Title of Invention

Synthetic capsaicinoid derivatives and feed comprising such compounds as growth promotors. Field of the invention and related prior art

The invention relates to synthetic capsaicinoid derivatives and feed comprising such compounds as growth promotors in animals and in particular in birds/chickens.

The growth rate of poultry and chickens is of great economic importance and for feeding of the world population. Even a small increase in the growth rate will have a significant influence for farmers and producers. aXichem is a global supplier of natural-analogue industrial compounds and its mission is to develop, patent and market natural-analogue substances. aXichem is focusing on

phenylcapsaicin, an analogue of natural capsaicin and derivatives thereof. Since

phenylcapsaicin is synthetically produced, the substance offers a wide range of benefits compared with natural capsaicin, which is extracted from chili peppers and therefore has a varying quality. Synthetic capsaicinoids can be prepared with high purity and in good yields and quantity and at a reasonable cost. Additionally, the available amounts of naturally occurring capsaicin is limited. The standardized natural capsaicin extract contains at least 3 isomers, which all have different chemical properties but which are difficult to differentiate between. It may therefore be difficult to obtain capsaicin extracts which have sufficiently uniform purity and composition for the intended use.

Phenylcapsaicin is not environmentally harmful and is thus a viable alternative with a wide range of applications in a variety of areas. aXichem holds European (EP1670310) and US patents (US7446226) on their synthetic products their synthesis and use as antifouling agent (WO2005025314). These synthetic low toxicity analogs of capsaicin can by synthezised with high purity with a reasonable cost and in high yield. Phenylcapsaicin has proved to be less toxic and far less irritating than natural capsaicin.

Summary of the invention

The invention relates to at least one compounds with the general formula (1)

wherein R is a substituent selected from the group of Ci-Cis alkyl, trifluoromethyl, C3-C12 cycloalkyl, phenyl, phenoxy, phenylthio, halogen; or tautomers or salts thereof, for use as a growth promotor.

The invention also relates to compositions and feed comprising compounds of formula (1) according to claim 1.

Types of feed and birds are found in the claims 7 and 8. Detailed description of the invention

The invention relates to at least one compounds with the general formula (1)

wherein R is a substituent selected from the group of Ci-Cis alkyl, trifluoromethyl, C3-C12 cycloalkyl, phenyl, phenoxy, phenylthio, halogen; or tautomers or salts thereof, for use as a growth promotor.

A preferred compound according to claim 1, is phenylcapsaicin.

The invention also relates to a composition comprising a compound according to claim 1 or 2 for use as a food ingredient.

The invention also relates to a feed, comprising 2-500 mg/kg of a compound of formula 1 according to claim 1, preferably 2 to 100 mg/kg, more preferably 2-20 mg/kg.

The invention also relates to a compound according to claim 1 for uses as a growth promotor at a dose of 5 to 200 mg/kg body weight. The invention also relates to a compound, composition or feed according to any of the previous claims for use in bird feed as a growth promotor.

The invention also relates to bird feed comprising compounds or compositions according to any of the previous claim, wherein the feed is selected from one or more of the group consisting of seeds, corn, worms, millet, oat, peanuts and in the form of pellets, slurry, drinking water and emulsions.

The invention also relates to bird feed according to claim 7 , wherein the bird feed is used for birds selected from the group consisting of poultry, in particular broiler chicken; egg producing birds, in particular chickens; turkey; ostrich; quail; grouse; ducks; geese; wild birds; tame birds and breeding birds.

Synthetic capsaicinoids are well-suited to a variety of applications, including marine antifouling paint, pest control in forestry and agriculture and certain pharmaceutical applications. Phenylcapsaicin has also demonstrated potential properties as an anti-microbial agent in feed for commercial poultry production.

Another use of capsaicin related compounds is as rodent (mammal) repelling agents in outdoor feeding of birds and other animals. Synthetic derivatives produced in high purity and high quantity and to a reasonable cost will be superior to extracts from peppers in this use.

