherichia coli (E. Coli)  is widely utilised due to its highly explored  genetics , widely available genetic tools for  gene expression , accurate profiling and its ability to grow in inexpensive  media  at high cell densities. [ 49 ]


E. Coli is mostly appropriate for structurally simple proteins owing to its inability to carry out  post-translational modifications , lack of  protein secretary system  and the potential for producing  inclusion bodies  that require additional solubilisation. [ 48 ] [ 49 ] [ 50 ]  Regarding application,  E.Coli  is being utilised as the  expression system  of the  dengue vaccine . [ 10 ]


Yeast  matches  bacterial cells'  cost-effectiveness, efficiency and technical feasibility. [ 48 ]  Moreover,  yeast  secretes  soluble   proteins  and has the ability to perform  post-translational modifications  similar to  mammalian  cells. [ 50 ]


Notably, yeast incorporates more  mannose  molecules during  N-glycosylation  when compared with other  eukaryotes , [ 51 ]  which may trigger  cellular conformational stress responses . Such responses may result in failure in reaching native protein conformation, implying potential reduction of  serum half-life  and  immunogenicity . [ 48 ]  Regarding application, both the  hepatitis B virus surface antigen  ( HBsAg ) and the  virus-like particles  ( VLPs ) of the major capsid protein L1 of  human papillomavirus  type 6, 11, 16, 18 are produced by  Saccharomyces cerevisiae . [ citation needed ]


Mammalian  cells are well known for their ability to perform therapeutically essential  post-translational modifications  and express properly folded,  glycosylated  and functionally active proteins. [ 49 ] [ 52 ] [ 53 ]  However, efficacy of mammalian cells may be limited by  epigenetic   gene silencing  and  aggresome  formation (recombinant protein aggregation). [ 49 ]  For mammalian cells, synthesised proteins were reported to be secreted into chemically defined media, potentially simplifying protein extraction and purification. [ 48 ]


The most prominent example under this class is  Chinese Hamster Ovary (CHO)  cells utilised for the synthesis of recombinant  varicella zoster virus  surface glycoprotein (gE) antigen for  SHINGRIX . [ 7 ]   CHO cells  are recognised for rapid growth and their ability to offer process versatility. They can also be cultured in suspension-adapted culture in protein-free medium, hence reducing risk of  prion -induced contamination. [ 48 ] [ 49 ]


The  baculovirus - insect  cell  expression system  has the ability to express a variety of  recombinant proteins  at high levels and provide significant eukaryotic protein processing capabilities, including  phosphorylation ,  glycosylation ,  myristoylation  and  palmitoylation . [ 54 ]  Similar to  mammalian  cells, proteins expressed are mostly  soluble , accurately folded, and biologically active. [ 55 ]  However, it has slower growth rate and requires higher cost of  growth medium  than  bacteria  and  yeast , and confers  toxicological  risks. [ 48 ]  A notable feature is the existence of elements of control that allow for the expression of secreted and  membrane -bound proteins in Baculovirus-insect cells. [ 48 ] [ 54 ]


Licensed recombinant subunit  vaccines  that utilises  baculovirus - insect  cells include  Cervarix  (papillomavirus C-terminal truncated major capsid protein L1 types 16 and 18) [ 48 ] [ 56 ]  and  Flublok Quadrivalent  ( hemagglutinin  ( HA ) proteins from four strains of  influenza viruses ). [ 6 ]


Throughout history, extraction and  purification  methods have evolved from standard  chromatographic methods  to the utilisation of  affinity tags . [ 57 ]  However, the final extraction and purification process undertaken highly depends on the chosen  expression system . Please refer to subunit expression and synthesis for more insights. [ citation needed ]


Adjuvants  are materials added to improve  immunogenicity  of recombinant subunit  vaccines . [ 58 ]


Adjuvants increase the magnitude of  adaptive response  to the  vaccine  and guide the activation of the most effective forms of  immunity  for each specific  pathogen  (e.g. increasing generation of T cell memory). [ 58 ] [ 59 ] [ 60 ] [ 61 ]  Addition of adjuvants may confer benefits including dose sparing and stabilisation of final vaccine formulation. [ 58 ] [ 61 ]


Appropriate adjuvants are chosen based on safety, tolerance, compatibility of antigen and  manufacturing  considerations. [ 58 ]  Commonly used adjuvants for recombinant subunit  vaccines  are  Alum  adjuvants (e.g.  aluminium hydroxide ),  Emulsions  (e.g.  MF59 ) and  Liposomes  combined with  immunostimulatory  molecules (e.g. AS01 B ). [ 58 ] [ 60 ]


