This method was developed on the premise that essential oil vapours exert critical biological activity. These methods offer rapid screening protocols for the antimicrobial assessment of plant essential oils. It has been suggested that essential oils in the vapour phase possess the greatest degree of antimicrobial activity since the active constituents are highly volatile in nature and thus, the vapour is therefore the contributing attribute for its biological activity. [ 8 ]  Each individual constituent has differing volatility, therefore when the mixtures are introduced into a free, non-saturated state in a closed micro-environment; the volatile constituents begin to disperse at differing rates in the vapour phase within the headspace according to their degree of volatility until they reach equilibrium. [ 11 ]


Preliminary research involving the essential oils of  citronella  yielded promising results when tested against a selection of Gram-positive bacteria and Gram-negative bacteria. The use of such extracts can be explored especially in the development of cost-effective treatments of  respiratory illness , specifically of those caused by bacterial or fungal infection. Preliminary tests exhibit complete inhibition of the growth of certain bacterial strains. [ 1 ]


In a separate research, the micro-atmosphere method was used to investigate the anti-fungal efficacy of the essential oils from the  cinnamon , a plant belonging to the genus  Cinnamomum .


The procedure was employed to ensure that the active films, the material containing the active compound, does not come into direct contact to the tested fungal suspension. [ 12 ]


The micro-atmosphere method was performed, in order to evaluate the indirect effects of active films against  P. digitatum . Addition of 0.5% cinnamon essential oil, led to 12% inhibition of fungal growth. Higher anti-fungal effects were obtained by adding higher amounts of the essential oil. An inhibition of fungal growth between 28% and 50% were observed for the films incorporated with 1.5% and 3% essential oil, respectively.


In this particular research, the potential anti-fungal agent was subjected to both  disk diffusion test  and the micro-atmosphere method for comparison and to get an idea of how the active compound may be utilized. The active compound exhibited higher anti-fungal effects in the disc diffusion test compared to the micro-atmosphere assays. This could be attributed to the fact that in the disc diffusion test, both direct contact and migration of active compounds from the film to the outside induced the observed antimicrobial effects. On the other hand, only the migration of the volatile compounds to the headspace, may cause the anti-fungal effect in the micro-atmosphere method. [ 13 ]


Neosaxitoxin  (NSTX) is included, as other  saxitoxin -analogs, in a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs). The parent compound of PSTs, saxitoxin (STX), is a tricyclic perhydropurine alkaloid, which can be substituted at various positions, leading to more than 30 naturally occurring STX analogues. All of them are related imidazoline guanidinium derivatives. [ 3 ]


NSTX, and other PSTs, are produced by several species of marine dinoflagellates (eukaryotes) and freshwater cyanobacteria, blue-green algae (prokaryotes), which can form extensive blooms around the world. [ 4 ]  Under special conditions, during  harmful algal blooms  (HAB) or  red tide , all these toxins may build up in filter-feeding shellfish, such as mussels, clams and oysters, and can produce an outbreak of  Paralytic Shellfish Poisoning  (PSP). [ 5 ]


Saxitoxin analogues associated to PSP can be divided into three categories: [ 6 ]


NSTX is quite similar to saxitoxin, like all the neurotoxins associated to PSP, the only difference is that NSTX shows one hydroxyl group bonded to nitrogen "1", where saxitoxyn contains one hydrogen. [ 7 ]


This purine is highly hydrophilic [ 8 ]  and thermostable, it is not destroyed by cooking. [ 9 ]  Moreover, is very stable in usual storage, specially in acidic condition. [ 10 ]


NSTX blocks the extracellular portion, [ 11 ]  the outer vestibule, [ 12 ]  of some voltage gated sodium channels in a very powerful and reversible manner, without affection of other ion channels.


"Voltage-gated", also called "voltage-sensitive" and "voltage-dependent"  sodium channel  also known as "VGSCs" or "Nav channel" are crucial elements of normal physiology in a variety of animals, including flies, leeches, squid and jellyfish, as well as mammalian and non-mammalian vertebrates. This large integral membrane protein plays an essential role in the initiation and propagation of action potentials in neurons, myocytes and other excitable cells. [ 13 ]


Nav channels form the basis for  electrical excitability  in animals. Nav channels evolved from Ca 2+  channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na +  and Ca 2+ . Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. [ 14 ]


Sodium-channel proteins in the mammalian brain are composed of an association that include one alpha subunit and one or more auxiliary beta subunits. Nine types of alpha subunits have been described (Na v 1.1 to Na v 1.9), and a tenth related isoform (Na x ) may also play some role as a Nav channel. Based in this information, ten Nav classes can be described: Na v 1.1 to Na v 1.9, and Na x . [ 15 ]


Former five, [ 16 ]  but more recently, six [ 17 ]  neurotoxin receptor sites have been recognized between the seven receptor site [ 18 ]  located in the vertebrate sodium channel receptor alpha subunit:


NSTX and other site 1 blockers have high affinity (very low dissociation constant) and high specificity for Nav channels. The action of NSTX produces minimal effect on cardiac Nav, where it exhibits about 20–60 fold lesser affinity than in Nav channels from rat skeletal muscle and rat brain. [ 19 ]  Most data emphasize the role of "STX resistant" Na v  channel 1.5 in human heart. [ 20 ] [ 21 ]


Toxins such as neosaxitoxin and tetrodotoxin have less affinity for most cardiac Nav channels than for most Nav channels in nerve tissue. Moreover, NSTX is so active on nerve Nav channel than is roughly a million-fold more potent than lidocaine. [ 22 ]


This mechanism of action can produce two well known kinds of effects in humans:


It can be approximately described using one of the classical model of neurotoxic disease, known from ancient times as red tide, the most harmful algal bloom (HAB). This well known clinical model is the "paralytic shellfish poisoning". [ 23 ]


Of course, there are great differences between different algal blooms, [ 24 ] [ 25 ] [ 26 ] [ 27 ]  because of the mix of species included in each HAB, usually related to environmental conditions; [ 28 ]  because of the levels and quality of PSTs produced in each HAB, that may be modulated by concurrent microorganism; [ 29 ] [ 30 ] [ 31 ] [ 32 ]  and, last but not least, because of the specific properties of each kind of PST, for example:


In spite of its heterogeneous and poorly understood epidemiology, the clinical picture of PSP could be useful to anticipate clinical effects of systemic NSTX.


Usually, the victims of mild and severe acute intoxications eliminate the toxin in urine during the first 24 hours after ingestion, and improve to full recovery in the first day of intrahospital care (when vital support is provided in a timely manner). [ 40 ]


When outbreaks of PSP occur in remote locations, where medical assistance is limited, reported lethality is under 10% in adults, but can reach 50% in children younger than six years old. This difference could be secondary to dissimilar doses and composition of involved mixes of PSTs; delay in medical support; or some kind of susceptibility of children. [ 41 ]  More recent information suggest that lethality could be around 1% of symptomatic patients, [ 42 ]  including cases where air transportation was required from remote locations of Alaska. [ 43 ]


Electrophysiologic observations demonstrated sub clinical abnormalities lasting for some days [ 44 ]  or weeks [ 45 ]  after clinical recovery .


Some evidence suggest the presence of metabolic pathways for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. [ 46 ]


Forensic analysis of fatalities after severe cases, conclude that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound. [ 47 ]


Considering the heterogeneous nature of toxins mixes contained in contaminated bivalve molluscs, the safe limit of toxin content in shellfish adequate for human ingestion is expressed in "saxitoxin equivalents". According to the Food and Agriculture Organization of the United Nations (FAO) and European Parliament, this limit is 80 microgram of saxitoxin equivalent per 100 gram of mussel meat (each mussel weights around 23 g). [ 48 ] [ 49 ]  The U.S. Food and Drug Administration extends the same definition to "fish" quality, but the term "fish" refers to fresh or saltwater fin fish, crustaceans, other forms of aquatic animal life other than birds or mammals, and all mollusks; and incorporate the use of "ppm" as another measure for saxitoxin equivalent concentration in mentioned foods. [ 50 ]


Paradoxically, the chronic and/or repeated exposure to marine seafood toxins, which is a much more realistic phenomenon, has not been fully examined. [ 51 ] [ 52 ]  One study in rats exposed to chronic (12 weeks) NSTX administration demonstrated some reduction in water and food intake, and a mild degree of transient cholestasis, probably associated to fasting, without other abnormalities. [ 53 ]


This action has been demonstrated in animals [ 54 ]  and humans. [ 55 ] [ 56 ] [ 57 ] [ 58 ] [ 59 ]


The medical use of the NSTX anesthetic effect is supported by three reasons:


In conclusion, NSTX is a well defined molecule with a long-lasting and sometimes dangerous relationship with human subjects. Recent investigations suggest a clinical application as a new local anesthetic that sounds "too good to be true", but more investigation is required. [ 86 ]


Norethisterone acetate  ( NETA ), also known as  norethindrone acetate  and sold under the brand name  Primolut-Nor  among others, is a  progestin  medication which is used in  birth control pills ,  menopausal hormone therapy , and for the treatment of  gynecological disorders . [ 1 ] [ 2 ] [ 3 ] [ 4 ]  The medication available in low-dose and high-dose formulations and is used alone or in combination with an  estrogen . [ 5 ] [ 4 ] [ 6 ] [ 7 ]  It is  ingested orally . [ 6 ]


Side effects  of NETA include  menstrual irregularities ,  headaches ,  nausea ,  breast tenderness ,  mood  changes,  acne ,  increased hair growth , and others. [ 6 ]  NETA is a progestin, or a  synthetic   progestogen , and hence is an  agonist  of the  progesterone receptor , the  biological target  of progestogens like  progesterone . [ 1 ]  It has weak  androgenic  and  estrogenic  activity and no other important  hormonal  activity. [ 1 ] [ 8 ]  The medication is a  prodrug  of  norethisterone  in the body. [ 9 ] [ 10 ]


NETA was  patented  in 1957 and was introduced for medical use in 1964. [ 11 ] [ 12 ]  It is sometimes referred to as a "first-generation" progestin. [ 13 ] [ 14 ]  NETA is marketed widely throughout the world. [ 4 ]  It is available as a  generic medication . [ 15 ]


NETA is used as a  hormonal contraceptive  in combination with  estrogen , in the treatment of  gynecological disorders  such as  abnormal uterine bleeding , and as a component of  menopausal hormone therapy  for the treatment of  menopausal   symptoms . [ 4 ]


NETA is available in the form of  tablets  for use  by mouth  both alone and in combination with  estrogens  including  estradiol ,  estradiol valerate , and  ethinylestradiol . [ 16 ] [ 4 ]   Transdermal patches  providing a combination of 50 μg/day estradiol and 0.14 or 0.25 mg/day NETA are available under the brand names CombiPatch and Estalis. [ 16 ] [ 4 ]


NETA was previously available for use by  intramuscular injection  in the form of  ampoules  containing 20 mg NETA, 5 mg  estradiol benzoate , 8 mg  estradiol valerate , and 180 mg  testosterone enanthate  in  oil solution  under the brand name  Ablacton  to suppress  lactation  in  postpartum  women. [ 17 ] [ 18 ] [ 19 ] [ 20 ]


