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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

Background

Nonclinical studies have shown netazepide (YF476) to be a potent, selective, competitive and orally active gastrin receptor antagonist.

Aim

To administer to humans for the first time single oral doses of netazepide, to assess their tolerability, safety, pharmacokinetics and effect on 24-h gastric pH.

Methods

We did two randomised double-blind single-dose studies in healthy subjects. The first (n = 12) was a six-way incomplete crossover pilot study of rising doses of netazepide (range 0.5–100 mg) and placebo. The second (n = 20) was a five-way complete crossover study of netazepide 5, 25 and 100 mg, ranitidine 150 mg and placebo. In both trials we collected frequent blood samples, measured plasma netazepide and calculated pharmacokinetic parameters. In the comparative trial we measured gastric pH continuously for 24 h and compared treatments by percentage time gastric pH ≥4.

Results

Netazepide was well tolerated. Median t max and t ½ for the 100 mg dose were about 1 and 7 h, respectively, and the pharmacokinetics were dose-proportional. Netazepide and ranitidine each increased gastric pH. Onset of activity was similarly rapid for both. All netazepide doses were more effective than placebo (P ≤ 0.023). Compared with ranitidine, netazepide 5 mg was as effective, and netazepide 25 and 100 mg were much more effective (P ≤ 0.010), over the 24 h after dosing. Activity of ranitidine lasted about 12 h, whereas that of netazepide exceeded 24 h.

Conclusions

In human: netazepide is an orally active gastrin antagonist, and gastrin has a major role in controlling gastric acidity. Repeated-dose studies are justified. NCT01538784 and NCT01538797.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

Cholecystokinin (CCK) and gastrin are polypeptide hormones found in the brain and other tissues.[1] CCK stimulates CCK1 (also called CCK-A) receptors in the gall bladder and pancreas causing contraction and exocrine secretion respectively. The C-terminal octapeptide is necessary to elicit the full response.[2] CCK also acts on receptors distinct from CCK1 receptors, designated CCK2 (CCK-B) or gastrin receptors, which require only the C-terminal tetrapeptide to elicit a full response.[3] The C-terminal tetrapeptide is common to both CCK and gastrin.

Cloning has shown that CCK2 receptors in the brain and other tissues are identical.[4] CCK stimulates CCK2 receptors in the brain, whereas gastrin, which is secreted by G cells in the gastric antrum,[5] stimulates gastric acid secretion and gastrointestinal cell growth, especially growth of the enterochromaffin-like (ECL) cells in the gastric mucosa.[6]

Food,[7] gastric distension[8] and an increase in gastric pH[9] all cause secretion of gastrin into the circulation. Circulating gastrin stimulates gastrin receptors on ECL cells in the gastric fundus to secrete histamine,[10] which in turn stimulates adjacent gastric oxyntic cells to secrete acid into the stomach lumen. Gastric acid secretion is controlled by (H+, K+)-ATPase (the proton pump) in response to stimulation of histamine H2-receptors[11] or muscarinic M3-receptors.[12]

Initially, acid is neutralised by the buffering capacity of food. But as acid secretion continues and digestion proceeds and the gastric contents move into the duodenum, the buffering capacity of the food diminishes and intragastric pH falls. Falling pH stimulates D cells in the gastric antrum to secrete somatostatin, a hormone that switches off gastrin secretion.

Acid secretion is also under nervous control. Food causes central stimulation of the vagus, which leads to gastrin release. The vagus also controls acid secretion via somatostatin.