Delivery systems  are primarily divided into polymer-based  delivery systems  ( microspheres  and  liposomes ) and live  delivery systems  ( gram-positive bacteria ,  gram-negative bacteria  and  viruses ) [ citation needed ]


Vaccine antigens  are often encapsulated within  microspheres  or  liposomes . Common microspheres made using  Poly-lactic acid (PLA) [ 62 ]  and  poly-lactic-co-glycolic acid (PLGA) [ 62 ]  allow for controlled  antigen  release by degrading in vivo while  liposomes  including multilamellar or unilamellar vesicles allow for prolonged release. [ 60 ]


Polymer-based  delivery systems  confer advantages such as increased resistance to degradation in  GI tract , controlled  antigen  release, raised particle uptake by  immune cells  and enhanced ability to induce  cytotoxic T cell  responses. [ 60 ]  An example of licensed recombinant vaccine utilising  liposomal  delivery is  Shringrix .


Live  delivery systems , also known as  vectors , are cells modified with  ligands  or  antigens  to improve the  immunogenicity  of recombinant subunits via altering  antigen presentation ,  biodistribution  and trafficking. [ 63 ]  Subunits may either be inserted within the carrier or  genetically engineered  to be expressed on the surface of the  vectors  for efficient presentation to the  mucosal immune system . [ 46 ]


Recombinant subunit  vaccines  are safe for administration. [ 67 ] [ 68 ]  However, mild local reactions, including  induration  and  swelling  of the injection site, along with  fever ,  fatigue  and  headache  may be encountered after vaccination. [ 67 ] [ 69 ] [ 70 ]  Occurrence of severe  hypersensitivity  reactions and  anaphylaxis  is rare, [ 71 ]  but can possibly lead to  deaths  of individuals.  Adverse effects  can vary among populations depending on their  physical health  condition, age,  gender  and  genetic  predisposition. [ 72 ] [ 73 ]


Recombinant subunit  vaccines  are  contraindicated  to people who have experienced  allergic reactions  and  anaphylaxis  to  antigens  or other components of the  vaccines  previously. [ 74 ] [ 75 ]  Furthermore, precautions should be taken when administering  vaccines  to people who are in  diseased  state and during  pregnancy , [ 74 ]  in which their injections should be delayed until their conditions become stable and after childbirth respectively.


ENGERIX-B  (produced by GSK) and  RECOMBIVAX HB  (produced by merck) are two recombinant subunit  vaccines  licensed for the protection against  hepatitis B . Both contain  HBsAg  harvested and purified from  Saccharomyces cerevisiae  and are formulated as a suspension of the  antigen  adjuvanted with  alum . [ 76 ] [ 77 ]


Antibody  concentration ≥10mIU/mL against  HBsAg  are recognized as conferring protection against hepatitis B infection. [ 76 ] [ 77 ]


It has been shown that primary 3-dose  vaccination  of healthy individuals is associated with ≥90% seroprotection rates for  ENGERIX-B , despite decreasing with older age. Lower seroprotection rates are also associated with presence of underlying  chronic diseases  and  immunodeficiency . Yet, GSK HepB still has a clinically acceptable  safety profile  in all studied populations. [ 78 ]


Cervarix ,  GARDASIL  and  GARDASIL9  are three recombinant subunit  vaccines  licensed for the protection against  HPV  infection. They differ in the  strains  which they protect the patients from as  Cervarix  confers protection against type 16 and 18, [ 56 ]   Gardasil  confers protection against type 6, 11, 16 and 18, [ 79 ]  and Gardasil 9 confers protection against type 6, 11, 16, 18, 31, 33, 45, 52, 58 [ 5 ]  respectively.  The  vaccines  contain purified  VLP  of the major capsid L1 protein produced by recombinant  Saccharomyces cerevisiae . [ citation needed ]


It has been shown in a 2014 systematic quantitative review that the bivalent HPV vaccine ( Cervarix ) is associated with  pain  (OR 3.29; 95% CI: 3.00–3.60),  swelling  (OR 3.14; 95% CI: 2.79–3.53) and  redness  (OR 2.41; 95% CI: 2.17–2.68) being the most frequently reported adverse effects. For Gardasil, the most frequently reported events were  pain  (OR 2.88; 95% CI: 2.42–3.43) and  swelling  (OR 2.65; 95% CI: 2.0–3.44). [ 80 ]


Gardasil was discontinued in the U.S. on May 8, 2017, after the introduction of Gardasil 9 [ 81 ]  and Cervarix was also voluntarily withdrawn in the U.S. on August 8, 2016. [ 82 ]