Side effects  of NETA include  menstrual irregularities ,  headaches ,  nausea ,  breast tenderness ,  mood  changes,  acne ,  increased hair growth , and others. [ 6 ]


NETA is a  prodrug  of  norethisterone  in the body. [ 9 ]  Upon  oral ingestion , it is rapidly converted into norethisterone by  esterases  during  intestinal  and  first-pass   hepatic   metabolism . [ 10 ]  Hence, as a prodrug of norethisterone, NETA has essentially the same effects, acting as a potent  progestogen  with additional weak  androgenic  and  estrogenic  activity (the latter via its  metabolite   ethinylestradiol ). [ 1 ] [ 8 ]


In terms of dosage equivalence, norethisterone and NETA are typically used at respective dosages of 0.35 mg/day and 0.6 mg/day as  progestogen-only contraceptives , and at respective dosages of 0.5–1 mg/day and 1–1.5 mg/day in combination with ethinylestradiol in  combined oral contraceptives . [ 8 ]  Conversely, the two drugs have been used at about the same dosages in  menopausal hormone therapy  for the treatment of  menopausal   symptoms . [ 8 ]  NETA is of about 12% higher  molecular weight  than norethisterone due to the presence of its C17β  acetate   ester . [ 2 ]   Micronization  of NETA has been found to increase its potency by several-fold in animals and women. [ 21 ] [ 22 ] [ 23 ] [ 24 ]  The  endometrial transformation  dosage of micronized NETA per cycle is 12 to 14 mg, whereas that for non-micronized NETA is 30 to 60 mg. [ 21 ]


NETA metabolizes into  ethinylestradiol  at a rate of 0.20 to 0.33% across a dose range of 10 to 40 mg. [ 26 ] [ 27 ]  Peak levels of ethinylestradiol with a 10, 20, or 40 mg dose of NETA were 58, 178, and 231 pg/mL, respectively. [ 26 ] [ 27 ]  For comparison, a 30 to 40 μg dose of oral ethinylestradiol typically results in a peak ethinylestradiol level of 100 to 135 pg/mL. [ 27 ]  As such, in terms of ethinylestradiol exposure, 10 to 20 mg NETA may be equivalent to 20 to 30 μg ethinylestradiol and 40 mg NETA may be similar to 50 μg ethinylestradiol. [ 27 ]  In another study however, 5 mg NETA produced an equivalent of 28 μg ethinylestradiol (0.7% conversion rate) and 10 mg NETA produced an equivalent of 62 μg ethinylestradiol (1.0% conversion rate). [ 25 ] [ 28 ]  Due to its estrogenic activity via ethinylestradiol, high doses of NETA have been proposed for add-back in the treatment of  endometriosis  without estrogen supplementation. [ 26 ]  Generation of ethinylestradiol with high doses of NETA may increase the risk of  venous thromboembolism  but may also decrease  menstrual bleeding  relative to progestogen exposure alone. [ 27 ] [ 28 ]


NETA has  antigonadotropic  effects via its progestogenic activity and can dose-dependently suppress  gonadotropin  and  sex hormone  levels in women and men. [ 1 ] [ 29 ] [ 30 ] [ 31 ]  The  ovulation -inhibiting dose of NETA is about 0.5 mg/day in women. [ 1 ]  In healthy young men, NETA alone at a dose of 5 to 10 mg/day orally for 2 weeks suppressed testosterone levels from ~527 ng/dL to ~231 ng/dL (–56%). [ 30 ]


NETA, also known as norethinyltestosterone acetate, as well as 17α-ethynyl-19-nortestosterone 17β-acetate or 17α-ethynylestra-4-en-17β-ol-3-one 17β-acetate, is a progestin, or  synthetic  progestogen, of the  19-nortestosterone  group, and a synthetic  estrane   steroid . [ 2 ] [ 5 ]  It is the C17β  acetate   ester  of norethisterone. [ 2 ] [ 5 ]  NETA is a  derivative  of  testosterone  with an  ethynyl group  at the C17α position, the  methyl group  at the C19 position removed, and an acetate ester attached at the C17β position. [ 2 ] [ 5 ]  In addition to testosterone, it is a combined derivative of  nandrolone  (19-nortestosterone) and  ethisterone  (17α-ethynyltestosterone). [ 2 ] [ 5 ]


Chemical syntheses  of NETA have been published. [ 32 ]


Schering AG  filed for a patent for NETA in June 1957, and the patent was issued in December 1960. [ 11 ]  The drug was first marketed, by  Parke-Davis  as  Norlestrin  in the  United States , in March 1964. [ 11 ] [ 12 ]  This was a  combination formulation  of 2.5 mg NETA and 50 μg  ethinylestradiol  and was indicated as an  oral contraceptive . [ 11 ] [ 12 ]  Other early brand names of NETA used in oral contraceptives included  Minovlar  and  Anovlar . [ 11 ]


Norethisterone acetate  is the  INN Tooltip International Nonproprietary Name ,  BANM Tooltip British Approved Name , and  JAN Tooltip Japanese Accepted Name  of NETA while  norethindrone acetate  is its  USAN Tooltip United States Adopted Name  and  USP Tooltip United States Pharmacopeia . [ 2 ] [ 5 ] [ 4 ]


NETA is marketed under a variety of brand names throughout the world including Primolut-Nor (major), Aygestin ( US Tooltip United States ), Gestakadin, Milligynon, Monogest, Norlutate ( US Tooltip United States ,  CA Tooltip Canada ), Primolut N, SH-420 ( UK Tooltip United Kingdom ), Sovel, and Styptin among others. [ 2 ] [ 5 ] [ 4 ]


NETA is marketed in high-dose 5 mg oral tablets in the  United States  under the brand names Aygestin and Norlutate for the treatment of gynecological disorders. [ 35 ]  In addition, it is available under a large number of brand names at much lower dosages (0.1 to 1 mg) in combination with  estrogens  such as  ethinylestradiol  and  estradiol  as a  combined oral contraceptive  and for use in  menopausal hormone therapy  for the treatment of  menopausal   symptoms . [ 7 ]


NETA has been studied for use as a potential  male hormonal contraceptive  in combination with  testosterone  in men. [ 36 ]


The  n -octanol-water partition coefficient,   K ow  is a  partition coefficient  for the two-phase system consisting of  n -octanol  and water. [ 1 ]   K ow  is also frequently referred to by the symbol P, especially in the English literature. It is also called  n -octanol-water partition ratio . [ 2 ] [ 3 ] [ 4 ]


K ow  serves as a measure of the relationship between  lipophilicity  (fat solubility) and  hydrophilicity  (water solubility) of a substance. The value is greater than one if a substance is more soluble in fat-like solvents such as n-octanol, and less than one if it is more soluble in water. [ citation needed ]


If a substance is present as several  chemical species  in the octanol-water system due to  association  or  dissociation , each species is assigned its own  K ow  value. A related value, D, does not distinguish between different species, only indicating the concentration ratio of the substance between the two phases. [ citation needed ]


In 1899,  Charles Ernest Overton  and  Hans Horst Meyer  independently proposed that the tadpole toxicity of non-ionizable organic compounds depends on their ability to partition into lipophilic compartments of cells. They further proposed the use of the partition coefficient in an olive oil/water mixture as an estimate of this lipophilic associated toxicity.  Corwin Hansch  later proposed the use of n-octanol as an inexpensive synthetic alcohol that could be obtained in a pure form as an alternative to olive oil. [ 5 ] [ 6 ]


K ow  values are used, among others, to assess the environmental fate of  persistent organic pollutants . Chemicals with high partition coefficients, for example, tend to accumulate in the fatty tissue of organisms ( bioaccumulation ). Under the  Stockholm Convention , chemicals with a log  K ow  greater than 5 are considered to bioaccumulate. [ 7 ]


Furthermore, the parameter plays an important role in drug research ( Rule of Five ) and  toxicology .  Ernst Overton  and  Hans Meyer  discovered as early as 1900 that the  efficacy of an anaesthetic  increased with increasing  K ow  value (the so-called  Meyer-Overton rule ). [ 8 ]


K ow  values also provide a good estimate of how a substance is distributed within a  cell  between the lipophilic  biomembranes  and the aqueous  cytosol . [ citation needed ]


Since it is not possible to measure  K ow  for all substances, various models have been developed to allow for their prediction, e.g.  Quantitative structure–activity relationships  (QSAR) or  linear free energy relationships  (LFER) [ 9 ] [ 10 ]  such as the  Hammett equation . [ 9 ]


A variant of the  UNIFAC  system can also be used to estimate octanol-water partition coefficients. [ 11 ]


Values for log  K ow  typically range between -3 (very hydrophilic) and +10 (extremely lipophilic/hydrophobic). [ 12 ]


The values listed here [ 13 ]  are sorted by the partition coefficient. Acetamide is hydrophilic, and 2,2′,4,4′,5-Pentachlorobiphenyl is lipophilic.


The  p-i concept  refers to the pharmacological interaction of drugs with immune receptors. It explains a form of  drug hypersensitivity , namely  T cell  stimulation, which can lead to various acute inflammatory manifestations such as  exanthems ,  eosinophilia and systemic symptoms ,  Stevens–Johnson syndrome ,  toxic epidermal nercrolysis , and complications upon withdrawing the drug.


The p-i concept links  pharmacology  with  immunology : It implies that drugs bind directly, as an  off-target activity  to  immune receptors  which results in various forms of T cell stimulations. P-i thus starts with an off-target pharmacological activity of the drug followed by a cascade of immunological events which always starts with T cell activation, even if the drug did not bind to the T cell itself but to an  antigen presenting cell  (APC).


The drug bindings occur by  non-covalent bonds  (e.g.  Hydrogen bonds ,  electrostatic interactions ,  van der Waals forces ) to some of the highly polymorphic  T cell receptors  for  antigen  (TCR) and / or  human leukocyte antigens  (HLA). The binding occurs mostly on the cell surface and is labile, reversible and transient. It interacts with the crucial molecules of antigen dependent T cell activation, which may alter the self-HLA to make it look like an allo-HLA-allele, to which T cells strongly react; Some drug binding to TCR itself may – together with HLA-peptide interaction – elicit TCR-CDR signalling or alter the TCR conformation, thereby enhancing its interaction with HLA-peptide (allogeneic effect). Certain drugs may not only interact with the immune receptors on the surface but also inside the cell ( endoplasmic reticulum  e.g.  Abacavir  to HLA-B*57:01). This may cause a change of presented peptides (altered peptide model).


The polymorphism of the immune receptors explains to a large extent the notoriously unpredictable “ idiosyncrasy ” of drug hypersensitivity reactions (DHR), as some of the individually distinct protein sequences may bind the drug better than others. Thereby only those individuals react to the drug which express the fitting protein sequence, e.g. a certain HLA-allele.


Until now, only  αβ TCR  and  HLA-class I and II  proteins were described as target structures for p-i mediated drug hypersensitivity, but it is likely that other immune receptors ( γδ-TCR , HLA-Class Ib, etc.) are also possible off target structures.