Figures

Figure 1. The predicted growth curves describing broiler mass increase, show that broilers on a diet containing 10 ppm phenylcapsaicin (dot) differs significantly from broilers fed control feed without phenylcapsaicin (square). No differences were found between the 10 and 20 ppm (triangular) treatment groups nor between the 20 ppm and the control group (squares). The mean broiler masses of the 10 ppm treatment group (orange dots) were always higher than the mean of the control group (square) from day 10 and onwards. In general, the 20 ppm means (triangular) are also lower than for the 10 ppm group but this is not statistically significant. The grey vertical dots show individual broiler mass samples for the control (light grey), 10 ppm (grey) and 20 ppm (dark grey) treatment groups, respectively.

Figure 2. Broilers fed a diet containing 10 ppm phenylcapsaicin increase in mass faster than broilers in the control group and the difference in mean body mass of broilers between these two treatment groups increase over time. The open circles show the difference in mean broiler mass (10 ppm - control) at any given sampling day, and the solid line shows the linear rate of increase for these data. The broken line at y = 0 shows the expected line giving no difference between treatments. The slopes of these two lines are significantly different (F=34.8, df=7, p<0.001), demonstrating an increase in growth in broilers fed the diet added 10 ppm phenylcapsaicin.

Experimental

A study with a total of 9000 broilers of the breed Partridge K90 (both males and females) where used in a broiler feed study for verification of the increasing growth rate.

There were 3000 individuals in the control group (t=2.84590, df=653, p=0.0046, figure 1) and 3000 individuals in each of the groups feed with a diet of 10 ppm phenylcapsaicin and 20 ppm phenylcapsaicin respectively.

Mean broiler masses at day 59 were 1551, 1521 and 1470 g for the 10 ppm, 20 ppm and control groups, respectively.

The growth of the broilers in the 20 ppm treatment group was not significantly different from neither the 10 ppm nor the control group.

The mean difference in broiler mass between the 10 ppm and control group at day 59 was 81 g, i.e. the increase in mean broiler mass of the 10 ppm group compared to the control group was 5.5 %. However, this difference is not statistically different when considering only the final broiler masses between these feed groups.

At sampling days 10-59 the mean broiler masses of the 10 ppm treatment group was consistently higher than the mean masses of the control group, and this difference in mean broiler mass between the 10 ppm and control groups increased over time (figure 2). This increase in mean difference between the two treatment groups is significantly different from the null-hypothesis expected if there was no effect of adding 10 ppm phenylcapsaicin to the feed on broiler growth (F=34.8, df=7, p<0.001).

The described differences in broiler growth between the 10 ppm and control groups are believed to be smaller than expected (conservative estimates) in the current study due to the following reasons:

• The mean body mass of broilers in the control group were c. 5 % larger (not significant) than for the broilers in the 10 ppm treatment group at the beginning of the feed study (day 3). In most biological systems such a 'head start' is expected to increase during a growth study. This was not found in this study.

• The cold spell is expected to result in reduced growth, as resources otherwise used for growth must be used in thermoregulation to maintain core body temperature.

• The treatment of broilers with antibiotics removes the cost of bacterial growth otherwise expected to differ between the phenylcapsaicin and control groups, thus reducing the expected differences between treatment groups.

• The population of broiler in the present study consists of both males and females. Males have been shown to have increased growth rates compared to females and this results in a sample with higher variance. Increased variance makes it more difficult to get statistically different results between treatments.

These results demonstrate, in a full size feeding experiment, that 10 ppm phenylcapsaicin has the potential to be added as a growth factor in poultry feed and suggest that this effect should be higher than the observed 5.5 % increase.

Toxicity studies

Comparative toxicity data between capsaicin and phenylcapsaicin:

Capsaicin and Phenylcapsaicin are rapidly absorbed from the gastro-intestinal tract and almost completely metabolized. The major route of excretion was biliary excretion and therefore, in the mass balance study, the majority of metabolites were detected in feces. Although the low recovery lowers the reliability of the study, derivation of reference values is still possible when considering a correction factor for systemic exposure. The metabolic pathways identified for Capsaicin and Phenylcapsaicin are identical, although the major metabolite is glucuronidated and oxidated for Phenylcapsaicin while the major metabolite of capsaicin is just glucuronidated. Tissue accumulation is unlikely to occur for both substances. In conclusion, the data support the read-across from capsaicin to close data gaps of

phenylcapsaicin.