Flublok Quadrivalent  is a licensed recombinant subunit  vaccine  for active  immunisation  against  influenza . It contains  HA  proteins of four  strains  of  influenza  virus purified and extracted using the  Baculovirus - insect   expression system . The four viral strains are standardised annually according to  United States Public Health Services (USPHS)  requirements. [ 6 ]


Flublok Quadrivalent  has a comparable safety profile to traditional trivalent and quadrivalent vaccine equivalents. Flublok is also associated with less local reactions (RR = 0.94, 95% CI 0.90–0.98, three RCTs, FEM, I2 = 0%, low‐ certainty evidence) and higher risk of  chills  (RR = 1.33, 95% CI 1.03–1.72, three RCTs, FEM, I2 = 14%, low‐certainty evidence). [ 83 ]


SHINGRIX  is a licensed recombinant subunit  vaccine  for protection against  Herpes Zoster , whose risk of developing increases with decline of  varicella zoster virus (VZV)  specific  immunity . The vaccine contains  VZV  gE antigen component extracted from  CHO cells , which is to be reconstituted with  adjuvant  suspension AS01 B . [ 7 ]


Systematic reviews  and  meta-analyses  have been conducted on the efficacy, effectiveness and safety of  SHINGRIX  in  immunocompromised  18–49 year old patients and healthy adults aged 50 and over. These studies reported  humoral  and  cell-mediated immunity  rate ranged between 65.4 and 96.2% and 50.0–93.0% while efficacy in patients (18–49 yo) with  haematological malignancies  was estimated at 87.2% (95%CI, 44.3–98.6%) up to 13 months post-vaccination with an acceptable  safety profile . [ 84 ] [ 85 ]


NUVAXOVID  is a recombinant subunit vaccine licensed for the prevention of  SARS-CoV-2 infection . Market authorization was issued on 20 December 2021. [ 86 ]  The vaccine contains the  SARS-CoV-2 spike  protein produced using the  baculovirus   expression system , which is eventually adjuvanted with the  Matrix M  adjuvant. [ 8 ]


While the practice of  immunisation  can be traced back to the  12th century , in which ancient  Chinese  at that time employed the technique of  variolation  to confer  immunity  to  smallpox  infection, [ citation needed ]  the modern era of vaccination has a short history of around 200 years. It began with the  invention  of a vaccine by Edward Jenner in 1798 to eradicate  smallpox  by injecting relatively weaker  cowpox  virus into the human body. [ citation needed ]


The middle of the 20th century marked the golden age of vaccine science. [ citation needed ]  Rapid technological advancements during this period of time enabled scientists to cultivate  cell culture  under controlled environments in laboratories, [ 87 ]  subsequently giving rise to the production of vaccines against  poliomyelitis ,  measles  and various  communicable diseases . [ citation needed ]  Conjugated vaccines were also developed using  immunologic  markers including capsular  polysaccharide  and  proteins . [ 87 ]  Creation of products targeting common illnesses successfully lowered infection-related  mortality  and reduced  public healthcare  burden.


Emergence of  genetic engineering techniques  revolutionised the creation of vaccines. By the end of the 20th century, researchers had the ability to create  recombinant vaccines  apart from traditional  whole-cell vaccine , for instance  Hepatitis B vaccine , which uses the viral  antigens  to initiate  immune responses . [ 87 ]


As the  manufacturing  methods continue to evolve, vaccines with more complex constitutions will inevitably be generated in the future to extend their therapeutic applications to both infectious and  non-infectious diseases , [ citation needed ]  in order to safeguard the health of more people.


Recombinant subunit  vaccines  are used in development for  tuberculosis , [ 9 ]   dengue fever , [ 10 ]   soil-transmitted helminths , [ 88 ]   feline leukaemia [ 89 ]  and  COVID-19 . [ 90 ]


Subunit vaccines are not only considered effective for SARS-COV-2, but also as candidates for evolving immunizations against malaria, tetanus, salmonella enterica, and other diseases. [ 11 ]


Research has been conducted to explore the possibility of developing a heterologous  SARS-CoV  receptor-binding domain (RBD) recombinant protein as a human  vaccine  against  COVID-19 . The theory is supported by evidence that  convalescent   serum  from  SARS-CoV  patients have the ability to neutralise  SARS-CoV-2  (corresponding virus for  COVID-19 ) and that amino acid similarity between  SARS-CoV  and  SARS-CoV-2 spike  and RBD protein is high (82%). [ 90 ]


Toxicity  is the degree to which a  chemical substance  or a particular  mixture  of substances can damage an  organism . [ 1 ]  Toxicity can refer to the effect on a whole organism, such as an  animal ,  bacterium , or  plant , as well as the effect on a substructure of the organism, such as a  cell  ( cytotoxicity ) or an organ such as the  liver  ( hepatotoxicity ). Sometimes the word is more or less synonymous with  poisoning  in everyday usage.