Originally, the immune stimulation by drugs were exclusively explained by the  hapten  concept, which was investigated in the early 1930s: [ 1 ] [ 2 ] [ 3 ] [ 4 ]  In these early studies it was found that drugs were too small to represent an antigen inherently. Only if they or their metabolites are haptens, thereby forming larger and stable drug-protein complexes, were they seen as new antigens. Such a formation of a complete antigen (drug-protein complexes, so-called  adducts ) were considered a necessary step to stimulate an immune response, since the drug alone remained unnoticed by the immune cells.


One classical clinical model for a hapten reaction is  contact dermatitis . This is a skin disease based on a localized immune reaction in the skin to a chemically reactive, topical applied compound, which binds by covalent bonds to a carrier protein; many of the contact sensitizers also have a toxic effect, which may be important for the  costimulation  of the immune system. Extensive  in vitro  and  in vivo  data support the hapten concept in contact dermatitis.


It was tempting to use the hapten model to explain generalized DH as well: The drug class most often involved in generalized DHR are  penicillins . When applied to the skin, they elicit contact dermatitis. Penicillins are classical haptens and penicillin modified proteins like  albumin  have been repeatedly found in patients after therapy. Thus it was assumed that the generalized DHR upon parenteral or oral application after penicillins is also due to the hapten-feature of this drug class.


The “hapten-concept” was soon extended to explain all immune mediated (adverse drug reactions): Only if the drug or a metabolite could act as hapten and bind covalently to proteins, then was the drug considered to be able to elicit immune reactions including generalized  exanthema ,  drug induced hepatitis ,  DRESS ,  SJS / TEN  etc. Consequently, during preclinical risk assessment of a new drug, the potential drug candidate may be screened carefully for hapten-features, and if the candidate drugs caused some adverse immune mediated reaction, it was linked to their hapten feature or, if the parent compound lacked hapten characteristics, a hapten feature of a drug metabolite as cause for the DHR was postulated. Importantly, the hapten theory as explanation for all generalized immune reactions was often disputed and hard to reconcile with many experimental or clinical findings.


The p-i concept represents an alternative explanation of immune stimulation by drugs in DHR. It implies that no formation of a new antigen (hapten-protein complex) is needed to elicit an immune activation: T cells are stimulated by the drug binding to immune receptors directly, which leads to conformational changes of HLA and/or TCR as well as to signalling by the  TCR-CD3 complex : The p-i concept was created and formulated by Prof. emeritus Werner J. Pichler in early 2000, based on studies with drug specific T cell clones, derived from patients with DHR. The underlying investigations were performed by various PhD students in Pichler's research group at the  Inselspital / University of Bern  in  Bern ,  Switzerland . The essential, initial finding was that T cells from patients with DH showed a specific reaction to the incriminated drug in vitro ( proliferation ,  cytotoxicity ,  cytokine  release), and that this drug dependent stimulation of immune cells relied on labile (=non-covalent) binding of drugs to cell surface proteins, namely on antigen presenting cells and T cells, which were present in the cell culture.


That a non-covalent, and drug binding was sufficient for T cell stimulation was shown by three main findings  underlying the lability of drug bindings: washing the cells in the cell culture (APCs, T cells) effectively removed the drugs, and the cells could not be stimulated any longer; This means that the drug was binding in a labile way, which was possible with the inert, parent compound; Transformation to a reactive metabolite was not needed to elicit a reaction:  blocking processing or metabolism by drugs did not interfere with reactivity and even fixing the antigen presenting cells by glutaraldehyde failed to eliminate T cell reactivity; And lastly by the speed of reaction: the reaction occurred within minutes, before metabolism could happen; addition of the drug to the cell culture containing drug reactive T cells resulted in a  Ca 2+  influx  in drug specific T cells within less than a minute.


Over the years this p-i concept could be confirmed by many functional and structural studies including  crystallography  which localized the precise region of the immune receptor (HLA-B*57:01, TCRVβ20, etc.), to which a particular drug binds. Since crucial and highly sensitive molecules of T cell activation are targeted, complex and highly variable immunological consequences can develop: different types of T cells are activated to a variable degree, leading in inflammatory consequences with a highly polymorphic clinical picture of acute symptoms, followed by different late appearing complications.


The modification of the self-HLA, of TCR, or of the TCR-peptide-HLA complex by non-covalent, bindings of drugs is a reversible, transient process, whose effect is highly dependent on the affinity of drug-protein interactions. The drug can bind first to the HLA-peptide complex (p-i HLA) or the TCR complex (p-i TCR). Sometimes the drug may be trapped in between TCR and HLA.


P-i HLA is often linked with a striking HLA-association of the DHR (shown for abacavir, carbamazepine, allopurinol, dapson, vancomycin etc.), since the drug binds to a certain HLA-allele with higher affinity than to other HLA-molecules. P-i TCR is less investigated. Drug binding to certain parts of the TCR Vβ chains may be sufficient for full activation, if interaction with HLA-peptide complexes is possible; other p-i TCR bindings may require additional T cell activation (e.g. by viral infection) to lead to clinical symptoms.


The in vitro analysis of p-i using drug specific T cell clones (TCC) or TCR-transfected cell lines generated from patients with DH revealed a strong stimulation: A high level of T cell mediated  cytotoxicity , a broad spectrum of secreted  cytokines  and polyclonality was observed; The p-i stimulation was unorthodox: some CD4+ T cells were uncharacteristically reacting to HLA-class I or CD8+ T cells to HLA-class II drug presentation or did not show strict HLA restriction, and some TCC were polyspecific (the reactive T cell clones reacted with various peptides): [ 5 ] [ 6 ] [ 7 ]  Altogether, the picture emerged that p-i induced T cell stimulations have features of allo-like immune stimulations (allo-stimulation).


The two main clinical outcomes of acute p-i reactions are MPE/DRESS on one hand, and SJS/TEN on the other hand. In MPE/DRESS patients, high numbers of circulating, atypical (activated) lymphocytes and high levels of various cytokines can be found in the circulation. [ 8 ] [ 9 ]  In vitro drug stimulation reveals a proliferating, high cytokine secreting, cytotoxic CD4 and CD8 T cell reaction to the incriminated drug, which sometimes can be detected for many years.
Quite in contrast, patients with SJS/TEN may show lymphopenia, just the blister fluids are full of mainly CD8/NK+, cytotoxic T cells, which are able to kill keratinocytes. [ 10 ] [ 11 ] [ 12 ]  Cytotoxic molecules ( granzyme B ,  perforin ,  granulysin ) can be detected in vivo in blood and the blister fluid in the first few days of the disease. [ 12 ] [ 13 ]  During the acute disease (1–2 weeks), the T cells still react in in vitro assays, but after 3–4 weeks, the CD8 cell compartment, which is considered to be the main responsible cell population for the disease, appears to be exhausted and are refractory to drug stimulation. [ 14 ] [ 15 ]  How this CD8 exhaustion is achieved is unknown.


If the p-i stimulation resulted in T cell expansion and activation (MPE, DRESS), some of the p-i-activated T cells might continue to react in the absence of drug: their TCR may be cross-reactive with unmodified, self HLA presenting exogenous peptides (mainly of herpes virus origin) or some self-peptides: this kind of cross-reactivity with exogenous or self peptides and self-HLA explain two late complications after severe DHR, mostly DRESS: one is viral reactivation: herpes viruses are permanently harboured in various cell types after infection (fibroblasts, endothelial cells, hematopoietic cells, brain cells, etc.) [ 16 ] [ 17 ] [ 18 ]  and are controlled by T cells. When these herpes-virus specific T cells are activated by p-i, they react with the herpes virus peptide expressing cells and damage them by their cytotoxic potential: [ 12 ] [ 19 ]  a consequence is the release virus particles into the circulation  [ 16 ] [ 17 ] [ 18 ]  and symptoms of viral reactivation (high virus load, possibly increase of liver enzymes and of activated lymphocytes) appear.


A second wave of peptide reactivity may end up in autoimmunity: the abnormal stimulation by p-i includes T cells from the naïve and memory T cell pool. It may include self-peptide reactive T cells, which, if the corresponding self-peptides are presented and are encountered, release cytokines and exert cytotoxicity - autoimmunity may arise. [ 20 ] [ 21 ]  As such self-peptide reactive T cells are present in relatively low amounts, they need >6–8 weeks to expand and appear after the virus-reactivations. Autoimmunity occurs in a minority of patients (<20%), and may is often manifested as autoimmune poly-endocrine syndrome. [ 20 ] [ 21 ] [ 22 ]


Multiple drug hypersensitivity (MDH): a further consequence of p-i stimulations like DRESS or severe MPE is MDH; such patients develop an additional DHR to a structurally different drug, with the same or different clinical manifestations. [ 23 ]  MDH occurs in ca. 20% of patients with DRESS, and can occur any time, from the start of DRESS (often to a combination therapy), during the initial activation, and can even appear years after the first DHR  [ 23 ] [ 24 ]


A main difficulty of DHR research and weak point of the p-i concept is the fact that it is cumbersome to demonstrate a p-i reactivity – namely that the T cell stimulation occurred due to non-covalent drug binding to immune receptors. In principle, a T cell mediated DHR was explained by p-i if the drug binding to the immune receptors was found to be labile.


To demonstrate the lability of drug binding, drug specific T cell lines, T cell clones and TCR hybridoma cells were required. [ 25 ] [ 26 ] [ 27 ]  Washing of the cell mix of drug, APC and T cells abrogated p-i reactivity, while T cell reaction to haptens persisted. The presentation of peptides by HLA on APC takes > 4hr pulsing (uptake of hapten modified protein, processing and presentation), and may also require metabolism, if the stimulating drug was chemically inert and not a hapten. Therefore, an immediate reactivity of T cells (e.g. measured by rapid Ca 2+  influx) as well as reactivity to the drug in the presence of protein and metabolism inhibitors or by using glutaraldehyde-fixed antigen presenting cells was interpreted as p-i reaction. [ 25 ] [ 26 ] [ 27 ]  P-i reactivity was demonstrated for a number of drugs ( SMX ,  lidocain ,  lamotrigine ,  carbamazepine , various  radio contrast media ,  quinolones ,  vancomycin ,  dapsone , etc.), including some drugs which can act via p-i or as hapten ( piperacillin ,  flucloxacillin ,  amoxicillin ,  cephalosporins ,  monobactams ). In these cases the p-i reactivity was responsible for more severe reactions like hepatitis in flucloxacillin/B*57:01 carriers or DRESS with amoxicillin and piperacillin [ 27 ]


DRESS/SJS/TEN (severe DHR) and HLA-linked DHR are p-i. Initially it was thought that p-i reactions were the exception while the hapten mechanism represented the main cause of systemic T cell mediated DHR. Conversely it seems to be the opposite as p-i appears to be the main mechanism in T cell mediated DHR: Whenever the mechanism was investigated how drugs cause severe DHR (DRESS, SJS/TEN), it was always found to be due to p-i. [ 25 ] [ 26 ]


It is unclear whether the majority of severe MPE is due to p-i. As the in vitro analysis of amoxicillin induced MPE (analysis of >150 amoxicillin induced MPE) regularly reveals high secretions of IL-5, IL-13, IFNg, granzyme B and granulysin upon drug exposure (very similar to DRESS cases), the in vitro drug stimulation in MPE is often strong and includes the secretion of Th1, Th2, and cytotoxic cytokines simultaneously. It is actually often stronger and broader than the cytokine secretion upon tetanus control, and is reminiscent of an in vitro mixed leukocyte reaction (MLR). [ 27 ]  Further work is needed, but most MPE appear to be mediated by p-i.