Comparison of toxicokinetic and metabolism data

Capsaicin Phenylcapsaicin

Recovery of administered radioactivity after 168 h (24 h)(mass balance) urine 15.3% (13.8%) 3.3% (2.8%) faeces 32.3% (25.5%) 50.9% (43.1%)

Recovery within 7 days 47.6 % 54.1%

carcass 3.2% 4.2 %

Expired air Not assessed Not assessed

Tmax in tissues and blood, 0.5 h 0.5 h

plasma

The 14C located on the carbonyl group could easily be lost from the fatty acid chain and transferred to 14C-acetyl coenzyme A upon hydrolysis of the amide linkage, which means the radioactivity generated from 14C could be transferred to numerous endogenous substances. The author of the study concluded that therefore un-assigned radioactive peaks on the radiochromatograms are most likely endogenous substances which acquired their radioactivity from the biochemical metabolism of 14Clabeled fatty acid chain. Such metabolism of relabeled fatty acid chain could also explain the low recovery in the mass balance studies, as the biochemical product of fatty acid chain could either be numerous 14C -endogenous substances which are finally exhaled as 14C02. Low radioactivity in carcasses at the end of the study support thatl4C -phenyl-capsaicin and 14C-capsaicin was biotransformed to other relabeled volatile organic compounds in multiple steps and these metabolites were gradually released as expired air via the lung. If needed correction of the internal reference values (AEL) using the total recovery (50%) seems to be valid as the majority of metabolites are excreted in bile. Only 2-3% parent were detected in bile. Thus systemic availablity of the a.s. is likely. A complete absorption of Capsaicin was also postulated by

Donnerer et al. 1990. Naunyn-Schmiedeberg 's Arch. Pharmacol., 342, 357-361)

Metabolism data

Cumulative radioactivity in 47.1% 72.3%

bile (24 h)

Parent compound in bile (% 2 % 0.4%

of radioactive dose in bile)

Major tissues with abundant stomach , small intestine and stomach , small intestine and radioactivity liver liver

Total metabolites 50 27

Metabolic pathway hydrolysis, deamination, hydrolysis, deamination, hydrogenation, dehydrogenation

dehydrogenation, ,glucuronidation,

oxygenation, oxygenation,

glutathionation, demethylation, glutathionation,

glucuronidation and demethylation and sulfation sulfation

Major metabolite M42 (31.8% of the M18 (61.97% of the

radioactive dose in bile), radioactive dose in bile), glucuronidation glucuronidation and

oxygenation

Structure of major

metabolite metabolites

Blood to plasma ratio 0.61 - 1.17 0.55- 0.88

(0.5-24 h)

Acute Oral Tox - QECD 423:

2000 mg/Kg is applied by oral without produce mortality. Based on the test results, the test substance is unclassified according to the GHS. The acute oral toxicity LD50 value: LD50 > 5000 mg/Kg.

Acute Dermal Irritation/Corrosion - QECD 404:

The test item 0,5 mL was applied to the a gauze patch, then cover the gauze patch on a small area (approximately 6 cm ² ) of skin, which is held in place with non-irritating tape. The test item is non-irritationg to the rabbits skin within an observation period of up to 14 days starting after an exposure time of 4 hours.

Acute Eye Irritation/Corrosion - QECD 405:

0,1 ml of the test item was placed in the conjunctival sac of one eye of rabbit. The test substance is slightly irritant to the rabbit eye within an observation period of up to 7 days without washout after the test substance was placed in the conjunctival sac of one eye.

Acute Inhalation Tox - QECD 403. 2009:

Under the condtions of this study the single exposure acute inhalation LC50 of

phenylcapsaicin is not greater than 5.65 mg/L in male and female rats based on active substance.