A central concept of  toxicology  is that the effects of a  toxicant  are  dose -dependent; even  water  can lead to  water intoxication  when taken in too high a dose, whereas for even a very toxic substance such as  snake venom  there is a dose below which there is no detectable toxic effect. Toxicity is species-specific, making cross-species analysis problematic. Newer  paradigms  and metrics are evolving to bypass  animal testing , while maintaining the concept of toxicity endpoints. [ 2 ]


In  Ancient Greek  medical literature, the adjective  τοξικόν  (meaning "toxic") was used to describe substances which had the ability of "causing death or serious debilitation or exhibiting symptoms of infection." [ 3 ]  The word draws its origins from the Greek noun τόξον  toxon  (meaning "arc"), in reference to the use of bows and  poisoned arrows  as weapons. [ 3 ]


English-speaking American culture has adopted several figurative usages for  toxicity , often when describing harmful inter-personal relationships or character traits (e.g. " toxic masculinity "). [ 4 ]


Humans have a deeply rooted history of not only being aware of toxicity, but also taking advantage of it as a tool. Archaeologists studying bone arrows from caves of Southern Africa have noted the likelihood that some aging 72,000 to 80,000 years old were dipped in specially prepared poisons to increase their lethality. [ 5 ]  Although scientific instrumentation limitations make it difficult to prove concretely, archaeologists hypothesize the practice of making poison arrows was widespread in cultures as early as the paleolithic era. [ 6 ] [ 7 ]  The San people of Southern Africa have managed to preserved this practice into the modern era, with the knowledge base to form complex mixtures from poisonous beetles and plant derived extracts, yielding an arrow-tip product with a shelf life beyond several months to a year. [ 8 ]


There are generally five types of toxicities: chemical, biological, physical, radioactive and behavioural.


Disease-causing microorganisms and parasites are toxic in a broad sense but are generally called  pathogens  rather than toxicants. The biological toxicity of pathogens can be difficult to measure because the  threshold dose  may be a single organism. Theoretically one  virus , bacterium or  worm  can reproduce to cause a serious  infection . If a host has an intact  immune system , the inherent toxicity of the organism is balanced by the host's response; the effective toxicity is then a combination. In some cases, e.g.  cholera toxin , the disease is chiefly caused by a nonliving substance secreted by the organism, rather than the organism itself. Such nonliving biological toxicants are generally called  toxins  if produced by a microorganism, plant, or fungus, and  venoms  if produced by an animal.


Physical toxicants are substances that, due to their physical nature, interfere with biological processes. Examples include  coal  dust,  asbestos  fibres or finely divided  silicon dioxide , all of which can ultimately be fatal if inhaled. Corrosive chemicals possess physical toxicity because they destroy tissues, but are not directly poisonous unless they interfere directly with biological activity. Water can act as a physical toxicant if taken in extremely high doses because the concentration of vital ions decreases dramatically with too much water in the body. Asphyxiant gases can be considered physical toxicants because they act by displacing oxygen in the environment but they are inert, not chemically toxic gases.


Radiation can have a toxic effect on organisms. [ 9 ]


Behavioral toxicity refers to the undesirable effects of essentially therapeutic levels of medication clinically indicated for a given disorder (DiMascio, Soltys and Shader, 1970). These undesirable effects include anticholinergic effects, alpha-adrenergic blockade, and dopaminergic effects, among others. [ 10 ]


Toxicity can be measured by its effects on the target (organism, organ, tissue or cell).  Because individuals typically have different levels of response to the same dose of a toxic substance, a population-level measure of toxicity is often used which relates the probabilities of an outcome for a given individual in a population.  One such measure is the  LD 50 . When such data does not exist, estimates are made by comparison to known similar toxic things, or to similar exposures in similar organisms. Then, " safety factors " are added to account for uncertainties in data and evaluation processes. For example, if a dose of a toxic substance is safe for a laboratory rat, one might assume that one-tenth that dose would be safe for a human, allowing a safety factor of 10 to allow for interspecies differences between two mammals; if the data are from fish, one might use a factor of 100 to account for the greater difference between two chordate classes (fish and mammals). Similarly, an extra protection factor may be used for individuals believed to be more susceptible to toxic effects such as in pregnancy or with certain diseases. Or, a newly synthesized and previously unstudied chemical that is believed to be very similar in effect to another compound could be assigned an additional protection factor of 10 to account for possible differences in effects that are probably much smaller. This approach is very approximate, but such protection factors are deliberately very conservative, and the method has been found to be useful in a wide variety of applications.


