Evaluation of two carrier protein-angiotensin I conjugate vaccines to assess their future potential to control high blood pressure (hypertension) in man.

AIMS: We aim to modulate the renin-angiotensin system (RAS) by active immunization against angiotensin I hormone (AI), potentially providing a novel conjugate vaccine treatment for hypertension in man. METHODS: Immunization studies in rat and human subjects compare the effectiveness of tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) vaccines for immunotherapy following conjugation with an AI peptide analogue (AI). Cardiovascular responses were assessed in immunized rats and human subjects (two-dose trial only), following increasing i.v. infusions of either AI or angiotensin II hormone (AII). RESULTS: The AI-TT and AI-KLH conjugate vaccines induced an equivalent immune response, and inhibition of the pressor effects to exogenous AI in rats. Single-dose clinical trials with both conjugate vaccines only resulted in an immune response to the KLH carrier protein. A two-dose clinical trial of AI-KLH conjugate vaccine resulted in a significant immune response to AI. A shift in diastolic blood pressure (DBP) dose-response was demonstrated following challenge with AI and AII for the study volunteer showing the largest anti-AI IgG induction. CONCLUSION: KLH was shown to be a suitable alternative to TT as a carrier protein for AI, thus supporting continued evaluation of our AI-KLH conjugate vaccine for treatment of hypertension in man.

Active immunization with angiotensin I peptide analogue vaccines selectively reduces the pressor effects of exogenous angiotensin I in conscious rats.

1. Male, Sprague-Dawley rats were actively immunized with novel angiotensin vaccines, and their pressor responses to exogenous angiotensin I (AI) and angiotensin II (AII) were assessed in vivo. Serum antibody titres were also measured. 2. The most effective vaccine consisted of an AI analogue conjugated with a tetanus toxoid carrier protein and adjuvanted with aluminium hydroxide. When this vaccine was injected on days 0, 21 and 42, pressor responses to AI on day 63 were significantly inhibited (maximum, 8.9 fold shift), but responses to AII were unaffected. The anti-angiotensin antibody titre was increased 32,100 fold, and, uniquely, these antibodies also cross-reacted with angiotensinogen. 3. These findings indicate that active immunization against AI may be a useful approach for treating cardiovascular disorders involving the renin-angiotensin system.

Empirical scoring functions. II. The testing of an empirical scoring function for the prediction of ligand-receptor binding affinities and the use of Bayesian regression to improve the quality of the model.

This paper tests the performance of a simple empirical scoring function on a set of candidate designs produced by a de novo design package. The scoring function calculates approximate ligand-receptor binding affinities given a putative binding geometry. To our knowledge this is the first substantial test of an empirical scoring function of this type on a set of molecular designs which were then subsequently synthesised and assayed. The performance illustrates that the methods used to construct the scoring function and the reliance on plausible, yet potentially false, binding modes can lead to significant over-prediction of binding affinity in bad cases. This is anticipated on theoretical grounds and provides caveats on the reliance which can be placed when using the scoring function as a screen in the choice of molecular designs. To improve the predictability of the scoring function and to understand experimental results, it is important to perform subsequent Quantitative Structure-Activity Relationship (QSAR) studies. In this paper, Bayesian regression is performed to improve the predictability of the scoring function in the light of the assay results. Bayesian regression provides a rigorous mathematical framework for the incorporation of prior information, in this case information from the original training set, into a regression on the assay results of the candidate molecular designs. The results indicate that Bayesian regression is a useful and practical technique when relevant prior knowledge is available and that the constraints embodied in the prior information can be used to improve the robustness and accuracy of regression models. We believe this to be the first application of Bayesian regression to QSAR analysis in chemistry.

From toxicological problem to therapeutic use: the discovery of the mode of action of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), its toxicology and development as a drug.

NTBC is a triketone with herbicidal activity that has been shown to have a novel mode of action by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase in plants. Early studies on the toxicity of this compound found that rats treated with NTBC developed corneal lesions. Investigations aimed at understanding the mechanistic basis for the ocular toxicity discovered that the rats developed tyrosinaemia and excreted large amounts of 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate, owing to inhibition of the hepatic enzyme 4-hydroxyphenylpyruvate dioxygenase. The corneal lesions resemble those seen when rats are fed a diet supplemented with tyrosine, leading us to conclude that the ocular toxicity seen with NTBC is a consequence of a marked and sustained tyrosinaemia. Studies in collaboration with Professor Sven Lindstedt showed that NTBC was a potent inhibitor of purified human liver 4-hydroxyphenylpyruvate dioxygenase. This interaction lead to the concept of using NTBC to treat patients with tyrosinaemia type 1, to block or reduce the formation of toxic metabolites such as succinylacetoacetate in the liver. Zeneca Agrochemicals and Zeneca Pharmaceuticals made NTBC available for clinical use and, with the approval of the Swedish Medical Products Agency, a seriously ill child with an acute form of tyrosinaemia type 1 was successfully treated in February 1991. Subsequently, other children with this inborn error of metabolism in Sweden and other countries have been treated with NTBC. The drug is now available to those in need via Swedish Orphan AB.

Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes.

This paper describes the development of a simple empirical scoring function designed to estimate the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to estimate lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalises flexibility. The coefficients of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coefficients obtained are stable to changes in the composition of the training set. The function is also tested on two test sets containing a further 20 and 10 complexes, respectively. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.

PRO_SELECT: combining structure-based drug design and combinatorial chemistry for rapid lead discovery. 1. Technology.

This paper describes a novel methodology, PRO_SELECT, which combines elements of structure-based drug design and combinatorial chemistry to create a new paradigm for accelerated lead discovery. Starting with a synthetically accessible template positioned in the active site of the target of interest, PRO_SELECT employs database searching to generate lists of potential substituents for each substituent position on the template. These substituents are selected on the basis of their being able to couple to the template using known synthetic routes and their possession of the correct functionality to interact with specified residues in the active site. The lists of potential substituents are then screened computationally against the active site using rapid algorithms. An empirical scoring function, correlated to binding free energy, is used to rank the substituents at each position. The highest scoring substituents at each position can then be examined using a variety of techniques and a final selection is made. Combinatorial enumeration of the final lists generates a library of synthetically accessible molecules, which may then be prioritized for synthesis and assay. The results obtained using PRO_SELECT to design thrombin inhibitors are briefly discussed.

Design of active analogues of a 15-residue peptide using D-optimal design, QSAR and a combinatorial search algorithm.

This report describes the rational design of novel analogues of a 15-residue antibacterial peptide CAMEL0. A constrained D-optimal design was carried out to derive a training set of 60 analogues. Partial least squares (PLS) models describing quantitative structure-activity relationships (QSARs) were initially derived for the peptides using two published and one novel parameter set. The novel "Design parameters' were based on key structural features identified in hypothetical models of the mechanisms by which peptides interact with cell membranes. In an extension of the PLS method, influence statistics were used to decrease the weighting of compounds having a large effect on model predictions. A combinatorial search algorithm was developed which used PLS models as predictors to select a test set of 39 peptides with high predicted potencies. Within this set, the most potent analogue CAMEL135, which contained seven point mutations from CAMEL0, was identified. For a panel of 24 bacteria, the mean MIC value of CAMEL135 was approximately half of that for CAMEL0. For the parameter sets tested, covariance functions derived from Z-scales gave highest Q2-values for the training set, whilst the model using the the 'Design parameters' gave least error when predicting the activity of the test set. The predictive ability of a third published set of peptide parameters was found to compare favourably with that of the parameters used in the design. Analysis of the PLS models indicates that hydrophobicity and amphipathicity are the most important features influencing activity for this class of compound.

Statistical comparison of established T-cell epitope predictors against a large database of human and murine antigens.

Identification of T-cell epitopes within a protein antigen is an important tool in vaccine design. The T-cell epitope prediction schemes often are exploited by workers but have proved unreliable in comparison with experimental techniques. We compared published T-cell epitope predictors against two databases of human and murine T-cell epitopes. Each predictor was assessed against random cyclic permutations of epitopes in order to determine significance. Predictor performance was expressed in terms of two parameters, specificity and sensitivity. Specificity is an expression of the quality of predictions, whereas sensitivity is an expression of the quantity of epitopes predicted. Against the human data set, the strip-of-hydrophobic helix algorithm [Stille et al., Molec. Immun. 24, 1021-1027 (1987)] was the only significant predictor (p < 0.05), whereas against murine data only, the Roth2 pattern [Rothbard and Taylor, EMBO J. 7, 93-100 (1988)] was significant (p < 0.05). Not only were the majority of algorithms no better than random against both data sets, against the murine data two schemes were significant (p < 0.05) anti-predictors. This report indicates which predictors are relevant statistically and is the first to describe anti-predictors which can themselves be useful in the identification of T-cell epitopes.