Skin Sensitisation - QECD 406. 1992:

All animals survived throughout the test period without showing any clinical signs of toxicity. Under the conditions of the present study it can be stated that the test item Phenylcapsaicin caused no reactions identified as sensitisation at the tested concentration. According to Commission Regulation (EU) No. 286/2011 as well as GHS (Globally Harmonized

Classification System) the test item Phenylcapsaicin has no obligatory labelling requirement for skin sensitisation and is unclassified.

Reverse Mutation Assay - QECD 471, 1997:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Phenylcapsaicin did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Phenylcapsaicin is considered to be non-muytagenic in this bacterial reverse mutation assay.

Further experiment

The effects of dietary inclusion of 10 and 20 ppm phenylcapsaicin, the active ingredient of aXiphen®, on broiler chicken growth performance, Salmonella caecal colonisation and translocation to the spleen, and Salmonella in floor samples are described.

Methods

3000 Guangdong Partridge K90 broilers were divided into three equal groups and given a dietary inclusion of 0, 10 and 20 ppm phenylcapsaicin added to the normal feed. The broilers were kept in 15 sections, 5 sections for each feed treatment, in a single, cageless poultry house, and followed through the normal 60 days raising period of this variety of broiler chicken. The divisions were designed so that physical contact between broilers, or between broilers and droppings in different sections, was impossible.

Ten broilers from each section and all treatments were weighed weekly for growth

performance analysis (weekly sample). Body weight gain, feed intake, feed conversion ratio and mortality were calculated for the entire experimental period (0 - 60 days of age). Masses of the remaining broilers at broiler age 60 days were also reported (remaining population), but due to a misunderstanding it was reported in bulk and not individually. Statistical analysis can thus not be performed on these data, but total mean broiler mass and daily feed conversion ratio is reported for each diet group.

Five of the ten broilers, arbitrarily chosen, were sacrificed and analysed for caeca and spleen Salmonella numbers. From each section, Salmonella numbers was also analysed from five floor faeces samples weekly. No Salmonella was administered in this study and the prevalence of Salmonella in caeca, spleen and floor samples are the result of normal occurrence and transmission in a population of domestic broilers.

Main findings

Growth performance

The dietary inclusion of phenylcapsaicin significantly increased broiler body

mass.

Broiler mass: Mean body mass were 1828 g (weekly sample) and 1871 g (remaining population) for the 10 ppm group and 1710 g (weekly sample) and 1716 g (remaining population) for the control broilers. The mean body weight of the 20 ppm group were 1760 g and 1860 g for the weekly sample and remaining population, respectively.

For the weekly broiler sample at broiler age 60 days, and compared to the control group, broilers on the 10 ppm phenylcapsaicin diet showed a statistically significant increase (p = 0.002) in body mass of 118 g (6.9%). The increase for the remaining population was even larger at 155 g (9.0%). Corresponding increases for 20 ppm group were 50 g (2.9%) and 144 g (8.4%) for the weekly sample and remaining population, respectively. The differences in mean broiler masses between the weekly samples and the remaining population is probably due to random sampling effects when drawing the broilers in the weekly sample from the main population.

For the 20 ppm group, the 50 g mean increase in broiler mass of the weekly sample at day 60 is an underrepresentation of the real value, as described by the 144 g mean increase in the remaining broiler population at day 60. This increase in mean broiler mass for the remaining population of the 20 ppm group is larger than the statistically significant 118 g increase for the weekly sample of 10 ppm group. Following the logic of set theory, it is thus concluded that that the increase in mean broiler masses of the 10 ppm (155 g) and 20 ppm (144 g) groups of the remaining populations are both statistically significantly larger than the control broilers, as demonstrated for the weekly sample of the 10 ppm group (118 g) at broiler age 60 days.