Assessing all aspects of the toxicity of cancer-causing agents involves additional issues, since it is not certain if there is a minimal effective dose for  carcinogens , or whether the risk is just too small to see. In addition, it is possible that a single cell transformed into a cancer cell is all it takes to develop the full effect (the "one hit" theory).


It is more difficult to determine the toxicity of chemical mixtures than a pure chemical because each component displays its own toxicity, and components may interact to produce enhanced or diminished effects. Common mixtures include  gasoline ,  cigarette smoke , and  industrial waste .  Even more complex are situations with more than one type of toxic entity, such as the discharge from a malfunctioning sewage treatment plant, with both chemical and biological agents.


The preclinical toxicity testing on various biological systems reveals the species-, organ- and dose-specific toxic effects of an investigational product. The toxicity of substances can be observed by (a) studying the accidental exposures to a substance (b) in vitro studies using cells/ cell lines (c) in vivo exposure on experimental animals. Toxicity tests are mostly used to examine specific adverse events or specific endpoints such as cancer, cardiotoxicity, and skin/eye irritation. Toxicity testing also helps calculate the No Observed Adverse Effect Level (NOAEL) dose and is helpful for clinical studies. [ 11 ]


For substances to be regulated and handled appropriately they must be properly classified and labelled. Classification is determined by approved testing measures or calculations and has determined cut-off levels set by governments and scientists (for example,  no-observed-adverse-effect levels ,  threshold limit values , and  tolerable daily intake  levels).  Pesticides  provide the example of well-established  toxicity class  systems and  toxicity labels . While currently many countries have different regulations regarding the types of tests, numbers of tests and cut-off levels, the implementation of the  Globally Harmonized System [ 12 ] [ 13 ]  has begun unifying these countries.


Global classification looks at three areas: Physical Hazards (explosions and pyrotechnics), [ citation needed ]  Health Hazards [ citation needed ]  and  environmental hazards . [ citation needed ]


The types of toxicities where substances may cause lethality to the entire body, lethality to specific organs, major/minor damage, or cause cancer. These are globally accepted definitions of what toxicity is. [ citation needed ]  Anything falling outside of the definition cannot be classified as that type of toxicant. [ citation needed ]


Acute toxicity looks at lethal effects following oral, dermal or inhalation exposure. It is split into five categories of severity where Category 1 requires the least amount of exposure to be lethal and Category 5 requires the most exposure to be lethal. The table below shows the upper limits for each category.


Note: The undefined values are expected to be roughly equivalent to the category 5 values for oral and dermal administration. [ citation needed ]


Skin corrosion and irritation are determined through a skin patch test analysis, similar to an  allergic inflammation patch test . This examines the severity of the damage done; when it is incurred and how long it remains; whether it is reversible and how many test subjects were affected.


Skin corrosion  from a substance must penetrate through the epidermis into the dermis within four hours of application and must not reverse the damage within 14 days.  Skin irritation  shows damage less severe than corrosion if: the damage occurs within 72 hours of application; or for three consecutive days after application within a 14-day period; or causes  inflammation  which lasts for 14 days in two test subjects.  Mild skin irritation  is minor damage (less severe than irritation) within 72 hours of application or for three consecutive days after application.


Serious  eye  damage  involves tissue damage or degradation of vision which does not fully reverse in 21 days. Eye irritation involves changes to the eye which do fully reverse within 21 days.


An Environmental hazard can be defined as any condition, process, or state adversely affecting the environment. These hazards can be physical or chemical, and present in air, water, and/or soil. These conditions can cause extensive harm to humans and other organisms within an ecosystem.


The EPA maintains a list of priority pollutants for testing and regulation. [ 15 ]


Workers in various occupations may be at a greater level of risk for several types of toxicity, including neurotoxicity. [ 16 ]   The expression "Mad as a hatter" and the "Mad Hatter" of the book  Alice in Wonderland  derive from the known occupational toxicity of  hatters  who used a toxic chemical for controlling the shape of hats. Exposure to chemicals in the workplace environment may be required for evaluation by industrial hygiene professionals. [ 17 ]


Hazards in the arts have been an issue for artists for centuries, even though the toxicity of their tools, methods, and materials was not always adequately realized. Lead and cadmium, among other toxic elements, were often incorporated into the names of artist's oil paints and pigments, for example, "lead white" and "cadmium red".