Importantly, all drugs which develop DHR and have a strong HLA-linkage (e.g. allopurinol/oxypurinol and B*58:01) stimulate via p-i. Protein reactions are not HLA restricted: A protein is large and is processed into various small peptides. These peptides (including the hapten-modified peptides) fit into different HLA alleles and not in only one HLA like observed with drugs. Thus, the DHR-HLA linkage seen with certain drugs can only be explained by direct drug binding to an allele-typic region of the HLA-molecule. This was also confirmed in structural and computational studies. Importantly, if a drug can be stimulatory by p-i or by hapten mechanism (SMX/SMX-NO, beta-lactams), the severe T cell mediated symptoms are mediated by p-i and are HLA-allele restricted, [ 28 ]  while the hapten-reactions are not. Thus, the list of drugs acting via p-i and causing DHR is now longer than the list of hapten-like drugs (table).


First hapten, then non covalent drug binding: A strong argument for a hapten-mechanism underlying DHR was the ability of the drug to cause all, namely IgG, IgE and T cell mediated DHR, since these different immune reactions required the immunogenic presentation of the drug in various ways. This is best achieved using hapten (and thus antigenic) features of a drug. Indeed, the classical hapten-drugs beta-lactam antibiotics, SMX-NO, or PPI are able to induce all forms of Gell and Coombs immune stimulations, while e.g. classical and exclusive p-i drugs like carbamazepine or abacavir induce only T cell reactions, but never anaphylaxis. [ 29 ]  Importantly, an ability to act as hapten does not rule out that non-covalent binding like in p-i plays a role in DHR. Actually, during a DHR the type of drug-protein binding may change: A drug may act as hapten in the induction phase causing asymptomatic immunity, but the effector mechanism of immunoglobulin-reactions and some severe T-cell mediated DHR may actually be due to non-covalent drug bindings. Thus, beta-lactam antibiotics – the classical hapten-drugs - are the main elicitor for "fake antigen" reactions, drug induced immune  thrombocytopenia  (DITP) and p-i stimulations, which are all based on non-covalent drug-protein interactions and are not antigen induced.


The polyclonal T cells response stemming from the memory T cell pool includes T cells which are primed by prior immune responses. An important role play herpes viruses (HHV6, CMV, EBV, Herpes simplex I), where a relatively large amount of T cells are involved in the control of these herpes viruses. Indeed, herpes virus reactivation is so common, that it is part of the Japanese definition of DRESS. [ 30 ]   Since the precursor frequency of such herpes virus specific T cells is high (up to 10% of the CD8+ T cells in the elderly can be devoted to herpes virus control, [ 30 ] [ 31 ]  symptoms due to such T cells appear already after ca. 2–6 weeks.


Penetration enhancers  (also called chemical penetration enhancers, absorption enhancers or sorption promotors) are chemical compounds that can facilitate the penetration of  active pharmaceutical ingredients  (API) into or through the poorly permeable biological membranes. These compounds are used in some pharmaceutical formulations to enhance the penetration of APIs in  transdermal drug delivery  and transmucosal drug delivery (for example, ocular, nasal, oral and buccal). [ 1 ]  They typically penetrate into the biological membranes and reversibly decrease their barrier properties.


Human skin  is a very impermeable membrane that protects the body from ingress of harmful substances and prevents water loss from underlying organs. However, this seriously limits the use of skin as a site for drug administration. One of the approaches to facilitate  transdermal drug delivery  is the use of penetration enhancers. Many different compounds have been explored as potential penetration enhancers to facilitate transdermal drug delivery. These include  dimethylsulphoxide ,  azones  (such as  laurocapram ), pyrrolidones (for example  2-pyrrolidone ), alcohols ( ethanol  and  decanol ),  glycols  (for example  propylene glycol ), surfactants,  urea , various hydrocarbons and terpenes. [ 2 ] [ 3 ] [ 4 ]  Different potential skin site and modes of action were identified for penetration enhancement through the skin. In some cases, penetration enhancers may disrupt the packing motif of the  intercellular lipid matrix  or  keratin  domains. In other cases, drug penetration to the skin is facilitated because the penetration enhancer saturates the tissue and becomes a better system to dissolve the molecules of API.


Topical administration to the eye is usually characterised by very poor  drug bioavailability  due to several natural defence mechanisms, including  nasolacrymal drainage ,  blinking , and poor permeability of the  cornea . Enhancement of the corneal permeability to drug molecules is one of the strategies to improve the efficiency of topical drug delivery to the eye. Several classes of compounds have been researched as potential penetration enhancers through ocular membranes. These include  chelating agents ,  cyclodextrins , surfactants,  bile acids  and salts, and  crown ethers . [ 5 ]  There are also reports on the use of cell penetrating peptides and chitosan as penetration enhancers in ocular drug delivery. [ 6 ]  The most commonly used penetration enhancers in ocular formulations are benzalkonium chloride and ethylenediamine tetraacetate (EDTA).  Benzalkonium chloride  is often used as an antimicrobial preservative in eye drops [ 7 ]  and EDTA is used as a chelating agent.


Cyclodextrins,  chitosan , some  surfactants ,  bile acids  and salts,  sodium tauro-24,25-dihydro-fusidate , and  phospholipids  were reported as penetration enhancers in nasal drug delivery both for humans and equines. [ 8 ]  Chitosan is one of the most widely researched penetration enhancers in nasal drug delivery and it enhances the penetration of drugs by opening the  tight junctions  in the cell membranes. [ 9 ]


Penetration enhancers have been applied to improve the absorption of poorly permeable, hydrophilic drugs or macromolecules. [ 10 ]  Permeation enhancers that have been used successfully for oral drug development include medium-chain fatty acids like caprylic acid [ 11 ]  or caprate, or its amino acid ester like Salcaprozate sodium (SNAC). [ 12 ]  The above-mentioned permeation/penetration enhancers have a surfactant-like activity where they perturb the intestinal epithelium, promoting transcellular or paracellular absorption. [ 13 ]


Peripherally acting  μ-opioid receptor   antagonists  ( PAMORAs ) are a class of  chemical compounds  that are used to reverse  adverse effects  caused by  opioids  interacting with  receptors  outside the  central nervous system  (CNS), mainly those located in the  gastrointestinal tract . PAMORAs are designed to specifically inhibit certain  opioid receptors  in the gastrointestinal tract and with limited ability to cross the  blood–brain barrier . Therefore, PAMORAs do not affect the  analgesic  effects of opioids within the central nervous system. [ 1 ]


Opioid drugs are known to cause  opioid-induced constipation  (OIC) by inhibiting  gastric emptying  and decreasing  peristaltic  waves leading to delayed absorption of medications and more water absorption from the  feces . That can result in hard and dry stool and  constipation  for some patients. [ 2 ]


OIC is one of the most common  adverse effects  caused by opioids, so the discovery of PAMORAs can prevent the effects that often compromise  pain management . [ 3 ]


Methylnaltrexone bromide  was the first medication in the  drug class  approved by the  FDA . [ 4 ]  It was discovered in 1979 by Leon Goldberg, a  pharmacologist  at the  University of Chicago . Having witnessed the suffering of a dying friend with OIC, Goldberg tested various  derivatives  of  naltrexone , a drug known to block the effects of opioids. His objective was to find a drug that could not pass the  blood brain barrier , without affecting the analgesic effects of the opioids. After Goldberg died, his colleagues at the university continued to develop the compound. It was approved by the FDA in April 2008, originally for OIC in adult patients with advanced illness and later in adult patients with chronic noncancer pain. [ 5 ]


In the late 1970s, Dennis M. Zimmerman and his co-workers from  Lilly Research Laboratories , Indiana, did research on structural concepts for narcotic antagonists defined in a 4-phenylpiperidine series. [ 6 ]  They reported  N -methyl- trans -3,4-dimethyl-4-phenylpiperidine to be pure opioid receptor  antagonist  with a new  pharmacophore . To increase the  potency  they attached a  phenolic group  to the  aromatic ring ,  N -methyl- trans -3,4-dimethyl-4-(3-hydroxyphenyl)piperidine. That structure was used to design and develop other opioid receptors antagonists such as  alvimopan . [ 5 ]  Alvimopan was approved later in 2008 for in-hospital use to increase the  gastrointestinal function  following a partial large or  small bowel  resection with primary  anastomosis .  Naloxegol  was approved in September 2014 and  naldemedine  in March 2017, both for the treatment of OIC in adult patients with chronic cancer. [ 7 ] [ 8 ] [ 9 ] [ 10 ]


PAMORAs act by inhibiting the binding of opioids  agonist  to the  μ-opioid receptor  (MOR). The objective of PAMORAs treatment is to restore the  enteric nervous system  function (ENS). The MOR is found in several places in the body and PAMORAs is a  competitive antagonist  for binding to the receptor. The MORs in the gastrointestinal tract are the main receptors that PAMORAs are intended to block and prevent the binding of opioid agonists. [ 11 ]  PAMORAs are used in the treatment of opioid-induced bowel dysfunction (OIBD), a potential adverse effect caused by chronic opioid use. PAMORAs act on the three  pathophysiological  mechanisms of this adverse effect. They act on  gut motility ,  gut secretion  and  sphincter  function. [ 12 ]


PAMORAs effect on gut motility is that it can increase the resting tone in the circular muscle layer. The antagonist enhances the effect on tonic inhibition of the  muscle tone . This will normalize the tone in the circular muscle layer and therefore prevent opioid-induced rhythmic contractions.  When these two factors are combined, it results in decreased  transit time . Impliedly these effects will decrease the passive absorption of fluids which helps with decreasing OIBD symptoms such as constipation,  gut spasm  and  abdominal cramp . [ 13 ]


PAMORAs effect on gut secretion will help reverse the decreased  cAMP  formation that opioid agonists induce. [ 14 ]  Also, the antagonist will establish a normal secretion of  chloride . Opioids agonists can also reduce the  secretion  of  peptides  by increasing the  sympathetic nervous system  through the μ-receptors in the ENS, which can lead to drier and harder stool. PAMORAs work against it so the stool becomes softer and less dry. [ 13 ]


PAMORAs effect on the function of the  sphincter  is in theory to regulate the movement coordination. The antagonist can prevent  sphincter of Oddi dysfunction  that is caused by opioids. [ 15 ]  Antagonists can also reduce opioid-induced anal sphincter dysfunction. The dysfunction is tied to  straining ,  hemorrhoids  and incomplete emptying. [ 16 ]


Even though μ-opioid receptor (MOR) targeting drugs have been used for a long time, not much is known about the  structure-activity relationship  and the  ligand -receptor interactions on the basis of well-defined  biological effects  on receptor activation or inhibition. Also, the distinction in the receptor-ligand interaction patterns of agonists and antagonists is not known for sure. One theory states that the  morphinans  biological activity could be determined by the size of the N-substituents. For example, antagonists usually have larger substituents, such as  allyl - or  cyclopropyl methyl  at the morphinan nitrogen, while agonists generally contain a  methyl group . On the other hand, agonist activity is also shown in ligands with larger groups at the morphinan nitrogen, and therefore this hypothesis is challenged. [ 17 ]