Many gastrin antagonists have been described.[13, 14] Some have been studied in healthy subjects and patients, but none has progressed beyond early clinical development.[15] Netazepide (YF476) [(3R)-N-(1-(tert-butylcarbonylmethyl)-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl)-N′-(3-(methylamino)phenyl)urea] is a benzodiazepine derivative and a novel gastrin receptor antagonist.[16] The pharmacology of netazepide has been assessed in nonclinical studies,[17, 18] as follows. It caused concentration-dependent inhibition of specific binding of the ligand [125I]CCK-8 to rat and cloned human[19] gastrin receptors in vitro. The affinity of netazepide for rat gastrin receptors was 264 and 70 times higher than that of two other gastrin antagonists, L-365,260[20] and CI-988,[21] respectively, and 4100 times its affinity for rat pancreatic CCK1 receptors. Netazepide had very little affinity for various other receptors, such as histamine, muscarinic and benzodiazepine receptors.[17] In anaesthetised rats, intravenous netazepide caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, with an ED50 of 0.0086 µmol/kg, compared with an ED50 of 0.013 µmol/kg for intravenous famotidine. Intravenous netazepide at a high dose of 10 µmol/kg did not affect histamine- or bethanechol-induced acid secretion. In Heidenhain pouch dogs, intravenous and oral netazepide each caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, with ED50 values of 0.018 and 0.020 µmol/kg respectively.[17] ED50 for intravenous and oral famotidine, a histamine H2-receptor antagonist (H2RA), was 0.078 and 0.092 µmol/kg respectively. In dogs with a gastric fistula, intravenous netazepide dose-dependently inhibited pentagastrin-induced gastric acid secretion, with ED50 0.0023 µmol/kg. Also in gastric fistula dogs, oral netazepide, famotidine and the proton pump inhibitor (PPI), omeprazole, each dose-dependently inhibited peptone-induced gastric acid secretion, with ED50 0.11, 0.76 and 4.28 µmol/kg respectively.[18] On the basis of those results, netazepide was about 7 and 40 times more potent than famotidine and omeprazole respectively. Comparison of ED50 for intravenous and oral netazepide indicates that the oral bioavailability was 26–28% in rats and 27–50% dogs.

Thus, overall these nonclinical studies show netazepide to be a potent, highly selective, competitive and orally active antagonist of gastrin receptors. Indeed, netazepide has been described as the ‘gold standard’ for gastrin antagonists,[14] and has been used in many other nonclinical studies, mainly to assess the physiology and pathology of gastrin. A PubMed search for YF476 currently yields 27 references.

Netazepide was well tolerated in animal toxicology studies (Ferring, Investigator's Brochure 1997). In 13-week studies, the no-observable-adverse-effect level was 100 mg/kg/day in rats and in dogs. There were increases in gastric G cells and circulating gastrin, and a reduction in gastric ECL cells, consistent with antagonism of gastrin receptors by netazepide. Tests of teratogenicity and mutagenicity were negative. The favourable pharmacological and toxicological profiles of netazepide and its good oral bioavailability in animals justified studies of netazepide in human.

Here, we report the first administration of netazepide to human. We did two single-dose studies in healthy subjects. The first was a pilot study of single rising oral doses. The second was a placebo-controlled comparative study of single fixed oral doses of netazepide and ranitidine.[22]

The overall aim was to assess the safety, tolerability and pharmacokinetics (PK) of netazepide, and to compare the effect of netazepide on 24-h gastric pH with that of ranitidine. Also, we aimed to assess the relationship between the PK and pharmacodynamics (PD) of netazepide.

This article is the first of a series of three articles based on the early clinical pharmacology studies of netazepide, which hitherto have been published only as abstracts.[22-25]

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

We did the studies in accordance with the ICH Guideline for Good Clinical Practice and Declaration of Helsinki. Brent Ethics Committee approved the studies. Subjects gave written informed consent.

Registry and registration numbers: ClinicalTrials.gov NCT01538784 and NCT01538797.

Treatments

Yamanouchi Pharmaceutical Co, Japan, supplied netazepide (0.5, 5 and 25 mg) capsules and matching placebo capsules. The hospital pharmacy: supplied encapsulated ranitidine 150 mg tablets (Zantac; GlaxoSmithKline) and matching placebo capsules; packed and labelled treatments; and randomised subjects to treatments using sequentially numbered containers.

The nonclinical studies suggested that oral netazepide 10 mg would be as effective as oral ranitidine 150 mg in suppressing gastric acidity.[18] Therefore, for our two clinical studies we chose a range of single doses encompassing 10 mg.