20th-century printmakers and other artists began to be aware of the toxic substances, toxic techniques, and toxic fumes in glues, painting mediums, pigments, and solvents, many of which in their labelling gave no indication of their toxicity. An example was the use of  xylol  for cleaning  silk screens . Painters began to notice the dangers of breathing painting mediums and thinners such as  turpentine . Aware of toxicants in studios and workshops, in 1998 printmaker  Keith Howard  published  Non-Toxic Intaglio Printmaking  which detailed twelve innovative  Intaglio -type printmaking techniques including  photo etching ,  digital imaging ,  acrylic -resist hand-etching methods, and introducing a new method of  non-toxic   lithography . [ 18 ]


There are many environmental health mapping tools.  TOXMAP  is a Geographic Information System (GIS) from the Division of Specialized Information Services [ 19 ]  of the  United States National Library of Medicine  (NLM) that uses maps of the United States to help users visually explore data from the  United States Environmental Protection Agency 's (EPA)  Toxics Release Inventory  and  Superfund  programs. TOXMAP is a resource funded by the US Federal Government. TOXMAP's chemical and environmental health information is taken from NLM's Toxicology Data Network
(TOXNET) [ 20 ]  and  PubMed , and from other authoritative sources.


Aquatic toxicity  testing subjects key indicator species of  fish  or  crustacea  to certain concentrations of a substance in their environment to determine the lethality level. Fish are exposed for 96 hours while crustacea are exposed for 48 hours. While GHS does not define toxicity past 100 mg/L, the EPA currently lists aquatic toxicity as "practically non-toxic" in concentrations greater than 100 ppm. [ 21 ]


Note: A category 4 is established for chronic exposure, but simply contains any toxic substance which is mostly insoluble, or has no data for acute toxicity.


Toxicity of a substance can be affected by many different factors, such as the pathway of administration (whether the toxicant is applied to the skin, ingested, inhaled, injected), the time of exposure (a brief encounter or long term), the number of exposures (a single dose or multiple doses over time), the physical form of the toxicant (solid, liquid, gas), the concentration of the substance, and in the case of gases, the partial pressure (at high ambient pressure, partial pressure will increase for a given concentration as a gas fraction), the genetic makeup of an individual, an individual's overall health, and many others.  Several of the terms used to describe these factors have been included here.


Considering the limitations of the  dose-response  concept, a novel Abstract Drug Toxicity Index (DTI) has been proposed recently. [ 22 ]   DTI redefines drug toxicity, identifies hepatotoxic drugs, gives mechanistic insights, predicts clinical outcomes and has potential as a screening tool.


Venom in medicine  is the medicinal use of  venoms  for  therapeutic  benefit in treating  diseases .


Venom is any  poisonous  compound secreted by an animal intended to harm or disable another. When an organism produces a venom, its final form may contain hundreds of different  bioactive elements  that interact with each other inevitably producing its  toxic  effects. [ 1 ]  This mixture of ingredients includes various  proteins ,  peptides , and non-peptidic  small molecules . [ 2 ]  The active components of these venoms are isolated, purified, and screened in  assays .  These may be either  phenotypic assays  to identify component that may have desirable therapeutic properties ( forward pharmacology ) or target directed assays to identify their  biological target  and  mechanism of action  ( reverse pharmacology ). [ 3 ]


Venoms are  naturally occurring  substances that organisms evolved to deploy against other organisms, in defense or attack. [ 2 ]   They are often mixtures of   proteins  that act together or singly to attack their specific  targets  within the organism against which they are used, generally with high  specificity  and generally easily accessible through the vascular system. [ 2 ]   This has made venoms a subject of study for people who work in  drug discovery . [ 2 ]   With developments in  omic  technologies ( proteomics ,  genomics , etc.), researchers in this field became able to identify genes that produce certain elements in an animal's venom, as well as  protein domains  that have been used as building blocks across many species. [ 2 ]  In conjunction with methods of separation and purification of compounds, scientists are able to study each individual compound that exists within a venom "concoction", looking for compounds to serve as drug leads or other use. [ 2 ]  Each venomous organism produces thousands of different proteins giving access to millions of different molecules that still have potential uses. In addition, nature is continuously evolving; as prey develop resistance to these venoms, the predators also evolve as well, creating novel toxins that can continue to act upon its respective prey. [ 4 ]


