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Cyclic peptides have the potential to bind to challenging targets, which are undruggable with small molecules, but their application is limited by low membrane permeability. Here, using a series of cyclic pentapeptides, we showed that established physicochemical criteria of permeable peptides are heavily violated. We revealed that a dominant core conformation, stabilized by amides' shielding pattern, could guide the design of novel compounds. As a result, counter-intuitive strategies, such as incorporation of polar residues, can be beneficial for permeability. We further find that core globularity is a promising descriptor, which can extend the capability of standard predictive models.

Strategies of solubility enhancement and perspectives in solubility measurements of pharmaceutical compounds

Ten years ago, we issued an open prediction challenge to the cheminformatics community: would participants be able to predict the equilibrium intrinsic solubilities of 32 druglike molecules using only a high-precision (CheqSol instrument, performed in one laboratory) set of 100 compounds as a training set? The "solubility challenge" was a widely recognized success and spurred many discussions about the prediction methods and quality of data. We revisited the competition a second time recently and challenged the community to a different challenge, not a blind test this time but using a larger test set of molecules, gathered and curated from published sources (mostly "gold standard" saturation shake-flask measurements), where the average interlaboratory reproducibility for the molecules was estimated to be ∼0.17 log unit. Also, a second test set was included, comprising "contentious" molecules, the reported (mostly shake-flask) solubility of which had higher average uncertainty, ∼0.62 log unit. In the second competition, the participants were invited to use their own training sets, provided that the training sets did not contain any of the test set molecules. We were motivated to revisit the competition to (1) examine to what extent computational methods had improved in 10 years, (2) verify that data quality may not be the main limiting factor in the accuracy of the prediction method, and (3) attempt to seek a relationship between the makeup of the training set data and the prediction outcome.

Muscle atrophy and cachexia are common comorbidities among patients suffering from cancer, chronic obstructive pulmonary disease, and several other chronic diseases. The peptide hormone ghrelin exerts pleiotropic effects including the stimulation of growth hormone secretion and subsequent increase of insulin-like growth factor-1 levels, an important mediator of muscle growth and repair. Ghrelin also acts on inflammation, appetite, and adipogenesis and therefore has been considered a promising therapeutic target for catabolic conditions. We previously reported on the synthesis and properties of an indane based series of ghrelin receptor full agonists which led to a sustained increase of insulin-like growth factor-1 in a dog pharmacodynamic study. Herein we report on the identification of a series of pyrrolidine or piperidine based full agonists and attempted optimization to give compounds with profiles suitable for progression as clinical candidates.

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.

Sildenafil, the active ingredient of the drug developed by Pfizer for the treatment of erectile dysfunction was firstly synthesized in 1989 in the United Kingdom and since then it has become one of the most prescribed drugs for sexual performance in the western world with more than 2.7 million prescriptions in the US in 2021. Since its discovery, this drug compound has attracted the interest of formulators and crystallographers, with a high number of crystal forms of sildenafil being found and characterized, including polymorphs, hydrates, solvates, salts and cocrystals, converting it in one of the most promiscuous multicomponent crystal former drugs in the pharmaceutical sciences arena. In this minireview, the polymorph, pseudopolymorph and multicomponent solid forms landscape of sildenafil is presented through a comprehensive compilation of their 42 solid forms reported in literature.

Ten years ago we issued, in conjunction with the Journal of Chemical Information and Modeling, an open prediction challenge to the cheminformatics community. Would they be able to predict the intrinsic solubilities of 32 druglike compounds using only a high-precision set of 100 compounds as a training set? The "Solubility Challenge" was a widely recognized success and spurred many discussions about the prediction methods and quality of data. Regardless of the obvious limitations of the challenge, the conclusions were somewhat unexpected. Despite contestants employing the entire spectrum of approaches available then to predict aqueous solubility and disposing of an extremely tight data set, it was not possible to identify the best methods at predicting aqueous solubility, a variety of methods and combinations all performed equally well (or badly). Several authors have suggested since then that it is not the poor quality of the solubility data which limits the accuracy of the predictions, but the deficient methods used. Now, ten years after the original Solubility Challenge, we revisit it and challenge the community to a new test with a much larger database with estimates of interlaboratory reproducibility.