Methylnaltrexone bromide, naloxegol, and naldemedine all have similar structures, which is not far away from the chemical structure of  morphine  and other MOR-agonists. All contain a rigid  pentacyclic  structure that involves  benzene ring  (A),  tetrahydrofuran  ring (B), two  cyclohexane  rings (C and D) and a  piperidine  ring (E). [ 18 ]  The most important functional groups for the biological action of opioids are the  hydroxyl group  on the  phenol , N-methyl group,  ether  bridge between C4 and C5, the  double bond  between  carbon  number C7 and C8 and the hydroxyl groups at C3 and C6. The phenolic ring and its 3-hydroxyl group is vital for the analgesic effects as the removal of the OH group decrease the analgesic activity 10-fold. There is another principle for the hydroxyl group on C6 as the removal enhances its activity. The increased activity is mainly because of the increased  lipophilicity  and the increased ability to cross the blood–brain barrier. Naldemedine has the hydroxyl group while methylnaltrexone bromide has a  ketone  group and naloxegol has an  ester . The double bond between C7 and C8 is not required for the analgesic effect and reduction of the double bond will increase the activity. None of the antagonists has a double bond in their structure. The N-substituent on the skeleton is thought to determine the pharmacological behavior and its interaction with MOR. It is also thought to play a key role in distinguishing antagonists from agonists. Allyl group, a  methylcyclopropyl group  or a  methylcyclobutyl  as N-substituent groups are thought to lead antagonist activity. [ 19 ] [ 20 ] [ 21 ]


Agonists and antagonists form certain  chemical bonds  with  amino acids  that construct the MOR. The majority of antagonists, as well as agonists, are predicted to form  charged interaction  with Asp147 and a  hydrogen bond  with Tyr148. However, majority of antagonists also form additional  polar interactions  with other amino acid residues such as Lys233, Gln124, Gln229, Asn150, Trp318 and Tyr128. Only a small minority of agonists form the same additional polar interactions. Both agonists and antagonists are known to form hydrogen bonds with His297. [ 22 ]


It can be concluded that interactions with the amino acid residues, Asp147 and Tyr148 are essential for the  ligand  to bind to the receptor and the molecules that form additional polar interactions with other residues are more often antagonists than agonists. [ 17 ]


The N-substituent group can form  hydrophobic  bonds with Tyr326 and Trp293 and the aromatic and cyclohexane rings can form similar bonds to Met151. The backside of the ligand can also form a hydrophobic bond, but with Val300 and Ile296. [ 22 ]


Methylnaltrexone bromide is the  bromide  salt form of methylnaltrexone, a  quaternary  methyl derivative of  noroxymorphone . The methyl group and the quaternary salt formation increase the  polarity  and reduce the  lipid solubility  thereby restricts the blood–brain-barrier penetration. Methylnaltrexone has eight times higher  affinity  for MOR than for  κ-opioid receptor  (KOR) and  δ-opioid receptor  (DOR). [ 23 ]  Naltrexone forms interaction with Asp147 and Tyr148 along with a  hydrogen bond  with Lys233. [ 24 ]


Peripherally selective trans-3,4-dimethyl-4-(3-hydroxylphenyl)piperidine opioid antagonists were developed for the treatment of  gastrointestinal motility  disorder by Zimmerman and his coworkers. From that, they derived the 4-(3-hydroxyphenyl)-3,4-dimethylpiperidine scaffold with functional groups spanning various sizes, charge, and polarity to reach peripheral opioid receptor antagonism while decreasing CNS drug exposure. The  in vitro  μ-Ki,  in vivo   AD50 , and  ED50  and peripheral index (ratio) was examined for several selective analogs, and from that, they found out that the trans-3,4-dimethyl-4-(3-hydroxyphenyl) piperidine, Alvimopan, gave the best results. [ 5 ]  The large  zwitterionic  structure and the high polarity prevents Alvimopan from crossing the blood–brain barrier, potency at binding peripheral MORs is thereby 200 times that of central MORs. [ 25 ]


Naloxegol is a  polyethylene glycol -modified derivative of α- naloxol . Naloxegol has a similar form as  naloxone  as a  heteropentacyclic  compound both of which have an  allyl group  attached to the  amine  of the  piperidine  ring. However, naloxegol has a monomethoxy-terminated n=7  oligomer  of  PEG  connected to the 6-alpha-hydroxyl group of ɑ-naloxol via an  ether  linkage. The PEG moiety increases the  molecular weight  and therefore restricts the uptake of naloxegol into the  CNS . [ 26 ]  Furthermore, pegylated naloxegol becomes a  substrate  for the  P-glycoprotein   efflux  transporter that transports the compound out of the CNS. [ 27 ]


Naldemedine has a similar chemical structure as naltrexone but with an additional side chain that increases the  molecular weight  and  polar surface area  of the substance. Like naloxegol, naldemedine is a substrate of the P-glycoprotein efflux transporter. These properties result in less penetration into the CNS and decrease possible inference with the effects of opioid agonists. [ 28 ]
Naldemedine is a dual antagonist for MOR and DOR. Activation of the DOR has been known to cause nausea and/or vomiting, so a dual antagonist can decrease both OIC and nausea/vomiting. [ 29 ]


The  molecular weight ,  bioavailability ,  protein binding ,  elimination half-life , the  time to achieve maximum plasma concentration  and  binding affinity  are present in the table below. [ 26 ] [ 23 ]


Methylnaltrexone bromide has poor oral bioavailability, and for that reason, every other day it is administered  subcutaneously . About half of the dose is excreted in the urine and somewhat less in feces with 85% eliminated unchanged. [ 24 ]


Alvimopan has considerable low bioavailability (6%) due to its high  binding affinity  and low  dissociation rate . Essentially, alvimopan is mediated by  biliary secretion  with an average plasma clearance of 400 ml/min.  Metabolism  of alvimopan is via  intestinal flora  resulting in  hydrolysis  of alvimopan to the active amide metabolite (ADL 08-0011). However, the metabolite is considered clinically irrelevant due to its low binding affinity. [ 25 ]


When naloxegol is given with a fatty meal,  absorption  increases. Clearance is mostly via  hepatic metabolism  (P450-CYP3A) with unknown actions of the metabolites. Naloxegol has small fragments eliminated by  renal excretion . [ 30 ]


Naldemedine metabolites mainly via  CYP3A  to nor-naldemedine, it also metabolites via  UDP-glucuronosyltransferase  1A3 to naldemedine 3-G, but in a lesser extent. Those metabolites are both opioid receptor antagonists but are less potent than the  parent compound . [ 33 ]


Axelopran  is an oral PAMORA which is under development by Theravane Biopharma. It has completed phase II in  clinical trials  in more than 400 patients with OIC. Axelopran has a different chemical structure from other PAMORAs but with a similar  mechanism of action . It acts as an antagonist for MOR, KOR and DOR, but with higher affinity for MOR and KOR than for DOR. Like other PAMORAs, the main goal is the treatment of OIC. [ 34 ]
Axelopran is also being investigated in fixed-dose combination (FDC) with  oxycodone . It is done by using spray coating technology to create an FDC of axelopran and controlled-release oxycodone. [ 35 ]


There is a demand for optimization of the receptor  selectivity  and affinity accompanied by an exploration of candidate compounds regarding their  route of administration . These are the main objectives and future strategies for drug discovery and the development of PAMORAs.
Predominantly, the MORs exhibit functionally selective agonism. Therefore, future possible candidate compounds that target OIC are PAMORAs with optimized selectivity and affinity. [ 27 ]


Pharmacoepidemiology  is the study of the uses and effects of drugs in well-defined populations. [ 1 ] [ 2 ]


To accomplish this study, pharmacoepidemiology borrows from both  pharmacology  and  epidemiology . Thus, pharmacoepidemiology is the bridge between both pharmacology and epidemiology. Pharmacology is the study of the effect of drugs and clinical pharmacology is the study of effect of  drugs  on clinical humans. Part of the task of clinical pharmacology is to provide a risk benefit assessment by effects of drugs in patients: [ citation needed ]


Other parameters relating to drug use may benefit epidemiological methodology. Pharmacoepidemiology then can also be defined as the transparent application of epidemiological methods through pharmacological treatment of conditions to better understand the conditions to be treated. [ citation needed ]


Epidemiology  is the study of the distribution and determinants of diseases and other health states in populations. Epidemiological studies can be divided into two main types: [ citation needed ]


Pharmacoepidemiology benefits from the  methodology  developed in general epidemiology and may further develop them for applications of methodology unique to needs of pharmacoepidemiology. There are also some areas that are altogether unique to pharmacoepidemiology, e.g., pharmacovigilance. Pharmacovigilance is a type of continual monitoring of unwanted effects and other safety-related aspects of drugs that are already placed in current growing integrating markets. In practice, pharmacovigilance refers almost exclusively to spontaneous reporting systems which allow health care professionals and others to report adverse drug reactions to the central agency. The central agency combines reports from many sources to produce a more informative profile for drug products than could be done based on reports from fewer health care professionals. [ citation needed ]


In Australia, a 10% sample of all people eligible for government-subsidised medicines by the Pharmaceutical Benefits Scheme (PBS) are made available for research purposes.  Licences are held between Services Australia, who hold the data for the PBS, and academics at Monash University, University of New South Wales, University of South Australia and the University of Western Australia to use the 10% sample for research purposes. Research outputs from these data have to be approved by Services Australia prior to publication.  These data create a useful picture of all dispensed medicines in Australia and allow for pharmacovigilance and to explore trends in medicines usage. [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]


Pharmacological cardiotoxicity  is defined as  cardiac damage  that occurs under the action of a  drug . This can occur both through damage of  cardiac muscle  as well as through alteration of the  ion  currents of  cardiomyocytes . [ 1 ]


Two distinct drug classes in which cardiotoxicity can occur are in  anti-cancer  and  antiarrhythmic drugs .  Anti-cancer  drug classes that cause cardiotoxicity include  anthracyclines ,  monoclonal antibodies , and  antimetabolites . This form generally manifests as a progressive form of  heart failure , but can also manifest as an harmful  arrhythmia . [ 2 ]  In contrast, in  antiarrhythmic drugs , cardiotoxicity is due to a risk of  arrhythmias  resulting from treated-induced  ion current  imbalance. [ 3 ]


Other types of drugs are also known for cardiotoxicity, such as  clozapine  being associated with myocarditis. [ 4 ]


The cardiotoxicity of anticancer drugs has been well documented, with an entire sub-speciality of  cardio-oncology  dedicated towards investigating and treating these serious side effects. Two well known anticancer drug families that cause cardiotoxicity are  anthracyclines  and  monoclonal antibodies targeting HER2.  Other types of anticancer drugs that can lead to cardiotoxicity include alkylating agents such as  cyclophosphamide , BCR-ABL1 targeting receptor tyrosine kinases such as  imatinib , and VEGF antibodies such as  bevicizumab . [ 5 ]  This section of the article will focus on  anthracyclines  and  HER2 monoclonal antibodies  due to the prominence of cardiotoxicity in these compounds.