Study design

Pilot study

Before embarking on a controlled, randomised study of fixed doses, we did a single-dose rising study of netazepide 0.5, 5, 25, 50 and 100 mg by mouth. The design was double-blind, placebo-controlled and incomplete crossover in two groups of six healthy young men. One group received 0.5, 25 and 100 mg and the other 5, 50 and 100 mg. The ratio of netazepide:placebo was 4:2 at each dose level. Each subject was resident for two nights on three occasions and followed up 5–10 days after dosing. We assessed safety and tolerability by vital signs, ECG, safety tests of blood and urine and adverse events. We took blood at 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h after dosing, for assay of plasma netazepide.

Comparative study

The second study was single-dose, double-blind, double-dummy, randomised and five-way complete crossover in design in 20 healthy young men or women using a reliable method of contraception. Each subject received five treatments by mouth: netazepide 5, 25 or 100 mg; ranitidine 150 mg and placebo. Each subject was resident for two nights on five occasions and was followed up 5–10 days after the last dose. There were 7 days between doses. We assessed safety and tolerability as in the pilot study, took blood samples at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after dosing, and collected all urine from 0 to 24 h after dosing, for netazepide assay. We measured gastric pH continuously for 0.5 h before and 24 h after dosing. Subjects fasted overnight before each dose, which they took with 100 mL water at the same time (08:00–09:00 h) on each occasion. They rested on their beds for 6 h after dosing and were ambulant thereafter. They ate standard meals at 4, 9, 13 and 22 h after dosing, and drank 150 mL water at 2, 6, 8 and 11 h after dosing. We prohibited alcoholic and caffeinated drinks and smoking while subjects were resident.

Plasma concentrations of netazepide (YF476)

We collected blood in lithium-heparin tubes, and separated plasma by centrifugation (4 °C; 800 G for 10 min) and plastic pipette within 15 min of sampling. We stored plasma and urine samples at –20 °C until assay. Huntingdon Life Sciences, England, measured plasma netazepide by a validated liquid chromatographic-tandem mass spectrometric method.[26] The calibration line was linear over the range 0.1 (limit of quantification) to 25.0 ng/mL. Intra- and inter-batch precision was <14% and intra- and inter-batch accuracy was <11% over the entire range.

Measurement of ambulatory gastric pH

We inserted a disposable single-channel antimony internal reference pH electrode, with surface markings of 1 cm (Zinetics Medical, Salt Lake City, UT, USA), for a distance of 45–50 cm through the subject's nostril into the stomach. We monitored pH as the sensor passed down the oesophagus, through the gastro-oesophageal sphincter, and into the stomach. Entry of the electrode into the stomach was confirmed by a sharp fall in pH. Next, we withdrew the electrode through the sphincter into the oesophagus, and then repositioned it in the stomach; a sharp rise or fall in pH identified the point at which the sensor crossed the sphincter. We advanced the electrode to a final position 10 cm beyond that point. We recorded intragastric pH every 6 s via a menu-driven compact solid-state recorder (Flexilog 2020 24 h recorder; Oakfield Instruments, Oxon, UK); we calibrated the recorder before starting the recording, using pH 4 and 7 buffers. We activated an event marker on the recorder when the electrode was in position, and immediately after dosing. We uploaded recorded data to the Flexisoft II analysis software (Oakfield Instruments) operating within the Microsoft Windows environment, and exported it to other software packages as required.

Statistics

Sample size

For the comparative study, based on our previous experience, 20 subjects were deemed enough to detect 15% increase in pH with 80% power, assuming within-subject coefficient of variation of 0.15 and 5% significance.

Pharmacokinetics

We used WinNonLin to derive pharmacokinetic parameters by standard noncompartmental analysis, and sas for Windows for statistical analysis (Statistical Analysis System, SAS Institute Inc., Cary, NC, USA). We took maximum plasma concentrations of netazepide (C max), and the times at which they occurred (t max), directly from the experimental data. We calculated area under the plasma concentration-time curve over 0–24 h (AUC 0–24 h) using the linear trapezoidal rule, and area under the plasma concentration-time curves to infinite time (AUC 0–∞) using the equation: AUC 0–∞ = AUC 0–24 h + Ĉ lastz, where Ĉ last is the predicted plasma concentration of the last measurable sample, and λz is the terminal rate constant, determined by log-linear regression analysis of those points that constitute the final, linear phase of the plasma concentration-time curve. We calculated terminal half-life (t ½) as ln2/λz. For the comparative study, we used analysis of variance (anova) to assess dose-proportionality of C max and AUC 0–∞. Before doing so, we normalised both to a 100 mg dose and log-transformed them. We included subject and dose as effects in the anova model.