Feed conversion: Mean daily feed consumption was 77.91 g, 80.34 g and 82.66 g for the control, 10 ppm and 20 ppm groups over the time of the study, respectively, with a statistically significant differences between treatment groups (p < 0.01). The feed conversion ratios over the 60 days raising period for the weekly samples were 2.73, 2.63 and 2.82 for the control, 10 ppm and 20 ppm groups, respectively, with an almost statistical difference between the 10 and 20 ppm groups (p = 0.06). The corresponding feed conversion ratios for the remaining population of broilers were 2.72, 2.58 and 2.67 for the control, 10 ppm and 20 ppm groups, respectively. No statistical analysis could be performed on these data due to missing individual measurements. However, because the differences between treatment groups are smaller than between the weekly samples, it seems safe to conclude that there are no differences in feed conversion ratios between treatment groups. Nevertheless, numerically there is a 5,1% reduction in feed conversion ratios between the 10 ppm and control group that could be of importance to the industry if confirmed by future studies.

Mortality: Morality was reduced over the time of the study for broilers in the 20 ppm feed group compared to the control group (p = 0.02). The total number of 'natural' broiler deaths was 72 (0.72%) in the control group and 57 (0.57%) and 43 (0.43%) for the 10 ppm and 20 ppm groups, respectively, with a 21% and 40% reduction between the control and the 10 and 20 ppm groups, respectively. This reduction in mortality was caused by differences in the number of deaths during the last two weeks of the experiment (broiler age 46 to 60 days). In general, highest mortalities in broiler chickens are found early and late in the raising period, and the results from the present study suggests that dietary inclusion of phenylcapsaicin reduce the mortality late in the raising period. However, this result has to be repeated in future studies before firm conclusions on the mortality reducing effects of aXiphen® can be drawn.

Salmonella

Dietary inclusion of both 10 and 20 ppm phenylcapsaicin reduced the number of broilers with caecal and spleen Salmonella. Only dietary inclusion of 20 ppm phenylcapsaicin reduced the number of samples with Salmonella on housing floor.

Caeca: The presence of Salmonella in the caecum seems to be rather modest in the present study. Of the 225 broilers examined for each feed group, only 33 (14.6%), 30 (13.3%) and 42 (18.6%) broilers were found to contain Salmonella for the 10 ppm, 20 ppm and control groups, respectively, with no difference in total number of infected broilers between treatments. At day 60, the numbers of infected broilers were 2 (8%), 0 (0%) and 7 (28%) for the 10 ppm, 20 ppm and control groups, respectively, with a significant effect of effect of treatment. The number of infected broilers is statistically significantly lower for the 20 ppm group compared to the control group (p = 0.01). No statistical significant differences were found between neither the 10 ppm and control groups nor between the 10 and 20 ppm groups. This effect of phenylcapsaicin on the number of broilers with caecal Salmonella is also found in statistical significant differences in the distribution of infected broilers between the treatment groups over the time of the study. For all treatment groups, the proportions of infected broilers at day 11 were above 76% and then dropped off in the middle of the time series. At the end of the experiment (day 60), the number again increased for the control group but continued to decline for both the 10 ppm and 20 ppm groups. The finding of differences in both the numbers and the distributions of broilers with Salmonella both support, and together strengthen, the conclusion that dietary inclusion of phenylcapsaicin reduces the number of broilers with caecal Salmonella compared to the controls

When looking at the number of Salmonella colony forming units (CFU) in the broilers, the data set is dominated by the absence of Salmonella in the vast majority of samples.

Consequently, no differences in the amount of Salmonella between treatment groups were found during the time of the study when including all broilers analysed. However, when looking at the weekly mean and maximum CFU counts of the 10 ppm treatment group, they are consistently lower than the mean and maximum counts of the control group (p < 0.03). Correspondingly, the CFU means (p = 0.03), but not the maximums, of the 20 ppm group are also lower compared to the control group. It thus seems that dietary inclusion of

phenylcapsaicin reduces the number of broilers with caecal Salmonella but does not do not reduce the number of CFU in broilers with caecal Salmonella to the same extent.