The mechanism of anthracycline-induced cardiotoxicity is unknown and is under active research. However, multiple theories exist. One well supported mechanism is related to the production of superoxide anion  radicals  that in turn damage cardiac myocytes.  [ 6 ]  Recent research suggests that Top2b (topoisomerase-IIβ) helps mediate the production of oxygen radicals, representing a potential biomarker for this serious side effect. [ 7 ]    Other proposed mechanisms include interference with cardiac  ATP  production, mitochondria-related stress, and lipid peroxidation. [ 6 ]


On the other hand, the mechanism of  HER2  antibody cardiotoxicity is more well known.  [ 8 ]   HER2  is a protein expressed on the cell membranes of HER2 positive breast cancer cells.  However, HER2 is also expressed on the surface of cardiac myocytes. It is hypothesized that HER2 expressed in these cardiac cells have a cardioprotective mechanism, and the targeting of these proteins in this context leads to the cardiotoxicity associated with  HER2 monoclonal antibodies. [ 9 ]


The cardiotoxicity of anthracyclines can be classified into three categories: early, early onset chronic, and late onset chronic.  Early cardiotoxicity is rare, but manifests as arrthymias, myocarditis, and pericarditis. This type of toxicity occurs directly after treatment with anthracycline. Early onset chronic cardiotoxicity is defined as cardiotoxicity manifesting within one year of the completion of treatment, while late onset chronic cardiotoxicity occurs after one year.  [ 10 ]  The cardiotoxicity of anthracyclines is dose dependent.  At total exposure levels lower than 400 mg/m2, the incidence of heart failure is between 3%-5%.  At a exposure rate of 700 mg/m2, the heart failure rate is at 48%. [ 11 ]


Cardiotoxicity involving  HER2 monoclonal antibodies  manifests as decrease left ventricular ejection fraction and resulting heart failure. [ 12 ]  The cardiotoxicity of  HER2 monoclonal antibodies  is dose independent. [ 13 ]


The immediate intervention for the development of cardiotoxicity is discontinuation of the drug. Preventative measures for anthracycline induced cardiomyopathy include  dexrazoxane , which is the only preventative drug approved by the FDA for prevention of anthracycline cardiomyopathy. [ 14 ]   Overall, there are no specific treatments targeted towards the cardiotoxicity of anticancer drugs. Rather, treatment is of the resultant heart failure. This often takes the form o f ACE inhibitors  or  beta blockers .  [ 15 ]


Antiarrhythmics  are broad class of drugs that are used treat heart rhythm irregularities.  [ 16 ]  Utilizing the Vaughan-Williams (VW) system, antiarrhymic drugs are classified into four main classes based on their mechanism of action. Class I antiarrhymics lead to blockage of sodium channels. Class II antiarrhymatics are  beta-adrenoceptor blockers . Class III antiarrhymics act as  potassium channel blockers , while Class IV antiarrhymics are  non-dihydropyridine calcium channel blockers . While the effects of these drugs may be antiarrhymic, they can also be proarrhymic in other contexts.


The pharmacological cardiotoxicity of antiarrhymic compounds is related to their electrophysiological mechanism. In particular, because antiarrhymics drugs act on the opening/closing of ion channels, the modification of the electrical currents can lead to adverse cardiac events such  torsade de pointes  or  ventricular fibrillation . Due to the case-by-case basis in which these medication lead to cardiotoxicity and the development of specific adverse rhythms, it has become increasingly important to assess compounds in a preclinical environment (See  Pharmacological cardiotoxicity#In Silico Cardiotoxicity Assessment ).


The manifestation of antiarrhymic cardiotoxicity may manifest as worsening of the pre-existent arrhythmia or the development of a new arrhythmia.


Female sex at birth has been associated with an increased risk of the development of new arrhythmia, and other risk factors include age, kidney disease, drug-drug interactions, and other underlying heart problems. [ 17 ]


Like with anticancer drugs, the most common intervention for the development of cardiotoxicity is discontinuation of the causative drug. Individual risk factors, such as risk of arrhythmia re-emergence, are considered when deciding final courses of action. Adjacent devices, such as pacemakers, or ablation therapy may also be considered as alternatives to medical treatment for the primary arrhythmia. [ 18 ]


The treatment of  torsade de pointes  is typically with intravenous magnesium sulfate, which helps stabilize cardiac membranes. [ 19 ]  For  ventricular fibrillation  cases, either/or  defibrillation ,  amiodarone , or  epinephrine  is used dependent on the ACLS algorithm.  [ 20 ]


In the last years, in silico models have aided scientists and clinicians to cure several diseases. [ 21 ]  Computational modeling in particular has helped scientists to alter parameters that otherwise could have not been investigated. [ 21 ]


In the field of  electrophysiology , pharmacological cardiotoxicity can be carried out by leveraging specific computational models. Recently, it has become possible to analyze the pharmacological effect on  atria  and  ventricles  separately. [ 22 ] [ 23 ]


Since the two cardiac chambers are very different each other and play a key role both on a functional and anatomical basis, suitable computational models have to be accounted for to describe their different behavior. During the years, several models have been developed to best characterize and replicate the cellular action potential behavior of the most relevant anatomical region of the heart, such as Courtemanche model for atria or O'Hara model for ventricles. [ 22 ] [ 23 ]


In this way, it has been possible to create a virtual cellular population of cardiomyocytes and vary their  conductances  that are related to the main ionic currents which contribute to the action potential morphology, and is reflective of a specific anatomical region of the heart. [ 24 ] [ 25 ]


In order to create a stable population of cellular  action potentials , several biomarkers have been developed to best characterize the instability of cellular action potentials. Examples of biomarkers reported include: [ 24 ]


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 A
 P

 D

 90

 −
 A
 P

 D

 50

 {\displaystyle Triangulation=APD_{90}-APD_{50}}


A
 P
 A
 =

 V

 M
 a
 x

 −

 V

 0

 {\displaystyle APA=V_{Max}-V_{0}}


Once the cellular population is stable, all action potential are then compared to physiological data related to the most relevant anatomical regions to appropriately filter the action potential, aiming to consider just the physiologically relevant ones. [ 27 ]


At the atrial level, clusterization occurs with data associated to: [ 27 ]


According to  pharmacokinetic  and  pharmacodynamic  ideals, pharmacological action is integrated in the model. By means of specific electrical stimuli protocols, [ 28 ]  the pharmacological effect of a new drug can be investigated in a completely safe, and controlled computational environment, providing preliminary important considerations concerning the cardiotoxicity of new pharmacological compounds. [ 29 ]


According to the outcome of the simulations, several aspects can be investigated to identify the proarrhythmicity of a new pharmacological compound. [ 30 ] [ 31 ]  The typical changes, known as repolarization abnormalities, that are considered pro-arrhythmic include: [ 31 ]


Simulation can be carried out at different effective plasmatic therapeutic level of the drugs to identify the level at which cardiotoxicity cannot be neglected. The data collected could be finally managed to create a score system aimed to define the torsadogenic risk, namely the risk of inducing torsade de pointes, of the new drugs. [ 32 ] [ 33 ]


A possible torsade de point risk score to assess cardiotoxicity could be: [ 33 ]

 T
 d
 P
 R
 S
 =

 ∑

 c

 (

 W

 c

 ⋅
 n
 R

 A

 c

 )

 N
 ⋅

 ∑

 c

 W

 c

 )

 {\displaystyle TdPRS={\frac {\sum _{c}(W_{c}\cdot nRA_{c})}{N\cdot \sum _{c}W_{c})}}}


where

 ∑

 c

 {\displaystyle \sum _{c}}

  is the sum of all concentrations, [C] is the concentration taken into account,

 W

 c

 =

 E
 F
 T
 P
 C

 [
 C
 ]

 {\displaystyle W_{c}={\frac {EFTPC}{[C]}}}

  ,

 N

 {\displaystyle N}

  is the total number of models in the population, and

 n
 R

 A

 c

 {\displaystyle nRA_{c}}

  represents the number of models showing repolarization abnormalities. [ 33 ]


More detailed computation simulations can be carried out accounting for not cellular models, but taking into consideration the functional  syncytium  and enabling the cells to mutually interact, the so-called electrotonic coupling. [ 34 ]


In case of tissue simulation or in wider cases, such as in whole organ simulations, all the cellular models are note applicable anymore, and several corrections have to be made. Firstly, the governing equations can not be just  ordinary differential equations , but a system of  partial differential equations  has to be accounted for. [ 35 ]  A suitable choice may be the monodomain model: [ 36 ]


▽
 ⋅
 (
 D
 ∇
 V
 )
 =
 (

 C

 m

 ∂
 V

 ∂
 t

 +

 I

 i
 o
 n

 (
 V
 ,
 u
 )
 )

 {\displaystyle \triangledown \cdot (D\nabla V)=(C_{m}{\frac {\partial V}{\partial t}}+I_{ion}(V,u))}

 i
 n

 {\displaystyle in}

 Ω

 {\displaystyle \Omega }


n
 ⋅
 (
 D
 ∇
 V
 )
 =
 0

 {\displaystyle n\cdot (D\nabla V)=0}

 i
 n

 {\displaystyle in}

 ∂
 Ω

 {\displaystyle \partial \Omega }


where

 D

 {\displaystyle D}

  is the effective conductivity tensor,

 C

 m

 {\displaystyle C_{m}}

 is the capacitance of the cellular membrane,

 I

 i
 o
 n

 {\displaystyle I_{ion}}

  the transmembrane ionic current,

 Ω

 {\displaystyle \Omega }

  and

 ∂
 Ω

 {\displaystyle \partial \Omega }

  are the domain of interest and its boundary, respectively, with

 n

 {\displaystyle n}

  the outward boundary of

 ∂
 Ω

 {\displaystyle \partial \Omega }

 . [ 36 ]


Photopharmacology is an emerging multidisciplinary field that combines  photochemistry  and  pharmacology . [ 1 ]  Built upon the ability of light to change the  pharmacokinetics  and  pharmacodynamics  of bioactive molecules, it aims at regulating the activity of drugs in vivo by using light. [ 2 ]  The light-based modulation is achieved by incorporating molecular photoswitches such as azobenzene and diarylethenes or photocages such as o-nitrobenzyl, coumarin, and BODIPY compounds into the pharmacophore. [ 3 ]  This selective activation of the biomolecules helps prevent or minimize off-target activity and systemic side effects. Moreover, light being the regulatory element offers additional advantages such as the ability to be delivered with high spatiotemporal precision, low to negligible toxicity, and the ability to be controlled both qualitatively and quantitatively by tuning its wavelength and intensity. [ 4 ]


Though photopharmacology is a relatively new field, the concept of using light in therapeutic applications came into practice a few decades ago.  Photodynamic therapy  (PDT) is a well-established clinically practiced protocol in which  photosensitizers  are used to produce singlet oxygen for destroying diseased or damaged cells or tissues. [ 2 ]   Optogenetics  is another method that relies on light for dynamically controlling biological functions especially brain and neural. [ 4 ]  Though this approach has proven useful as a research tool, its clinical implementation is limited by the requirement for genetic manipulation. Mainly, these two techniques laid the foundation for photopharmacology. Today, it is a rapidly evolving field with diverse applications in both basic research and clinical medicine which has the potential to overcome some of the challenges limiting the range of applications of the other light-guided therapies.