Pharmacodynamics

For the comparative study, we analysed pH data in four intervals: 0–4, 4–9, 9–13 and 13–24 h after dosing. We calculated for each interval: AUC; time pH ≥4 and percentage time pH ≥4. We calculated AUC using the trapezoidal rule. The primary outcome measure was percentage time gastric pH ≥4. We regarded all other pH parameters as secondary. To test for differences among treatments, we subjected each pharmacodynamic parameter to anova using a model that included effects for subject, period, treatment, first-order carryover and treatment-period interaction. To stabilise variance and make data conform to a normal distribution, we log-transformed AUC and arcsin-transformed percentages.[27] If treatment was significant in the overall model, we did pairwise comparisons between each active treatment and placebo. In addition, we compared with ranitidine any netazepide doses that were significantly different from placebo. All tests were two-sided, and significance level was α = 0.05; we made no adjustments for multiple comparisons.

For graphical presentation, we calculated median pH for successive 2-min periods for individual subjects to produce smoothed pH-time data. We calculated medians because the data were not normally distributed.

Pharmacokinetic/pharmacodynamic relationship

For the comparative study, we used two methods to assess the relationship between PK and PD of netazepide. First, we investigated the dose-response relationship by calculating Pearson correlation coefficients between plasma C max or AUC 0–∞ (with and without logarithmic transformation) and time pH ≥4 in 24 h. Second, as a measure of the PD effect of netazepide, we subtracted each subject's pH after placebo at each time point (‘baseline’) from the corresponding pH after each active treatment. Then we modelled the median of those differences by fitting a fourth-order polynomial curve; we used that equation to simulate a pH profile for each dose. We used a two-compartmental oral model to simulate a profile of netazepide plasma concentrations, which we used with the modelled pH profile to find the parameter that best described the PK/PD relationship.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

For the pilot study, we enrolled 15 men, of whom 3 dropped out for social reasons and 12 (mean age 27 years; mean weight 77 kg) completed it. For the comparative study, we enrolled 21 subjects, of whom 1 dropped out for social reasons and 20 (11 men; 9 women; mean age 25 years; mean weight 75 kg) completed it. There were full datasets for statistical and pharmacokinetic analyses.

Tolerability and safety

All treatments were well tolerated. Any adverse events were minor, transient and occurred across treatments. There were no clinically relevant changes in any of the safety assessments.

Pharmacokinetics

Pilot study

Netazepide was rapidly absorbed; median t max for the five dose levels ranged from 0.5 to 0.88 h Table S1 and Figure S1. For the dose levels 0.5, 5, 25, 50 and 100 mg, mean C max was 2.9, 19.7, 93.6, 153.2 and 397.1 ng/mL, respectively, and mean AUC 0–24 h was 3.8, 32.0, 178.1, 283.4 and 658.2 ng.h/mL respectively. The plasma concentration-time curves suggest that netazepide has at least two elimination phases; the terminal elimination phase seems to begin about 4–12 h after dosing. Only after the 100 mg dose was the concentration of netazepide above the limit of reliable quantification at 24 h after dosing in every subject. It is likely therefore that t ½ values for lower doses do not reflect the terminal elimination phase. The short mean t ½ of about 2 h for the lower doses may represent a more rapid earlier phase of elimination, whereas the higher value of 6.6 h for the 100 mg dose is probably a more accurate estimate of the true terminal elimination half-life of netazepide. That conclusion is supported by the strongly bimodal distribution of the individual t ½; although the range of values was 1.5–6.9 h, no subject had a value in the range 2.8–4.8 h. Netazepide had dose-proportional PK in the dose range studied; statistical analysis showed no significant evidence against dose-proportionality for either C max (P = 0.91) or AUC 0–∞ (P = 0.97).