The earliest known use of venom in medicine dates back to 380 B.C. in ancient Greece. [ 5 ]   Aristotle 's "Historia Animalium", describes how venom can be used in the production of antidotes for the venom. [ 5 ]  During the height of the Roman empire, there is evidence of venom being added into medicine used to treat smallpox, leprosy, fever, and wounds. [ 5 ]  Despite this, early uses of venom were primarily involved in the process of making antidotes. This use of venom continued into the Middle Ages and well into the 19th century. The first modern study of venom in a medical light occurred in the late 19th century. A scientist, Albert Calmatte, injected animals with small amounts of venom, using their blood as the antidote. [ 5 ]


Captopril  emulates the function of the toxin found in Brazilian pit viper ( Bothrops jararaca ) venom and is generally accepted as the first venom "success" story.  Captopril is an  ACE inhibitor  (angiotensin-converting enzyme) that was approved by the FDA approved in April 1981. It lowers blood pressure by inhibiting the production of  angiotensin II  which acts in a pathway that leads to  vasoconstriction  which raises blood pressure. After the creation of this drug, many analogues ( enalapril ,  lisinopril ,  perindopril ,  ramipril , etc.) were produced. [ 6 ]


Ziconotide  is a synthetically made version of the ω- conotoxin  made by the  cone snail , [ 7 ]  that is used to treat severe pain and is delivered as an infusion into the  cerebrospinal fluid  using an  intrathecal   pump system . [ 8 ]   Ziconitide acts presynaptically on  N-type calcium channels , blocking the receptors of this channel with high selectivity and affinity, [ 7 ]


Eptifibatide  was modeled after a component in  southeastern pygmy rattlesnake  venom and is used in anticoagulation therapies in an effort to reduce the risk of  heart attacks ; it is used in only severe cases because of the possible side effect of  thrombocytopenia , a condition where platelets are unable to aggregate at all. [ 9 ] [ 10 ] [ 11 ]  Eptifibatide binds reversibly to platelets reducing the risk of  thrombosis . It is an antagonist of  glycoprotein IIb/IIIa . [ 9 ]


Exenatide  is a 39- amino-acid   peptide  that is a synthetic version of exendin-4, a  hormone  found in the saliva of the  Gila monster . [ 12 ]  It is used to treat  Type II Diabetes  as an adjunct to insulin and other drugs. [ 13 ] [ 14 ]  It is  GLP-1 receptor agonist  that was first isolated by John Eng in 1992 while working at the Veterans Administration Medical Center in the Bronx, New York. [ 12 ]


Batroxobin , is a  serine protease  found in  snake venom  produced by  Bothrops atrox  and  Bothrops moojeni , venomous species of  pit viper  found east of the Andes in South America. It cleaves  fibrinogen , similarly to  thrombin .  Batroxobin from  B atrox  is used as a drug called "Reptilase" that is used to stop bleeding,  while batroxobin from  B moojeni  is a drug called "Defibrase", used to break up blood clots.  It is also used in a system called "Vivostat", where a person's blood is taken just before surgery and exposed to batroxobin; the resulting clots are then harvested, and then dissolved, forming a  fibrin glue  that is then used on the person during the surgery. [ 15 ]


Advil  is primarily a brand of  ibuprofen  (a  pain reliever  in the  nonsteroidal anti-inflammatory drug  category). [ 1 ]  Advil has been called a "megabrand" because it offers various "products for a wide range of pain, head cold, and sleep problems." [ 2 ]


The brand first entered the American market in 1984 through  Whitehall [ 3 ]  (itself a division of  Wyeth , which was purchased by  Pfizer  in 2009), [ 4 ]  the same year ibuprofen gained  Food and Drug Administration  (FDA) approval for  over-the-counter  (OTC) sales in the United States (being available via prescription since 1974). [ 5 ]  Within ten years of having a market presence, it outsold  Bayer Aspirin  and was a fierce competitor to  Tylenol  (primarily a brand of  acetaminophen ). [ 6 ]  In the mid-1990s, for example, it held 13% of the multibillion-dollar over-the-counter American market for  analgesics . [ 7 ]


In 2023, there were 23 varieties of Advil available on the U.S. market including: [ 8 ]


Marketing campaigns for the brand (some including celebrities like  Regis Philbin ) have pushed slogans such as "Take Action. Take Advil." and have been presented under the premise of "True Advil Stories"; [ 9 ]  the brand has also been involved in sponsorship deals such as with  Major League Pickleball . [ 10 ]


Amrutanjan  is an  analgesic   balm  manufactured and distributed by  Amrutanjan Healthcare . [ 1 ]