Figure 1.  Schematic representation of the mechanism of (a) photopharmacology (b) photodynamic therapy, and (c) optogenetics.


The discovery of natural photoreceptors such as  rhodopsins  in the eye inspired the biomedical and pharmacology research community to engineer light-sensitive proteins for therapeutic applications. [ 2 ]  The development of synthetic  photoswitchable  molecules is the most significant milestone in the history of light-delivery systems. Scientists are continuing with their efforts to explore new photoswitches and delivery strategies with enhanced performance to target different biological molecules such as ion channels, nucleic acid, and enzyme receptors. Photopharmacology research progressed from in vitro to in vivo studies in a significantly short period of time yielding promising results in both forms. Clinical trials are underway to assess the safety and efficacy of these photopharmacological therapies further and validate their potential as an innovative drug delivery approach.


Molecular  photoswitches  are utilized in the field of photopharmacology, where the energetics of a molecule can be reversibly controlled with light to achieve spatial and temporal resolution of a particular effect. Photoswitches may function by undergoing  photoisomerization  through which light is used to conformationally adapt a molecule to a biological site, or through an environmental effect where an external factor such as a solvent effect or hydrogen bonding can selectively allow or quench an emissive state within a molecule. To visualize photophysical processes, a useful depiction is the  Jablonski diagram . This is a diagram which depicts electronic and vibrational energy levels within a molecule as vertical levels and shows the possible relaxation pathways from excited states. Typically, the ground state is referred to as S 0 , and is drawn at the bottom of the figure with nearby vibrational excitations just above it. An absorption will promote an electron into the S 1  state at any vibrational energy level, or into a higher order excited state if the absorbed energy has enough magnitude. The excited state can then undergo internal conversion which is the electronic relaxation to a lower state with the same vibrational energetics or vibrational relaxation within a state. This may be followed by an intersystem crossing wherein the electron undergoes a spin flip, or a radiative or nonradiative decay back to the ground state. [ 5 ]


One example of an organic compound that undergoes photoisomerization is  azobenzene . The structure is two phenyl rings joined with a N=N double bond and is the simplest aryl azo compound. Azobenzene and its derivatives have two accessible absorbance bands: the S 1  state from a n-π* transition which can be excited into using blue light, and the S 2  state from a π-π* transition that can be excited into using ultraviolet light. [ 6 ]  Azobenzene and its derivatives have two isomers, trans and cis. The trans isomer, having the phenyl rings on opposite sides of the azo double bond, is the thermally preferred isomer as there is less stereoelectronic distortion and more delocalization present. However, excitation of the trans isomer to the S 2  state facilitates a shift to the cis isomer. The S 1  absorption is associated with a conversion back to the trans isomer. In this way, azobenzene and its derivatives can act as reversible stores of energy by maintaining a strained configuration in the cis isomer. Modifications of the substituents on azobenzene allow the energetics of these absorptions to be tuned, and if they are engineered such that the two absorption bands overlap a single wavelength of light can be used to flip between them. There are a number of similar photoswitches which isomerize between E and Z configurations across an azo group (for instance, azobenzene and azopyrazole) or an ethylene bridge (for instance, stilbene and hemithioindigo). [ 7 ]


Alternatively, photoswitches may themselves be emissive and exhibit environmental control over their properties. One such example is a class of ruthenium polypyridyl coordination complexes. Typically they contain two bidentate bipyridine or phenanthroline ligands and an extended phenanthroline-phenazine bidentate ligand such as dipyrido[3,2-a:2,3-c]phenazine (dppz). [ 8 ]  These complexes have an accessible metal to ligand charge transfer excited state ( 1 MLCT) which undergoes rapid intersystem crossing to a  3 MLCT state due to the strong spin-orbit coupling of the ruthenium center. These excited states are localized on the extended ligand phenazine nitrogens, and emission occurs from the  3 MLCT state. Hydrogen bonding interactions such as the presence of water around these nitrogen atoms stabilizes the  3 MLCT state, quenching the emission process. Thus, by controlling whether an aqueous or otherwise protic polar solvent is present, emissive behaviors can be “turned on/off”, and alternation between “bright states” and “dark states” is facilitated. This light switch behavior makes these and similar complexes of recent interest in photopharmacological applications such as photodynamic therapy.


As previously mentioned, photopharmacology relies on the use of molecular photoswitches being incorporated into the structure of biologically active molecules which allows their potency to be controlled optically. [ 7 ]  They are introduced into the structure of bioactive compounds via insertion, extension, or bioisosteric replacement. [ 7 ]  These incorporations can be supported by structural considerations of the molecule or SAR (structure-activity relationship) analysis to determine the optimal position. [ 7 ]  Some examples of photoswitchable molecules commonly used in photopharmacology are azobenzenes, diarylethenes, and photocages. [ 9 ]


Azobenzenes are a class of photoswitchable molecules and are used in photopharmalogical applications for their reversible photoisomerization, as described in the previous section. An example of a photoswitchable molecule that uses azobenzene is phototrexate.  Phototrexate  is an inhibitor of human dihydrofolate reductase and is an analogue of  methotrexate , a chemotherapy agent. [ 10 ]  When in its photoactive cis form, phototrexate has been shown to be a potent antifolate and is relatively inactive when in the trans form. [ 10 ]  The azologization, or incorporation of azobenzene, of methotrexate allows for control of cytotoxic activity and is considered a step forward in developing targeted anticancer drugs with localized efficacy. [ 10 ]


Diarylethene  photoswitches have reversible cyclization and cycloreversion reactions that are photoinduced. [ 11 ]  They are a class of compounds that have aromatic functional groups bonded to each end of a carbon-carbon double bond. An example of this class of molecule that is used in photopharmacology is  stilbene . Under the influence of light, stilbene switches between its two isomers (E and Z).


Figure 4. Figure showing stilbene isomerizations under light from E to Z.


Diarylethenes have been shown to have some advantages over the more researched azobenzenes switches, such as thermal irreversibility, high photoswitching efficiency, favorable cellular stability, and low toxicity. [ 11 ]  Diarylethenes have been shown to have promise in fields other than photopharmacology as well. These fields include optical data storage, optoelectronic devices, supramolecular self-assembly and anti-counterfeiting. [ 11 ]


A class of substances known as photocages contain “photosensitive groups, also known as 'photoremovable protecting groups", from which target substances are released upon exposure to specific wavelengths of light”. [ 12 ]  The photosensitive groups physically and chemically protect the target from being released until the molecule undergoes photoreaction. [ 12 ]  Due to these interactions with light, they are commonly used molecules in photopharmacology. More recently, they have played an important role in photoactivated  chemotherapy  (PACT). In PACT, photocages utilize a photoremovable protecting group that protects cytotoxic drugs until the bond is cleaved via light interaction and the cytotoxic drug is released. [ 13 ]  Some well-known photocages include “o-nitrobenzyl derivatives, coumarin derivatives, BODIPY, xanthene derivatives, quinone and diarylenes derivatives”. [ 12 ]  However, there are limitations with using photocages in clinical applications as there are not many PPGs that can be used in vivo. This is due to PPG-payload conjugates needing to have acceptable solubility and biological inertness for biocompatibility and the need for efficient uncaging above 600 nm. [ 13 ]


Figure 5. Example of a photocage release system activated by NIR. [ 14 ]


Photopharmacology, the use of light to control the activity of drugs, has emerged as a promising approach to drug delivery and therapy. By harnessing the power of light, researchers can achieve precise control over drug release and activation, offering new possibilities for targeted and personalized treatments. This subsection explores the application of photopharmacology in drug delivery, focusing on recent advancements and potential clinical applications.


In one study, [ 15 ]  researchers designed HDAC inhibitors which can be activated or deactivated with light, providing precise therapeutic control. This approach could reduce the side effects of traditional chemotherapy by targeting inhibitors to specific body areas, potentially leading to more effective and personalized cancer treatments.


In another study, [ 16 ]  the researchers developed a strategy to attach a photoswitchable group to a common antibiotic; ciprofloxacin. By attaching the photoswitchable group, researchers can control the activity of ciprofloxacin with light. This approach could potentially lead to new ways of treating bacterial infections, with the ability to switch the antibiotic's activity on and off as needed.
In this paper [ 17 ]  an in vitro protocol to test different light wavelengths on human cancer cell lines is developed, finding that blue light most effectively inhibited cell growth. This suggests that photopharmacology could offer new cancer treatment options by targeting specific light wavelengths to modulate drug activity in tumor cells.


Another application of photopharmacology [ 18 ]  is developing a luminescent photoCORM grafted on carboxymethyl chitosan, which, when exposed to light, releases carbon monoxide (CO) to induce apoptotic death in colorectal cancer cells, demonstrating precise control over CO release for targeted cancer therapy.


Researchers developed a toolbox of photoswitchable antagonists that can interact with GPCRs, a class of proteins involved in various cellular processes. [ 19 ]  By using light to switch the activity of these antagonists, researchers can control the interaction between the antagonists and GPCRs in real time. This approach allows for precise modulation of GPCR activity, which could lead to new insights into cellular signaling pathways and potential therapeutic applications.


In another application [ 20 ]  by using light to control the assembly of nanopores, researchers can potentially regulate the flow of ions or molecules through these nanopores. This approach could have applications in various fields, including sensing, drug delivery, and nanotechnology.


Another paper [ 21 ]  reports on the use of photopharmacology to control drug activity; multifunctional fibers in the study deliver light and drugs to specific body areas. Implanted fibers activate light-responsive drugs, altering their structure, and offering precise drug delivery for conditions needing exact timing or dosage.


In another study, [ 22 ]  ligands were designed to switch their binding mode to G-quadruplex DNA upon exposure to visible light. This method could potentially modulate the activity of G-quadruplex DNA, crucial in gene expression and telomere maintenance, offering new therapeutic avenues, particularly in cancer treatment. The study underscores photopharmacology's promise in targeting specific DNA structures, suggesting G-quadruplex DNA as a viable target for future photopharmacological interventions.


Another study [ 23 ]  developed photoactivatable antibody-photoCORM conjugates targeting human ovarian cancer cells, releasing CO upon light exposure to diminish cell viability. This approach offers precise cancer cell targeting while minimizing harm to healthy tissue, showcasing the potential of photopharmacology in cancer therapy.


In another paper, [ 24 ]  a photoactivatable compound that binds to and modulates the activity of the CRY1 protein, regulating the mammalian circadian clock, was developed. By using light to control the compound's activity, researchers can potentially treat circadian rhythm disorders and related health conditions by modulating the function of CRY1. Photopharmacology involves using light to control the activity of drugs.


In another application [ 25 ]  researchers used photopharmacology to control drug release and focus on a drug interacting with tubulin, visualizing its release in real time with time-resolved serial crystallography. This technique offers insights into drug-tubulin interactions and demonstrates the potential for designing drugs with precise actions.