Although there was a greater proportionate increase in C max and AUC 0–∞ from 50 to 100 mg than from 25 to 50 mg, comparisons among treatments included both within- and between-subject comparisons, as the design was incomplete crossover. Netazepide had consistent bioavailability among subjects, as judged by AUC: the coefficient of variation for AUC 0–∞ was below 30% at all doses except the 0.5 mg dose. Variation was inevitably higher for that dose because many of the plasma concentrations were close to or below the limit of reliable quantification. C max was more variable than AUC 0–24 h, but the coefficient of variation in C max was still <60% for all doses.

Comparative study

Netazepide was rapidly absorbed: median t max after the 5, 25 and 100 mg doses ranged from 0.5 to 1 h Tables S1 and S2, and Figure S2. Elimination appeared to be biphasic; the terminal elimination phase began ~4–12 h after dosing. For the dose levels 5, 25 and 100 mg, mean C max was 19.3, 89.5 and 251 ng/mL, respectively, and mean AUC 0–24 h was 41.3, 193.2 and 611.7 ng.h/mL respectively.

As only a few blood samples were taken during the terminal phase of elimination and netazepide concentrations were below the limit of reliable quantification in some of them, the estimate of elimination half-life is unreliable after the lower doses. After the 100 mg dose, plasma concentrations were well above the limit of reliable quantification at all times up to 24 h, so the t ½ of 7.6 h is a more representative value. The values of t ½ for the 5 mg (2.1 h) and 25 mg (5.5 h) doses probably represent a faster initial phase of distribution and elimination, rather than the true terminal elimination half-life.

Netazepide had consistent bioavailability among subjects, as judged by the AUC: the coefficient of variation for AUC 0–24 h was <40% for all doses. Variability in C max was higher, with a coefficient of variation ranging from 41 to 87%.

The dose increased in the ratio 1:5:20, whereas AUC 0–∞ and C max increased in the ratio 1.0:4.7:15.9 and 1.0:4.6:13.0 respectively. Those increases did not deviate significantly from dose-proportionality (P = 0.073 and P = 0.158 respectively). Only 1–2% of netazepide was excreted unchanged in the urine over 24 h.

Pharmacodynamics

Time course of gastric pH

There were characteristic and predictable increases in gastric pH after placebo, corresponding to times at which subjects ate and drank Figure 1. Median gastric pH increased quickly after netazepide. After all meals, median gastric pH fell more slowly in subjects on netazepide than those on placebo. Even after breakfast, 24 h after dosing, it remained higher in subjects on netazepide than those on placebo. The trends were similar for all netazepide doses, although the effects on pH were most marked after 100 mg. Although median pH after ranitidine was high in the first 4 h after dosing, it fell more quickly than after netazepide and, from about 12 h after dosing, it was almost indistinguishable from that after placebo.

image

Figure 1. 24-h gastric pH (median; n = 20) after single oral doses of (a) netazepide 5 mg, (b) netazepide 25 mg, (c) netazepide 100 mg and (d) ranitidine150 mg. For clarity, the profile of each active treatment is compared with that of placebo. Subjects ate standard meals at 4, 9, 13 & 22 h after dosing, and drank water (150 mL) at 2, 6, 8 & 11 h after dosing.

Download figure to PowerPoint

Primary response variable

Netazepide had a clear effect on the primary response variable: it increased percentage time gastric pH ≥4 Table S2, and Figures 2 and S3. In all time intervals, mean percentage time pH ≥4 for netazepide doses was significantly higher than for placebo (P ≤ 0.023). In the interval 0–4 h, mean percentage time pH ≥4 for netazepide 5 mg was significantly lower than for ranitidine 150 mg (P = 0.043); however, neither netazepide 25 mg nor 100 mg was significantly different from ranitidine. Furthermore, with one exception, mean percentage time pH ≥4 was significantly higher for all netazepide doses than for ranitidine during all other time intervals (P ≤ 0.010). The exception was 13–24 h, during which mean percentage time pH ≥4 for netazepide 5 mg did not differ significantly from ranitidine.

image

Figure 2. Median (n = 20)% time gastric pH ≥4 during intervals 0–4, 4–9, 9–13, 13–24 & 0–24 h after single oral doses of netazepide (5, 25 & 100 mg), ranitidine (150 mg) and placebo.