The potential to predict Solvation Free Energies (SFEs) in any solvent using a machine learning (ML) model based on thermodynamic output, extracted from 3D-RISM simulations in water is investigated. The models on multiple solvents take into account both the solute and solvent description and offer the possibility to predict SFEs of any solute in any solvent with root mean squared errors less than 1 kcal/mol. Validations that involve exclusion of fractions or clusters of the solutes or solvents exemplify the model’s capability to predict SFEs of novel solutes and solvents with diverse chemical profiles. In addition to being predictive, our models can identify the solute and solvent features that influence SFE predictions. Furthermore, using 3D-RISM hydration thermodynamic output to predict SFEs in any organic solvent reduces the need to run 3D-RISM simulations in all these solvents. Altogether, our multi-solvent models for SFE predictions that take advantage of the solvation effects are expected to have an impact in the property prediction space.

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorab...

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

Cocrystals have been identified as a method for ensuring the delivery of poorly soluble drugs. Development of cocrystal phases presents many challenges, with the in vivo drug delivery benefits difficult to predict robustly. Additional to this, many of the benefits seen in the literature do not use formulation strategies which are representative of the clinical dosage form. Zafirlukast has been shown to possess poor development properties. It is poorly soluble and difficult to manufacture. Here we present a case study highlighting the benefits of cocrystals in this context, outlining the discovery, formulation and improved pharmacokinetics of mixed stoichiometry cocrystals of zafirlukast. This case study outlines methods, which could be broadly applied to other drug molecules, for determination of the properties of cocrystals through a suite of in vitro experiments that display a predictive pathway from physical form discovery to enhanced in vivo activity in animal models.

[Image: see text] The further optimization of a recently disclosed series of inverse agonists of the nuclear receptor RORC2 is described. Investigations into the left-hand side of compound 1, guided by X-ray crystal structures, led to the substitution of the 4-aryl-thiophenyl residue with the hexafluoro-2-phenyl-propan-2-ol moiety. This change resulted in to compound 28, which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement in vivo was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.

The discovery and detailed characterization of several new solid forms of a novel selective inhaled JAK-STAT inhibitor are described. Using a holistic cocrystallization screen approach to explore its formulation landscape, we decrease the risk of future potential development failures due to a nonoptimal pharmacokinetic lung profile or undesired lung effects in humans.

This commentary addresses data quality in equilibrium solubility measurement in aqueous solution. Broadly discussed is the “gold standard” shake-flask (SF) method used to measure equilibrium solubility of ionizable drug-like molecules as a function of pH. Many factors affecting the quality of the measurement are recognized. Case studies illustrating the analysis of both solution and solid state aspects of solubility measurement are presented. Coverage includes drug aggregation in solution (sub-micellar, micellar, complexation), use of mass spectrometry to assess aggregation in saturated solutions, solid state characterization (salts, polymorphs, cocrystals, polymorph creation by potentiometric method), solubility type (water, buffer, intrinsic), temperature, ionic strength, pH measurement, buffer issues, critical knowledge of the pKa, equilibration time (stirring and sedimentation), separating solid from saturated solution, solution handling and adsorption to untreated surfaces, solubility units, and tabulation/graphic presentation of reported data. The goal is to present cohesive recommendations that could lead to better assay design, to result in improved quality of measurements, and to impart a deeper understanding of the underlying solution chemistry in suspensions of drug solids.

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) of a compound is dependent on physicochemical properties such as molecular size, lipophilicity, and ionization state. However, much less is known regarding the relationship between ADMET and the molecular topology. In this study two descriptors related to the molecular topology have been investigated, the fraction of the molecular framework (f(MF)) and the fraction of sp(3)-hybridized carbon atoms (Fsp(3)). f(MF) and Fsp(3), together with standard physicochemical properties (molecular size, ionization state, and lipophilicity), were analyzed for a set of ADMET assays. It is shown that aqueous solubility, Caco-2 permeability, plasma protein binding, human ether-a-go-go-related potassium channel protein inhibition, and CYP3A4 (CYP = cytochrome P450) inhibition are influenced by the molecular topology. These findings are in most cases independent of the already well-established relationships between the properties and molecular size, lipophilicity, and ionization state.

Chemical evolution of a HTS-based fragment hit resulted in the identification of N -(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca v 3.2) inhibitor with in vivo antihypertensive effect in rats.

Solubility is a key physicochemical property of molecules. Serious deficiencies exist in the consistency and reliability of solubility data in the literature. The accurate prediction of solubility would be very useful. However, systematic errors and lack of metadata associated with measurements greatly reduce the confidence in current models. To address this, we are accurately measuring intrinsic solubility values, and here we report results for a diverse set of 100 druglike molecules at 25 degrees C and an ionic strength of 0.15 M using the CheqSol approach. This is a highly reproducible potentiometric technique that ensures the thermodynamic equilibrium is reached rapidly. Results with a coefficient of variation higher than 4% were rejected. In addition, the Potentiometric Cycling for Polymorph Creation method, [PC] (2), was used to obtain multiple polymorph forms from aqueous solution. We now challenge researchers to predict the intrinsic solubility of 32 other druglike molecules that have been measured but are yet to be published.