Spleen: Infection rates of the spleen also seem to be rather modest. Of the 225 spleens examined for each feed group during the study, only 20 (8.8%), 23 (10.2%) and 41(18.2%) broilers were found to contain Salmonella for the 10 ppm, 20 ppm and control groups, respectively. Nevertheless, there is an effect of treatment on the number of broilers with spleen Salmonella over the time of the study, with fewer broilers with infected spleens in both the 10 ppm and the 20 ppm (p < 0.02) groups compared to the control group. No differences were found between the 10 ppm and 20 ppm groups. At day 60, the numbers of infected broilers were 0 (0%), 0 (0%) and 3 (12%), with a statistical significant effect of treatment but no statistical significant difference between treatment groups. Over the time of the study, the distribution of the number of broilers with spleen Salmonella also differs between the control and the two phenylcapsaicin feed groups: In the control group the number of infected broilers slowly increase over the time of the study, while low initial numbers followed by a spike and then a decline were found in both the 10 and 20 ppm groups. The finding of differences in both the total numbers and numbers at day 60, and the distributions of broilers with

Salmonella over the time of the study, support, and together strengthen, the conclusion that dietary inclusion of phenylcapsaicin reduces the number of broilers with spleen Salmonella compared to the controls.

When analysing the number of Salmonella colony forming units (CFU) in the spleens, the data set is again dominated by absence of Salmonella in most samples and no differences in the amount of spleen CFU were found between treatment groups during the time of the study. Nevertheless, when comparing the weekly mean and maximum Salmonella counts between 10 ppm and control groups, phenylcapsaicin seemed to reduce the Salmonella infection; both the mean and maximum amount was lowest for the 10 ppm group for all but one of the sampling days (p = 0.08). No differences between the 20 ppm and the control groups were found. Again it seems that dietary inclusion of phenylcapsaicin reduces the number of broilers with spleen Salmonella but does not do not reduce the number of CFU in broilers with spleen Salmonella to the same extent.

Floor: The presence of Salmonella in floor samples was much higher than in both caeca and spleen. Of the 225 samples examined for each feed group over all sampling days, 126 (56%), 104 (46.2%) and 139 (61.7%) samples were found to contain Salmonella for the 10 ppm, 20 ppm and control groups, respectively. This gives a 9.4% and 25.2% reduction of samples with Salmonella in the 10- and 20 ppm groups compared to the control, and statistically significant reductions in Salmonella floor samples were found when comparing the 20 ppm group to both the 10 ppm and control groups (p < 0.05). No difference between the 10 ppm and control group was found. At day 60, the numbers of samples with Salmonella are 8 (32%), 6 (24%) and 10 (40%) for the 10 ppm, 20 ppm and control groups, respectively, with no effect of treatment between feed groups. The distribution in the number of floor samples with

Salmonella during the time of the study also seemed to differ between the 20 ppm and the control group (p = 0.08), with numbers of infected floor samples declining steeper in the 20 ppm group. No difference between the 10 ppm and control groups was found. For the 20 ppm group, the finding of differences in both the total number and the distribution of floor samples with Salmonella over the time of the study both support, and together strengthen, the conclusion that dietary inclusion of 20 ppm phenylcapsaicin reduces floor Salmonella compared to the controls.

When analysing the number of Salmonella colony forming units (CFU) in the floor samples, no differences between treatment groups were found. Nevertheless, the weekly mean, but not the maximum, CFU counts of the 10 ppm treatment group are consistently lower than the mean CFU counts of the control group (p < 0.001). No differences were found between the 20 ppm and control groups. It thus seems that dietary inclusion of 20 ppm, but not 10 ppm, phenylcapsaicin reduces the number of floor samples with Salmonella but does not reduce the number of CFUs in the floor samples.

Conclusion further experiment

The present study demonstrates that dietary inclusion of 10 or 20 ppm phenylcapsaicin is an effective growth promoter in commercial poultry production of the Guangdong Partridge K90 chicken by increasing broiler mass and reducing the number of Salmonella-infected broilers. It is thus concluded that the Salmonella reducing property of aXiphen® is transferable from laboratory to full-scale commercial poultry production facilities.