METHODS OF IMPROVING FISH FEED CONVERSION RATE AND SURVIVAL
20220167645 · 2022-06-02
Inventors
Cpc classification
A61P1/00
HUMAN NECESSITIES
A23K20/147
HUMAN NECESSITIES
A23K50/80
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to methods for improving fish feed conversion rates, health and survival by tachykinin antagonists. Specific fish feed consumptions comprising tachykinin antagonists are also provided.
Claims
1-40. (canceled)
41. A method of improving fish feed conversion rate (FCR), the method comprising administering to a fish population a composition comprising at least one tachykinin (TK) antagonist capable of binding to a piscine TK receptor and inhibiting its activity.
42. The method according to claim 41, wherein the reproductive system of the treated fish is not affected by the TK antagonist treatment.
43. The method according to claim 41, wherein the TK antagonist is a peptide-based molecule or a peptidomimetic.
44. The method according to claim 43, wherein the peptidomimetic is according to Formula I:
X.sub.1—NMeVal-X.sub.4-Leu-Met-Z (Formula I), wherein: the peptidomimetic consists of 5-10 amino acids; X.sub.1 is a stretch of 1-6 natural or non-natural amino acid residues and optionally an N-terminal capping moiety or modification; NMeVal is an N-methyl-Valine residue or N-methyl-D-Valine residue; X.sub.4 is —NH(CH.sub.2).sub.n—CO— wherein n is 2-6; and Z is the C-terminus of the peptidomimetic which may be amidated, acylated, reduced, or esterified.
45. The method according to claim 44, wherein X.sub.1 consists of 2 or 3 amino acid residues comprising an aromatic residue, a negatively charged residue and an N-terminus capping moiety.
46. The method according to claim 44, wherein the N-terminal capping moiety is a dicarboxylic acid residue.
47. The method according to claim 44, wherein the peptidomimetic consists of 5-10 amino acid residues comprising the sequence NMeVal-βAla-Leu-Met (SEQ ID NO. 7), and a sequence selected from the group consisting of: Succ-Asp-Phe; Succ-Asp-DPhe; Succ-Asp-Trp; Succ-Asp-DTrp; Succ-Asp-Ile-Phe; Succ-Asp-Ile-DPhe; Succ-Asp-Ile-Trp; Succ-Asp-Ile-DTrp; Succ-Asp-Ser-Phe; Succ-Asp-Ser-DPhe; Succ-Asp-Ser-Trp; Succ-Asp-Ser-DTrp, Succ-Glu-Phe; Succ-Glu-DPhe; Succ-Glu-Trp; Succ-Glu-DTrp; Succ-Glu-Ile-Phe; Succ-Glu-Ile-DPhe; Succ-Glu-Ile-Trp; Succ-Glu-Ile-DTrp; Succ-Glu-Ser-Phe; Succ-Glu-Ser-DPhe; Succ-Glu-Ser-Trp; and Succ-Glu-Ser-DTrp.
48. The method according to claim 44 wherein the peptidomimetic is selected from the group consisting of: TABLE-US-00014 (SEQ ID NO. 1, AN1) Succ-Asp-Ile-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 2, AN2) Succ-Asp-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 3, AN3) Succ-Asp-Ser-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 4, AN4) Succ-Asp-Ile-D-Trp-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 5, AN5) Succ-Asp-D-Trp-N(Me)Val-βAla-Leu-Met-NH.sub.2; and (SEQ ID NO. 6, AN6) Succ-Asp-Ser- D-Trp -N(Me)Val-βAla-Leu-Met-NH.sub.2; wherein Succ denotes a succinyl.
49. The method according to any one of claim 41, wherein the fish population comprises majority of fish aged 3-1200 dph.
50. The method according to claim 41, wherein the fish are edible fish.
51. The method according to claim 50, wherein the fish type is selected from the group consisting of: tilapia, carp, salmon, bass, catfish, and mullet.
52. The method according to claim 41, wherein the administering route is selected from parenteral administration, enteral administration, and administration by immersion.
53. The method according to claim 41, wherein the composition is administered to fish in a volume of water to be taken up by the gills or to be absorbed by the skin.
54. The method according to claim 53, wherein the composition is administered to fish as part of regular feed or water consumption.
55. The method according to claim 54, wherein the composition is administered as coating of feed pellets.
56. The method according to claim 55, wherein the feed pellets are coated with a final TK antagonist dose of 1-50 mg per one kg of feed.
57. The method according to claim 41, wherein the composition further comprises at least one nutrient or feed additive selected from the group consisting of color additive, taste additive, protein, carbohydrate, fat, mineral, and vitamin.
58. A method of improving at least one fish health parameter selected from the group consisting of: survival, skin condition, and resistance to pathogens; the method comprising administering to a fish population a composition comprising at least one tachykinin (TK) antagonist capable of binding to a piscine TK receptor and inhibiting its activity, wherein the TK antagonist is a peptide-based molecule or a peptidomimetic.