The appearance and disappearance of polymorphs is no longer a mysterious and inexplicable process. Although methods for polymorph control are still imperfect, there is a large armoury of methods that can be used to tackle this important and challenging problem. We survey the methods and their successes over the last few years.

The Potentiometric Cycling for Polymorph Creation [PC]2 has been applied to sparfloxacin, a third-generation fluoroquinolone antibiotic. Two different trihydrate phases precipitate from aqueous solution. X-ray data indicate that one of these is a previously unknown polymorph of sparfloxacin trihydrate. Because both forms crystallize from solution at the same time, the two crystalline forms are concomitant polymorphs that precipitate in a thermodynamically controlled ratio.

The solubility in water of diclofenac ({2-[(2,6-dichlorophenyl)amino]phenyl}acetic acid), a potent nonsteroidal anti-inflammatory drug, has been investigated. The various solid forms have been characterized by thermogravimetric analysis, differential scanning calorimetry, and X-ray diffraction. The commercially available form of diclofenac is the anhydrous sodium salt. This was recrystallized from ethanol and precipitated as a hydrate containing four diclofenac anions, four sodium cations, and nineteen water molecules per unit cell. This crystal structure is similar to but different from an earlier report of the structure. Crystals of the acid form of diclofenac were anhydrous and corresponded to an earlier crystal structure. Separate solubility measurements on all three of these solid forms of diclofenac gave consistent results for the intrinsic solubility. The aqueous solubility values reported in the literature for diclofenac are spread over a large range, with a factor of 100 separating the largest and the smallest. Our value is at the smaller end of this range. It is the only one supported by three independent procedures and rigorous characterization of the solid forms. The experimental conditions were precisely controlled.

We report a method to predict physico-chemical properties of druglike molecules using a classical statistical mechanics based solvent model combined with machine learning. The RISM-MOL-INF method introduced here provides an accurate technique to characterize solvation and desolvation processes based on solute-solvent correlation functions computed by the 1D Reference Interaction Site Model of the Integral Equation Theory of Molecular Liquids. These functions can be obtained in a matter of minutes for most small organic and druglike molecules using existing software (RISM-MOL) (Sergiievskyi, V. P.; Hackbusch, W.; Fedorov, M. V. J. Comput. Chem. 2011, 32, 1982-1992.). Predictions of caco-2 cell permeability and hydration free energy obtained using the RISM-MOL-INF method are shown to be more accurate than the state-of-the-art tools for benchmark datasets. Due to the importance of solvation and desolvation effects in biological systems, it is anticipated that the RISM-MOL-INF approach will find many applications in biophysical and biomedical property prediction.

Two new cocrystals of zafirlukast with piperazine, existing in five different solid forms, have been discovered during a cocrystal screening. The crystal structure of one of these forms has been determined by single crystal X-ray diffraction, and the stability landscape of the crystalline forms of the new cocrystal has been studied. In the present article, we extend the knowledge about the solid state of this important pharmaceutical drug for the treatment of asthma by reporting the crystal structures of two new solvates (acetonitrile and butanol) and the elusive anhydrous Form X, which have been solved by single crystal X-ray diffraction.

The title compound (systematic name: {5-[(7-chloroquinolinium-4-yl)amino]-2-hydroxy­benzyl}dimethylammonium dichloride dihydrate), C20H24ClN3O2+·2Cl−·2H2O, has one amodiaquinium dication, two Cl− anions and two water mol­ecules in the asymmetric unit. The crystal structure was solved by simulated annealing from laboratory X-ray powder diffraction data, with data collected at room temperature. Rietveld refinement of this model led to a final Rwp of 0.047 to 1.79 A resolution. A three-dimensional network of hydrogen bonding links the amodiaquinium cations via water mol­ecules and Cl− ions.

A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located.

The rates of thiolysis of cephalosporins were investigated by high-performance liquid chromatography and 1 H NMR spectroscopy. Thiols catalyse the hydrolysis through the formation of a thioester intermediate and the catalytically reactive form of the thiol is the thiolate anion. Variation of nucleophilic reactivity by changing the basicity of the thiolate anion generates a Bronstednuc value of 1.22 with cephaloridine, indicating that the breakdown of the tetrahedral intermediate is the rate-limiting step. The effect of C3 0 substituents on the rate of thiolysis of cephalosporins generates a large Hammettof ca 12, which is compatible with C—N bond fission occurring without protonation of the � -lactam nitrogen. Solvent kinetic isotope effects kH2O=kD2O of ca 1.1 also indicate that solvent water probably does not act as a general acid catalyst facilitating breakdown of the tetrahedral intermediate by protonating the departing amine. Copyright # 2004 John Wiley & Sons, Ltd.