The future of photopharmacology holds immense promise. It has the potential to revolutionize conventional drug therapy offering new avenues for precision medicine, treating neurological disorders, and in the field of  oncology  and  ophthalmology . [ 1 ]  Additionally, it holds promise for the field of  regenerative medicine  where photoswitches can be used to modulate the activity of signaling pathways for targeted tissue repair and regeneration. [ 3 ]


Photopharmacology will continue to grow and expand with the new discoveries and advances happening in other related fields such as synthetic chemistry, biology, nanotechnology, pharmacology, and bioengineering. While the potential of photopharmacology is vast, some challenges must be addressed to make it a clinical reality. One challenge is the development of stable and biocompatible photoswitches that are selective for their target receptors without cross-activity. [ 2 ]  It is particularly important that these photoswitchables have their absorbance bands fall within the wavelength range of 650 nm to 900 nm. [ 2 ]  Hence, optimum molecular designing of photoswitches is required to achieve the characteristics mentioned above and desired level of performance. At present, photopharmacology uses a rational drug design approach based on studying the structure-activity relationship, however, a phenotypic screening for photoswitchable drugs could also be beneficial.


In order to achieve good spatial-temporal control over drug activity there should be a significant difference between the activity of isomers. However, understanding the structural changes during the biological effects induced by photoswitching is limited. This scarcity of knowledge is also a challenge for the growth of this field, as it hampers the optimization of the activity and potency of the isomers to obtain the expected outcomes during applications. [ 3 ]


The biggest challenge in photopharmacology is finding appropriate and effective ways to deliver light to deep tissues in the body and tissues avoiding issues such as scattering and absorption. Various strategies have been attempted in this regard, one being the development of photoswitchable ligands that respond to deep-tissue penetrating wavelengths like red or infrared light. Moreover, some recent preclinical studies have spurred the development of wireless, compact or injectable, and remotely controllable devices capable of delivering light to neural tissues with minimal damage. [ 26 ]  There are novel optofluidic systems that can simultaneously regulate both drug delivery and light activity at specific sites. Although external delivery of light is the most preferred method, the use of internal exogenous light sources such as luminescent compounds where light would be delivered directly at the site of action. This could avoid the issues related to light penetration and also enhance the degree of selectivity. In addition, this creates the opportunity to use photopharmacology as a theranostic approach that combines targeted drug delivery and molecular imaging. [ 2 ]


In  pharmacology ,  pleiotropy  includes all of a drug's actions other than those for which the agent was specifically developed. [ 1 ]  It may include  adverse effects  which are detrimental ones, [ 1 ]  but is often used to denote additional beneficial effects. [ 2 ]


For example,  statins  are  HMG-CoA reductase  inhibitors that primarily act by decreasing cholesterol synthesis, but which are believed to have other beneficial effects, including acting as  antioxidants  and stabilizing  atherosclerotic plaques . [ 1 ]  Steroid drugs, such as  prednisone  and  prednisolone , have pleiotropic effects, including  systemic  ones, for the same reason that endogenous  steroid hormones  do: cells throughout the body have receptors that can respond to them, because the endogenous ones are  endocrine  messengers.


Another example is melatonin, which has a wide range of effects on biological systems on multiple scales, from modulating the circadian rhythm and inducing sleep via the activation of melatoninergic receptors, to recepto-independent antioxydative and anti-inflammatory effects over all organs down to cells. [ 3 ] [ 4 ]


Polymer-protein hybrids  are a class of  nanostructure  composed of  protein - polymer  conjugates (i.e. complexes composed of one protein attached to one or more  polymer chains ). [ 1 ] [ 2 ]  The protein component generally gives the advantages of biocompatibility and  biodegradability , as many proteins are produced naturally by the body and are therefore well tolerated and metabolized. [ 3 ]  Although proteins are used as targeted therapy drugs, the main limitations—the lack of stability and insufficient circulation times still  remain. [ 4 ]  Therefore, protein-polymer conjugates have been investigated to further enhance pharmacologic behavior and stability. [ 5 ]  By adjusting the chemical structure of the protein-polymer conjugates, polymer-protein particles with unique structures and functions, such as stimulus responsiveness, enrichment in specific tissue types, and enzyme activity, [ 6 ]  can be synthesized. Polymer-protein particles have been the focus of much research recently because they possess potential uses including bioseparations, imaging, biosensing, gene and drug delivery. [ 7 ]


Attaching a single polymer chain to a specific site away from the active center of the protein has less impact on protein activity compared with random attachments. [ 8 ] [ 9 ]  In practice, attaching a single polymer chain can be used to adjust chemical properties of the therapeutic protein. For example, conjugation of a single chain of the hydrophilic polyethylene glycol (PEG) can increase the hydrodynamic radius of the protein conjugate by 5-10 fold. [ 10 ]  Attachment to PEG was mainly achieved by covalent conjugation via the grafting to strategy, targeting chemo-selective anchor groups. Other polymers, such as oligosaccharides and polypeptides, offer different properties to the enzymes attached to them.


Researchers conjugated the thermo-responsive polymer poly(N-isopropylacrylamide) (pNIPAm) with the biotin-recognizing protein streptavidin close to its recognition site. [ 11 ]  At temperatures above the lower critical solution temperature (LCST), the polymer collapses and blocks the binding site, thus reversibly preventing biotin from binding to streptavidin. By copolymerization with two different thermosensitive polymers  poly(sulfobetaine methacrylamide)  (pSBAm) and pNIPAm together, researchers can control enzyme activity in a small temperature window. [ 12 ]


((N,N'-dimethylacrylamide)-co-4-phenylazophenyl acrylate) at the active site of endoglycanase creates a photoswitchable protein hybrid. [ 13 ]  The resulting hybrid catalyzes the hydrolysis of glycoside when irradiated by 350 nm UV light, but turns inactive under 420 nm visible light depending on the conformation of the conjugated polymer. [ 4 ]


A polymer shell is formed by conjugation of multiple molecules of polymers onto the protein core. The polymer shell can either protect the protein core from unwanted degradation or create desired interactive sites for guest molecules. The first generation of polymer shell protein core structures mainly used of Polyethylene glycol (PEG) chains to increase the hydrodynamic radius and reduce immune response to proteins. [ 14 ]  However, the PEG shell can reduce protein activity in the inner core. More advanced designs use biodegradable linkers to achieve programmed release of the protein core in specific tissues. Several therapeutic designs with biodegradable PEG shells are already being developed  in vivo . [ 15 ] [ 16 ] [ 17 ]


Direct conjugation of polymers ("grafting to" strategy) can efficiently construct a polymer shell with diverse polymer types, however, it has low polymer density, especially with large polymers. In contrast, "grafting from" strategy allows the formation of a dense and uniform polymer shell. The protein core can also function as a carrier for other therapeutic molecules, such as plasmid DNA. [ 18 ]


Dendrite polymer shells have a high volume to molecular weight ratio compared with traditional polymer shells. Using branched carbohydrates can give unique biological properties while maintaining molecular definition. [ 19 ] [ 20 ]


Although covalent conjugation has been the dominant strategy for constructing polymer-protein hybrids, noncovalent chemistry can add another level of complexity and provides the opportunity to create higher-ordered structures. Specifically, self-assembly by non-covalent interactions is progressing rapidly. [ 21 ] [ 22 ] [ 23 ]  Supra molecular self-assembly  can create  nanoparticles , vesicles/micelles, protein cages, etc. Metal-binding interactions, host-guest, and boronic acid-based chemistries are widely studied as non-covalent conjugation methods to create polymer-protein hybrids. [ 24 ] [ 25 ] [ 26 ]


Streptavidin is a protein purified from the bacterium  Streptomyces avidinii , which has a high affinity for biotin. By covalently linking streptavidin and polymers, well defined supramolecular constructs can be created due to the high specificity of


Streptavidin for both biotin and its analogues. [ 27 ]


Building upon the covalent core shell strategy, several polymer–streptavidin systems have been developed for affinity separation, bio-sensors and diagnostic applications due to the robust binding conditions and stability of the protein. [ 28 ]


Streptavidin can be used as a macro-initiator for  in situ ATRP , through grafting from strategy, a stoichiometrically well defined polymer-protein conjugate can be synthesized. Polymer streptavidin systems can also be empowered to cross the cellular membrane by conjugating with cell penetrating molecules such as peptides and membrane disturbing polymers.


Polymer streptavidin systems can also be modulated to respond to certain environmental changes such as pH. By incorporating pH responsive poly(propylacrylic acid) (PPAAc) into the system, tumor cell suppressor p53 and cytochrome C can be delivered into cancer cells efficiently. [ 29 ]


For biomolecules that are not hampered by the biotin-streptavidin interaction, iminobiotin, an analogue of biotin, has been applied as a pH-sensitive linker that allows the controlled and reversible assembly and intracellular release of cargo molecules in acidic intracellular compartments. [ 30 ]


Polymer-protein conjugates can also form a higher ordered supramolecular structure via self-assembly of amphiphilic polymers into micelles and microcapsules, which is one of the most promising strategies to generate drug delivery systems. Such systems have the innate advantage of rapid preparation, a high drug loading capacity, ease of surface decoration, and the potential to be stimuli responsive.


Micelles refers to a type of supramolecular structure consisting of amphiphilic molecules self-assemblies, usually hollow centered. Researchers successfully conjugated a diblock copolymer site specifically onto GFP, the resulting amphiphilic polymer-protein conjugate is capable of reversible self-assembly into micelles. [ 31 ]


In addition to retaining the native globular shape of proteins, the polypeptide backbone of denatured proteins can also be utilized to be conjugated with hydrophilic polymer chains to generate higher ordered structure through hydrophobic interactions. For example, nanoconjugates of poly-ethylene glycol(PEG) and denatured bovine serum albumin(BSA) will spontaneously self-assemble into a micellar structure, whose protein core can adsorb high numbers of hydrophobic drugs. [ 32 ]


An efficient way to synthesize protein-polymer hybrid nanoparticles is to take advantage of photoinitiated reversible addition−fragmentation chain transfer (RAFT) polymerization-induced self-assembly(PISA) by using multi-RAFT modified bovine serum albumin (BSA) as a macromolecular chain transfer agent. RAFT mediated growth of the PHPMA chains will graft from the BSA-RAFT, and increase the hydrophobicity of the star BSA−PHPMA conjugates.  At the critical aggregation concentration, they form nanoparticles due to the hydrophobic interactions. [ 33 ]  The resulting nanoparticles show excellent encapsulation capability for both hydrophobic and hydrophilic molecules, such as cancer drugs and DNA.


A rather easy method to prepare protein-polymer hybrid nanoparticles is nanoprecipitation. Spherical nanoparticles composed of BSA-PMMA with diameters of around 100 nm were obtained and the water insoluble chemotherapeutic drug camptothecin was encapsulated within the hydrophobic core consisting of PMMA. [ 6 ]  Such protein-polymer hybrid nanoparticles possess tunable sizes and surface charges, have attractive bio-compatibilities and allow efficient cell uptake. Camptothecin-encapsulated BSA-PMMA nanoparticles revealed enhanced anti-tumor activity both in vitro and in animals.