Amrutanjan was founded in 1893 by journalist and freedom fighter,  Kasinathuni Nageswara Rao . [ 2 ]  He popularized the balm by distributing it free-of-cost at music concerts. Even today, the words "Bombay" are inscribed along with Amrutanjan on the lid of its most well-known product, the pain balm. In 1936, Amrutanjan became a  public limited company  with the name Amrutanjan Limited. [ 3 ]


It is used as a balm for:


The brand is owned by the parent  Amrutanjan Healthcare . It is now headed by Sambhu Prasad, the grandson of Nageshwara Rao. [ 4 ]


Anacin  is an American brand of  analgesic  that is manufactured by  Prestige Consumer Healthcare . Anacin's active ingredients are  aspirin  and  caffeine . [ 1 ]


Anacin was invented by William Milton Knight and was first to be used  c.  1916  as stated in the patent. [ 2 ]  Trademarked in 1918, Anacin is one of the oldest brands of pain relievers in the United States. It originally contained  acetophenetidin  (phenacetin) and was promoted as "aspirin-free relief," but was reformulated in the 1980s following the FDA's ruling to withdraw phenacetin from the market in 1983 due to concerns over its carcinogenic properties. [ 3 ]


It was originally sold by the Anacin Co. ("Pharmaceutical Chemists") in  Chicago, Illinois .  American Home Products , now known as Wyeth, purchased the manufacturing rights in 1930. [ 4 ]  Anacin was reportedly their most popular product. [ 5 ]  Insight Pharmaceuticals acquired the brand in 2003. [ citation needed ]  In 2014,  Prestige Consumer Healthcare  signed an agreement with Insight to acquire the company; it was Prestige's largest acquisition to that point. [ 6 ]


In 1939, Anacin sponsored a daytime serial called  Our Gal Sunday . Their sponsorship spanned 18 of the program's 23 years on the air. [ 5 ]  Early Anacin radio commercials appeared in radio shows and dramas of the 1940s and '50s. These "formulaic" commercials usually claimed that Anacin was being actively prescribed by doctors and dentists at the time, treated " headaches ,  neuritis  and  neuralgia ", and that it contained "a combination of medically proven ingredients, like a doctor's prescription", without specifying those ingredients. Sometimes the announcer would mention that there were four active ingredients in Anacin, one of which was the medicine the consumer was already taking. It also claimed to help with depression. The announcer then reminded the listener that Anacin was available "at any drug counter", and "comes in handy (tin) boxes of 12 and 30, and economical family-size bottles of 50 and 100", usually spelling out its name at the end of the commercial. [ 7 ]


Anacin sponsored the first made-for-television  sitcom ,  Mary Kay and Johnny . Unsure of how many viewers would be watching when they sponsored the show in 1947, Anacin ran a simple test, offering a free mirror to the first 200 viewers to write for one. The offer drew over 9,000 responses, overwhelming the sponsor but proving television was a viable advertising medium. [ 8 ]


Anacin was also a leading sponsor of the television soaps  Love of Life ,  The Secret Storm  and the early years of  The Young and the Restless .


Anacin is one of the earliest and best examples of a concerted television marketing campaign, created for them in the late 1950s by  Rosser Reeves  of the  Ted Bates  ad agency.  Many people remember the commercials advertising "tension producing" situations, and the "hammers in the head" advertisement with the slogan "Tension. Pressure. Pain."


An Anacin advertisement in 1962 featured a mother trying to assist her grown daughter with various chores, such as preparing a meal. "Don't you think it needs a little salt?", the mother would say, only to have her nerve-racked daughter shout, "Mother, please, I'd rather do it myself!" As the mother wilted, the daughter would emote and rub her head, with her inner voice saying, "Control yourself! Sure, you've got a headache, you're tense, irritable, but don't take it out on her!"  Another commercial had a wife greeting her husband as he pulled into their driveway in his car; the husband responded by yelling "Helen, can't you keep Billy's bike  out of the driveway?!? " These advertisement scenarios became popular and were parodied a number of times, including in the  Allan Sherman  song "Headaches", the 1966 film  The Silencers  and the 1980 film  Airplane .


Anacin had a large billboard behind the center field fence of  Yankee Stadium  from the 1950s through 1973, until the stadium's 1974-75 renovation.


Anacin covers a family of pain relievers. There are currently two different formulations:


Anacin's side effects may include dizziness,  heartburn , irritability,  nausea , nervousness, rashes, hives, bloody stools, drowsiness, hearing loss, ringing in the ears, and trouble sleeping. [ 9 ]