The rate of aminolysis of benzylpenicillin and cephaloridine by hydroxylamine, unlike other amines, shows only a first order dependence on amine concentration. The rate enhancement compared with that predicted from a Bronsted plot for other primary amines with benzylpenicillin is greater than 10(6). This is much more than an alpha-effect and is compatible with rate-limiting formation of the tetrahedral intermediate due to a rapid intramolecular general acid catalysed breakdown of the intermediate. For cephaloridine, the rate enhancement is greater than 10(4) which demonstrates that beta-lactam C-N bond fission and expulsion of the leaving group at C3' are not concerted.

The thiolysis mechanism of benzylpenicillin has been determined by 1 H NMR and HPLC. The degradation of benzylpenicillin was accelerated when the thiol used presented a β-group capable of acting as a general acid catalyst, such as α-monothioglycerol, 2-mercaptoethanol and mercaptoethylamine. With these thiols, after the formation of the thioester, an intramolecular acyl transfer reaction occurs at a pH far below the pKa of the group acting as a general acid catalyst, which shows that the proton transfer has already occurred, probably in a concerted mechanism with the nucleophilic attack. Rate constants were calculated. The system can be taken as a simple model of the general acid catalyst in serine and cysteine proteases.

Polymorphism of drugs has been the subject of intense interest in the pharmaceutical industry for over forty years. Although identical in chemical composition, polymorphs differ in bioavailability, solubility, dissolution rate, chemical and physical stability, melting point, colour, filterability, density, flow properties, and many other properties. The difference in solubility is particularly important for pharmaceuticals, as it can affect drug efficacy, bioavailability and safety. Despite significant investment in processes to find all the possible polymorphs of active pharmaceutical ingredients (APIs), new polymorphs can suddenly appear without warning. Polymorphs tend to convert spontaneously from less stable to more stable forms, and, therefore, it is best to discover and characterize the stable form as early as possible. Ideally the most stable polymorph will be found while the drug candidate is still in the discovery process, so that this is the form used for subsequent testing. The most stable polymorph will be the least soluble and solubility may be a limiting factor in the efficacy of the API. Despite the huge importance of polymorphism in the properties of materials, however, there is no method that can produce all the stable polymorphs of a compound, or even one that can provide confidence that the most stable polymorph has been obtained. Here we describe a new method, `potentiometric cycling for polymorph creation (PC)2', which is able to generate the most stable polymorph in aqueous solution. This new method has been applied to sulindac, a non-steroidal anti-inflammatory drug, which also shows promise in anticancer treatment, producing two polymorphs of this API, including a new more stable one. By adjusting the conditions, this method is able to produce either polymorph exclusively.

The rate of degradation of 6-epi-ampicillin in acidic, neutral, and alkaline aqueous solutions was followed at 35 °C and an ionic strength of 0.5 mol dm-3 (KCl) by high-performance liquid chromatography (HPLC) and spectrophotometric assays. Pseudo-first-order rate constants were determined in a variety of buffer solutions, and the overall pH−rate profile was obtained by extrapolation to zero buffer concentration. The hydrolysis of 6-epi-ampicillin is subject to acid and hydroxide-ion catalysis and, for a penicillin, an unusual pH-independent reaction. Intramolecular general base-catalyzed hydrolysis by the side chain amido group is proposed to explain the enhanced rate of neutral hydrolysis of 6-epi-ampicillin and cephalosporins. The β-lactam of 6-epi-ampicillin also undergoes intramolecular aminolysis by nucleophilic attack of the 6-α side chain amino group to give a stable piperazine-2,5-dione derivative. The low effective molarity for intramolecular aminolysis of only 40 M is partly attributed to the u...

The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.