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Secondary response variables

Netazepide affected mean AUC of gastric pH similar to percentage time pH ≥4 Table S3. In all time intervals, AUC was significantly higher than for placebo (P ≤ 0.010). In the first 4 h after dosing, mean AUC of pH for netazepide 5 mg and 100 mg was significantly lower than that for ranitidine (P ≤ 0.032); however, it did not differ significantly between netazepide 25 mg and ranitidine. During all other time intervals, mean AUC of pH was significantly higher for all doses of netazepide than for ranitidine (P ≤ 0.032). There was no carryover effect for time pH ≥4 or for AUC of pH.

Pharmacokinetic/pharmacodynamic relationship

Pearson correlation coefficients among PK and PD parameters were weak; the best was between log AUC 0–∞ of netazepide and time pH ≥4, for which r 2 was only 0.16 Figures S4, S5 and S6.

The fourth-order polynomial curve fitted the placebo-adjusted intragastric pH data well; the trendline for netazepide 100 mg was r 2 = 0.91. Comparison of the modelled plasma concentrations of netazepide with the modelled pH difference between netazepide and placebo showed a delay between maximum plasma concentrations and maximum pH. However, when we calculated netazepide concentrations in the peripheral compartment, they fitted the modelled pH profile more closely, although there was still some delay.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

Single doses of netazepide were well tolerated and safe, and markedly increased gastric pH. Netazepide was rapidly absorbed, with t max ~1.0 h. Elimination appeared biphasic, with the terminal phase beginning 4–12 h after dosing. Plasma concentrations after netazepide 100 mg were well above the limit of reliable quantification at all times up to 24 h, so t ½ after that dose (~7 h) is probably a true reflection of the behaviour of netazepide. The lower t ½ after smaller doses probably reflects a faster initial phase of distribution and elimination rather than the true terminal elimination half-life. Netazepide had dose-proportional kinetics at the doses studied, and consistent bioavailability among subjects, as judged by AUC. Only 1–2% of the dose was excreted unchanged in urine, suggesting netazepide is cleared from plasma mainly by hepatic metabolism.

Gastric pH showed only the expected postprandial fluctuations after placebo, whereas all netazepide doses raised pH at some time after dosing. Mean percentage time pH ≥4 for all netazepide doses was significantly higher than that for placebo at all time intervals and for ranitidine at most time intervals. The effect of ranitidine was mainly in the first 4 h after dosing, whereas the effect of netazepide was more sustained and lasted at least 24 h, particularly after the higher doses. Overall, netazepide 5 mg was as effective as ranitidine 150 mg over the 24 h after dosing; netazepide 25 and 100 mg were much more effective.

There was only a weak correlation between plasma AUC and time pH ≥4 (r 2 = 0.16). It appears therefore that the effect of netazepide is dependent on other factors in addition to plasma concentrations. The modelled effect trendlines for netazepide 5, 25 and 100 mg showed little difference between the 25 and 100 mg doses, with 25 mg having a near-maximum effect. That suggests 25 mg is at or close to the top of the dose-response curve for suppression of basal and food-stimulated gastric acid secretion.

The effect on pH showed only a loose temporal relationship with plasma netazepide concentrations. As netazepide does not act in plasma, but via gastrin receptors on ECL cells in the stomach,[6] that is not surprising. Modelling of netazepide concentration in the second compartment led to a much better, although still not perfect, relationship with effect. The model we used was probably too simple because it assumes that only netazepide binds to gastrin receptors. In practice, gastrin also binds to the receptors, and it is possible that metabolites of netazepide may do so as well. Also, inhibition of gastric acid secretion, for example by H2RA or PPI, results in an increase in circulating gastrin. Although we did not measure circulating gastrin in our subjects, netazepide does increase it in rats.[28, 29] An increase in circulating gastrin in our subjects may have confounded the modelling results.