Characterization of the interaction of 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6,-tetramethyl- cyclohexane-1,3,5-trione with rat hepatic 4-hydroxyphenylpyruvate dioxygenase.

The synthetic beta-triketones are a novel family of chemicals, developed as herbicides that have activity on grass and broadleaf weeds and are selective in corn. Toxicological evaluation of a number of these chemicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). This paper describes the kinetics of inhibition of rat hepatic HPPD in vitro by the representative beta-triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6- tetramethylcyclohexane-1,3,5-trione (1). A marked inhibition of rat hepatic HPPD was observed when 1 was incubated with the enzyme for 3 min at 37 degrees C prior to the initiation of the enzyme reaction by the addition of substrate. In this system, a concentration of 200 nM 1 resulted in a > 90% loss of HPPD activity, and an apparent IC50 was established at approximately 50 nM. The rate constant for the inactivation of HPPD by 1 was (1.5 +/- 0.2) x 10(-5) s-1 nM-1 as determined by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzyme-inhibitor complex slowly dissociates, with approximately 5.5 +/- 0.6% of the enzyme activity being recovered by 6 h at 25 degrees C (t1/2, 25 degrees C, estimated at 101 +/- 14 h). In short, our studies establish 1 to be a tight-binding inhibitor of rat hepatic HPPD in vitro. This inhibition is characterized by the rapid inactivation of HPPD by the formation of an enzyme-inhibitor complex that dissociates extremely slowly with recovery of enzyme activity.

Application of benchmark dose risk assessment methodology to developmental toxicity: an industrial view.

The U.S. EPA first signalled its intention to use benchmark dose risk techniques in 1991. Subsequently, publication of draft Guidelines for the Risk Assessment of Reproductive Toxicity data indicated the Agency's intention for wide use of the technique. In developmental toxicity experiments, a number of factors need to be considered before attempting benchmark dose calculations, as compared to the conventional NOAEL approach. For example, care in the assessment of potential litter effects (the litter is the unit of such a study) on the data and whether the data are continuous (e.g. foetal body weight) or discontinuous (e.g. specific or grouped developmental defects where the abnormality is present or absent). Two examples of the use of the benchmark dose approach will be made. First, in the analysis of foetal body weight, where a benchmark dose estimate for an agent producing a 5% decrease in mean foetal weight may be calculated from a shift in the distribution of foetal weights between groups, or by conversion of data to reflect changes in the incidence of 'small' pups (i.e. those towards the extreme of the normal range). The second example involves studies conducted on the developmental toxicity of a triazole antifungal. In the first study, the agent was clearly teratogenic, but a NOAEL was not established and thus necessitated a second study. Analysis of benchmark does estimates (e.g. for % foetuses malformed) from the first study indicated that these were not significantly changed when the data from the second study were combined (i.e. the second study did not aid the risk assessment). The benchmark dose approach has significant scientific and practical advantages over the conventional NOAEL methodology in risk assessments derived from developmental toxicity studies.

Thresholds in chemical carcinogenesis.

It is common practice to base carcinogenic risk assessment on the view that there is no threshold for chemical carcinogenesis. In this context, the threshold is defined as a dose below which no effects are observed. Analysis of epidemiological and experimental data on chemical carcinogenesis confirms that no thresholds have been demonstrated in human or animal studies, including the ED01 study which used very large group sizes. Indeed, demonstrating a threshold by experiment is as difficult as proving a negative. Mechanistic data provide the justification for the assertion that thresholds do not exist in chemical carcinogeneses. This is commonly thought of as a dose-response relationship which is linear at low dose. It is noted that primary sites of action for other forms of toxicity--for example, inhibition of enzymes or occupation of receptors--also have a dose-response relationship which is linear at low dose. It is concluded that the use of differing methods of low-dose risk assessment for different toxicological endpoints is not justified. The same method should be used in order to provide symmetry in assessment of different risks; the influence of assumptions, such as the magnitude of safety factors or the mathematical model selected, should be clearly stated so that risk managers can make balanced judgments.

Retrospective appraisal of the relationship between skin irritancy and contact sensitization potential.

A retrospective analysis of the association between skin irritancy and the potential to cause contact sensitization has been performed employing a historical database for 50 chemicals and formulations. Correlations between the results of Draize skin irritation tests and skin sensitizing activity measured with the occluded patch test of Buehler have been examined. Weak, but nevertheless statistically significant, associations between contact sensitization and skin irritancy have been demonstrated. It is proposed that such correlations are consistent with the irritant properties of a material exerting an important influence on the extent to which contact sensitization is induced.

Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione.

The administration of the compound 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) to rats (10 mg/kg body wt) caused an elevation in the concentration of plasma tyrosine and gave products in urine that were identified as 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA). This observed chemically induced tyrosinemia established that this compound perturbs tyrosine catabolism and suggested that the causal effect is the inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD). This was confirmed when rat liver HPPD was found to be markedly inhibited by NTBC when the enzyme and chemical were incubated, in vitro, for 3 min at 37 degrees C prior to the initiation of the enzyme reaction by the addition of substrate. At 100 nM NTBC, approximately 90% of the enzyme activity was lost and an IC50 was calculated at approximately 40 nM. The inhibition of HPPD by NTBC (50 nM) is time-dependent; the enzyme activity was reduced by > 50% within 30 sec. Progress curve data of loss of enzyme activity with time gave a rate constant for the inactivation of rat liver HPPD [k*, formation of an HPPD-inhibitor (EI) complex] by NTBC of 9.9 +/- 2.5 x 10(-5) sec-1 nM-1. It was established that NTBC is not irreversibly bound in the EI complex but slowly dissociates with a recovery of enzyme activity of 13.7 +/- 1.0% over a 7-hr period (t1/2, 25 degrees C estimated at 63 hours). In comparison, the compound 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione (CMBC), an analog of NTBC, gave a similar rate for the inactivation of HPPD (k*, 3.3 +/- 0.8 x 10(-5) sec-1 nM-1), whereas 45 +/- 8% of the enzyme activity was recovered over a 7-hr period (t1/2, 25 degrees C approximately 10 hr). These studies establish that NTBC and CMBC are potent, time-dependent (tight-binding) reversible inhibitors of HPPD. The inhibition is characterized by a rapid inactivation of the enzyme by the formation of an HPPD-inhibitor complex that dissociates with recovery of enzyme activity. In vivo, the inhibition of HPPD causes a tyrosinemia that abates with the recovery of enzyme activity. The understanding of the mechanism by which NTBC perturbs tyrosine catabolism has led to the clinical use of this chemical as the first effective pharmacological therapy for the hereditary disorder tyrosinemia I.

Vehicle effects on in vitro percutaneous absorption through rat and human skin.

We studied the effects of three vehicles (propylene glycol, octanol and ethyl decanoate) with differing polarity on the in vitro percutaneous absorption of three chemicals (fluazifop-butyl, dimethyl phthalate and fomesafen sodium salt) with a range of physico-chemical properties. Absorption rate measurements were made from high vehicle volume (200 microliters/cm2) and low vehicle volume (< 10 microliters/cm2) applications. For the lipophilic fluazifop-butyl absorption rate was highest from the more polar vehicle propylene glycol, but this effect was only significant under high-volume conditions. There was a variable vehicle effect on absorption of the intermediate chemical dimethyl phthalate. The largest vehicle effect was seen for the more hydrophilic fomesafen sodium salt where absorption was fastest from the least polar vehicle ethyl decanoate. These results support the hypothesis that the absorption process can in part be predicted from a knowledge of solute solubility. Vehicle effects were greater from high volume applications than from those more comparable to occupational exposure conditions.

The predictive accuracy of in vitro measurements for the dermal absorption of a lipophilic penetrant (fluazifop-butyl) through rat and human skin.

The predictive accuracy of in vitro measurements in estimating dermal absorption has been evaluated in rat and human skin using fluazifop-butyl (FB), a lipophilic model compound, at dosage rates of 2.5, 25, and 250 micrograms/cm2. In vitro studies used rat and human epidermal membranes mounted in static diffusion cells with radiolabeled FB and receptor fluids of 50% aqueous ethanol (Aq Et), 6% polyethylene glycol 20 oleyl ether in saline (PEG), or tissue culture medium (TCM). In vivo rat studies with radiolabeled FB were carried out to parallel previously published human volunteer studies. For rat skin, in vitro measurements with all types of receptor fluid provide an adequate prediction (generally within a factor of 3) of in vivo absorption. Absorption data for human epidermal membranes with a receptor fluid of Aq Et were adequately predictive of the in vivo absorption. In contrast, membranes with PEG or TCM significantly underestimated the in vivo absorption. The results support the conclusion that in vitro studies are useful to predict in vivo dermal absorption in rat and man, when appropriate receptor fluids are used.