Oxadiazoles are five-membered heteroaromatic rings containing two carbons, two nitrogens, and one oxygen atom, and they exist in different regioisomeric forms. Oxadiazoles are frequently occurring motifs in druglike molecules, and they are often used with the intention of being bioisosteric replacements for ester and amide functionalities. The current study presents a systematic comparison of 1,2,4- and 1,3,4-oxadiazole matched pairs in the AstraZeneca compound collection. In virtually all cases, the 1,3,4-oxadiazole isomer shows an order of magnitude lower lipophilicity (log D), as compared to its isomeric partner. Significant differences are also observed with respect to metabolic stability, hERG inhibition, and aqueous solubility, favoring the 1,3,4-oxadiazole isomers. The difference in profile between the 1,2,4 and 1,3,4 regioisomers can be rationalized by their intrinsically different charge distributions (e.g., dipole moments). To facilitate the use of these heteroaromatic rings, novel synthetic routes for ready access of a broad spectrum of 1,3,4-oxadiazoles, under mild conditions, are described.

Solubility is a key physicochemical property of molecules. Serious deficiencies exist in the consistency and reliability of solubility data in the literature. The accurate prediction of solubility would be very useful. However, systematic errors and lack of metadata associated with measurements greatly reduce the confidence in current models. To address this, we are accurately measuring intrinsic solubility values, and here we report results for a diverse set of 100 druglike molecules at 25 °C and an ionic strength of 0.15 M using the CheqSol approach. This is a highly reproducible potentiometric technique that ensures the thermodynamic equilibrium is reached rapidly. Results with a coefficient of variation higher than 4% were rejected. In addition, the Potentiometric Cycling for Polymorph Creation method, [PC]2, was used to obtain multiple polymorph forms from aqueous solution. We now challenge researchers to predict the intrinsic solubility of 32 other druglike molecules that have been measured but are ...

The resistance of bacteria to the normally lethal action of beta-lactam antibiotics is largely due to the production of beta-lactamases that catalyze the hydrolysis of the beta-lactam. One class of these enzymes is a zinc-dependent metallo-beta-lactamase for which there are no clinically available inhibitors. The hydrolysis of cephalosporin beta-lactam antibiotics generates dihydrothiazines which subsequently undergo isomerization at C6 by C-S bond cleavage and through the intermediacy of a thiol. These thiols can be trapped by the beta-lactamase from Bacillus cereus, causing inhibition of the enzyme. The rate of production of the thiol corresponds to the rate of inhibition, and the inhibition constants are in the micromolar range but vary with the nature of the cephalosporin derivative. NMR studies have identified the structure of the thiols causing inhibition and also show that the thiol binds to the zinc ion, which in turn perturbs the metal-bound histidines. Inhibition is slowly removed as the thiol becomes oxidized or undergoes further degradation. The thiol intermediate generated from cephalothin is a slow binding inhibitor. There is no observed inhibition from the analogous degradation products from penicillins.

Nerve-growth factor (NGF), a member of the neurotrophin family, plays an important role in nociceptor function. Prompted by a previous uinexpected finding that NT-4/5, as well as NGF sensitizes single nociceptors to noxious heat, we have explored the relative potency of all neurotrophins in eliciting thermal hyperalgesia. NGF, brain-derived neurotrophic factor (BDNF), NT-4/5 and NT-3 were injected locally into the hind paw of rats, and the behavioral response to noxious heat was compared with that from the other paw that received an identical injection of vehicle. Like NGF, agonists of tyrosine kinaseB (trkB) receptors (NT-4/5 and BDN F) induced thermal hyperalgesia in the first 5 h after treatment (NT-4/5 > BDNF) but the effect had worn off by 24 h. In contrast, the trkC agonist NT-3 had no effect on the response to noxious heat. Electrophysiological recordings from single C-fibres in the in vitro skin-saphenous nerve preparation revealed sensitization to noxious heat stimuli after direct application of BDNF to the receptive field, as previously noted for NT-4/5, and in parallel with the behavioral findings. NT-3 was ineffective as in the behavioral studies. These results suggest that trkB agonists BDNF and NT-4/5 as well as the trkA agonist NGF can regulate nociceptive responses to noxious heat.

Thiolysis of benzylpenicillin has been investigated by HPLC and 1H-NMR techniques. Thiols catalyse the hydrolysis of benzylpenicillin through the formation of a thioester intermediate. The catalytically reactive form of the thiol has been demonstrated to be the thiolate anion. Variation of reactivity with changing basicity of the thiolate anion generates a Bronsted βnuc value of 0.96, indicating that the breakdown of the tetrahedral intermediate is the rate-limiting step, as occurs in aminolysis and alcoholysis. Solvent kinetic isotope effects of 2.2–2.4 indicate that the solvent, water, probably acts as a general acid catalyst in the breakdown of the tetrahedral intermediate. PM3 theoretical calculations support the proposal that breakdown of the tetrahedral intermediate is rate-limiting. The experimental activation energies for the thiolysis of benzylpenicillin vary from 6.9 to 10.4 kcal mol−1.