Although netazepide is a benzodiazepine derivative, and there are gastrin receptors in the brain, we did not formally test its effect on the central nervous system of our subjects because netazepide showed no activity in an animal model of anxiety, whereas L-365,260 did (Ferring, Investigator's Brochure 1997). However, no subject reported relevant symptoms after netazepide. Also, in a study using positron emission tomography,[11]C-netazepide penetrated the brain poorly in rats.[30]

Most of the gastrin receptor antagonists described have had problems with selectivity, potency or bioavailability.[13, 14, 31] Several have been tested in human, but none has been marketed. L-365,260 and CR-2194 were given to healthy subjects to assess their effect on pentagastrin-stimulated gastric acid secretion, but the results were disappointing.[32, 33] L-365,260 and CI-988 were assessed for anxiolytic activity in healthy subjects and patients.[34-39] Again, results were disappointing. More recently, JB95008, a gastrin antagonist with little oral bioavailability, was given by continuous intravenous infusion to patients with pancreatic cancer in two randomised controlled trials.[40, 41] JB95008 prolonged life compared with placebo, and was as effective as fluorouracil. Gastrin receptors are expressed on human pancreatic cancer cells, which grow when stimulated by gastrin.[42]

Experiments in healthy subjects, in which food or intravenous gastrin 17 was used to stimulate gastric acid secretion, indicate that gastrin accounts for about 90% of acid secreted after a meal, although the contribution varied among individuals.[43] Our results support those findings.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

Single oral doses of netazepide were well tolerated and safe, and caused dose-dependent sustained increases in gastric pH, consistent with antagonism of gastrin receptors, and highlighting the major role of gastrin in controlling gastric acidity in human. Netazepide is a tool for studying the physiology, pharmacology and pathology of gastrin, and a potential new treatment for acid-related conditions. Studies of repeated doses of netazepide are justified.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information

Declaration of personal interests: Netazepide (YF476) came from research by Ferring, Chilworth, England, in 1997.[16] Yamanouchi, Japan, partnered Ferring in the early development of netazepide (hence the code YF476). In 2006, Ferring licensed netazepide to Trio Medicines Ltd, a subsidiary of Hammersmith Medicines Research (HMR), a contract research organisation. Between those dates, Ferring licensed netazepide to two other companies. MB, SW, KD and TM are employees of HMR. OD is a former employee of HMR, and is now an employee of Novartis Pharma AG, Basle, Switzerland. AJ is an employee of Barts and The London School of Medicine and Dentistry, Queen Mary University of London, and has served as a speaker and consultant for and received research funding from Novartis. Declaration of funding interests: Ferring A/S, Indertoften 10, DK-2720 Vanlose, Denmark, funded the studies.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
apt5143-sup-0001-TableS1.tifimage/tif737KTable S1. Summary of pharmacokinetic parameters of plasma netazepide.
apt5143-sup-0002-TableS2.tifimage/tif742KTable S2. Back-transformed means (n = 20; 95% confidence interval) of percentage time pH ≥4.
apt5143-sup-0003-TableS3.tifimage/tif974KTable S3. Geometric means (n = 20; 95% confidence interval) of AUC for gastric pH.
apt5143-sup-0004-FigS1.tifimage/tif671KFigure S1. Pilot study: mean plasma concentrations of netazepide after single oral doses of 0.5, 5, 25, 50 and 100 mg.
apt5143-sup-0005-FigS2.tifimage/tif700KFigure S2. Comparative study: mean (±s.d.; n = 20) plasma concentrations of netazepide after single oral doses of 5, 25 and 100 mg.
apt5143-sup-0006-FigS3.tifimage/tif721KFigure S3. Box-whisker plots (n = 20) of % time gastric pH ≥4 during (a) 0–4 h, (b) 4–9 h, (c) 9–13 h, (d) 13–24 h and (e) 0–24 h after single oral doses of netazepide (5, 25 & 100 mg), ranitidine (150 mg) and placebo. Mean (+), median, interquartile range and range.
apt5143-sup-0007-FigS4.docxWord document20KFigure S4. Fourth-order polynomial trendlines for the increase in pH induced by netazepide 5, 25 & 100 mg, calculated as the median differences between netazepide and placebo.
apt5143-sup-0008-FigS5.docxWord document67KFigure S5. Modelled plasma concentrations of netazepide compared with placebo-adjusted increase in pH after netazepide 100 mg.
apt5143-sup-0009-FigS6.docxWord document19KFigure S6. Modelled concentrations of netazepide in the peripheral compartment compared with the placebo-adjusted increase in pH after netazepide 100 mg.

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