Calculation of benchmark doses from teratology data.

The benchmark dose approach has several potential advantages over the no observed adverse effect level (NOAEL) as a basis for risk assessment of toxic chemicals, based upon animal toxicity data. The practical use of the benchmark dose has been evaluated by applying dose-response models to an extensive historical database of teratology bioassays. Doses corresponding to 1 and 5% increases in incidence of lesions are calculated and compared to NOAELs. The statistical accuracy of these estimates was determined by calculating confidence intervals. The lower confidence limit on the 5% benchmark dose (LED05) is found to be comparable to the NOAEL for most datasets, and slightly higher on average. Benchmark doses at the 1% level could not be estimated accurately (i.e., they had wide confidence intervals) for a significant fraction of the datasets. LED01 values were lower on average than the NOAEL. Based on these results, it is concluded that benchmark doses for a 5% increases in incidence can be calculated for most datasets, and could be used as a satisfactory basis for risk assessment, e.g., to set reference doses or acceptable daily intakes. An exception occurs when the benchmark dose exceeds the highest dose of the study. This is only likely to occur when the chemical causes a small, but significant, increase in a finding that is uncommon in untreated animals.

A physiologically based mathematical model of dermal absorption in man.

A sound understanding of the mechanisms determining percutaneous absorption is necessary for toxicological risk assessment of chemicals contacting the skin. As part of a programme investigating these mechanisms we have developed a physiologically based mathematical model. The structure of the model parallels the multi-layer structure of the skin, with separate surface, stratum corneum and viable tissue layers. It simulates the effects of partitioning and diffusive transport between the sub-layers, and metabolism in the viable epidermis. In addition the model describes removal processes on the surface of the skin, including the effects of washing and desquamation, and rubbing off onto clothing. This model is applied to data on the penetration of the herbicide fluazifop-butyl through human skin in vivo and in vitro. Part of this dataset is used to estimate unknown model parameter values and the remainder is used to provide a partial validation of the model. Only a small fraction of the applied dose was absorbed through the skin; most of it was removed by washing or onto clothing. The model provides a quantitative description of these loss processes on the skin surface.

Concentration-flux relations for a multicellular biological membrane with metabolism.

A mathematical model is described for the simultaneous diffusion and metabolism of a chemical penetrating a multicellular biological membrane such as skin. Metabolism is assumed to follow saturable Michaelis-Menten kinetics, which leads to nonlinear relationships between the applied concentration and the metabolic and diffusive fluxes through the membrane. Approximate concentration-flux relations are derived under limiting conditions, and a computational method is described for the general case. The major barrier to dermal penetration of very lipophilic molecules is thought to be the viable tissues (viable epidermis and some of the dermis) underlying the stratum corneum, and some molecules are known to be metabolized by enzymes within these tissues. It is proposed to use the model to describe penetration and metabolism of such lipophilic molecules within the viable tissues of the skin.

Modelling dermal pharmacokinetics using in vitro data. Part I. Fluazifop-butyl in the rat.

The pharmacokinetics of the herbicide fluazifop-butyl have been determined in female rats following oral and intravenous dosing, and described by a mathematical model. Penetration of fluazifop-butyl through epidermal membranes has been determined using three different receptor fluids. It is demonstrated how this in vitro absorption data can be used with a pharmacokinetic model derived from oral and i.v. dosing studies to predict plasma concentrations and urinary excretion profiles following dermal dosing. Model predictions are compared with experimental measurements and found to be in good agreement.

Modelling dermal pharmacokinetics using in vitro data. Part II. Fluazifop-butyl in man.

In a previous paper it was demonstrated that dermal absorption of the herbicide fluazifop-butyl in the rat could be modelled by combining a knowledge of the pharmacokinetics following intravenous and oral dosing with in vitro measurements of dermal absorption. This paper demonstrates the validation of a similar model for the dermal absorption of fluazifop-butyl in man. Pharmacokinetic parameters derived from an oral dosing study are combined in a mathematical model with in vitro measurements of dermal absorption of fluazifop-butyl. Model predictions of the rate and extent of dermal absorption of fluazifop-butyl are compared with the results of dermal absorption studies in human volunteers. Good agreement is found between the model predictions and the experimental measurements. These results have implications for improved risk assessment. The model provides a tool for risk assessment based on both internal dose (e.g. peak plasma concentration, plasma area under the curve) as well as total absorbed dose. However, further work is required to evaluate whether the same techniques are applicable to a wider range of compounds.