59. The method according to claim 58, wherein the peptidomimetic is according to Formula I:
X.sub.1—NMeVal-X.sub.4-Leu-Met-Z (Formula I), wherein: the peptidomimetic consists of 5-10 amino acids; X.sub.1 is a stretch of 1-6 natural or non-natural amino acid residues and optionally an N-terminal capping moiety or modification; NMeVal is an N-methyl-Valine residue or N-methyl-D-Valine residue; X.sub.4 is —NH(CH.sub.2).sub.n—CO— wherein n is 2-6; and Z is the C-terminus of the peptidomimetic which may be amidated, acylated, reduced, or esterified.
60. The method according to claim 58 wherein the peptidomimetic is selected from the group consisting of: TABLE-US-00015 (SEQ ID NO. 1, AN1) Succ-Asp-Ile-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 2, AN2) Succ-Asp-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 3, AN3) Succ-Asp-Ser-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 4, AN4) Succ-Asp-Ile-D-Trp-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 5, AN5) Succ-Asp-D-Trp-N(Me)Val-βAla-Leu-Met-NH.sub.2; and (SEQ ID NO. 6, AN6) Succ-Asp-Ser- D-Trp -N(Me)Val-βAla-Leu-Met-NH.sub.2; wherein Succ denotes a succinyl.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0151] The present invention provides methods for improving fish feed conversion rate and survival and for increasing fish growth profitability. The invention is based on inhibition of tachykinin receptor activity in the GI track and in the nervous system of fish.
[0152] Without wishing to be bound to any mechanism of action, it is proposed that the TK antagonists act via at least one mechanism selected from the group consisting of: reducing gut motility, increasing feed retention time in the gut, modulating secretion of electrolytes or digestion fluids from gut epithelial cells, influencing proliferation of gut cells, mitigating the adverse effects of inflammation on gut function and affecting microbial population in the gut.
[0153] It is also proposed, that TK antagonists protect fish from stress-induces processes, thereby improving overall health and survival.
[0154] Without wishing to be bound to any mechanism of action, it is also proposed that treatment of fish with TK antagonists may also result in increase in intestinal size of the fish.
[0155] It should be understood that treated fish do not necessarily gain extra weight following treatment with a TK antagonist, but rather a lower amount of feed is required by a TK antagonist-treated fish, to gain the same weight as an untreated fish from the same species and age. Effect on FCR is not related to the fish sexual maturation state, both adult/sexually mature fish and young fish are affected by and eligible for such TK antagonist treatment. Improvement of fish FCR, overall health or survival according to the present invention does not involve alteration of any piscine reproduction system parameter.
[0156] A TK antagonist according to the invention is a compound capable of binding to a piscine tachykinin receptor and inhibiting its activity. TK antagonists of the present invention may inhibit at least one NK receptor type present in fish, including but not limited to NK1R, NK2R and NK3R, either by orthosteric or allosteric binding.
[0157] A TK antagonist according to the present invention may be a peptide-based molecule, such as peptide, polypeptide or peptidomimetic, or a non-peptidic molecule.
[0158] A TK antagonist according to the present invention may be synthesized by any method known in the art including synthetic and recombinant production methods (when a peptide containing only natural amino acids is produced). Peptide-based TK antagonists may be produced, according to some embodiments, using solid or liquid phase synthesis. The design and synthesis of several TK antagonist peptidomimetics is described for example in WO 2016/009439.
[0159] Feed conversion rate or ratio (FCR) is weight to weight ratio between feed consumed by fish in a specific period and fish body mass gained at this period therefore indicates the efficiency of a feed or a feeding strategy. Improvement of FCR means decrease in its value, namely a low FCR indicate that it takes less feed to produce one kilogram of fish than it would if the FCR were higher. The FCR allows for an estimate of the feed that will be required in a growing cycle and therefore assists in predicting the profitability of the production. Decreasing FCR values generally leads to increased profitability since feed is the highest expense in fish farming. FCR is influenced by several parameters, such as water quality, fish health, method of feeding, temperature etc., and is generally lower in small fish than in the same species of fish that is larger. Low FCR also result in less waste polluting the pond water. With better water quality, the carrying capacity of the pond is increased (https://pdf.usaid.gov/pdf_docs/PA00K8MQ.pdf).
[0160] The physiology underlining FCR improvement disclosed in the methods of the present invention can be broken into several successive steps: Feed Intake.fwdarw.Digestion.fwdarw.Absorption.fwdarw.Conversion. While digestion and absorption relate to gut physiology, conversion occurs mainly in the soma and is determined by the availability of metabolites and energy in growth processes on the expanse of other energy consuming processes (metabolism).
[0161] In the relationship between FCR and growth, two main dogmas are possible: a. Improved FCR value which is a manifestation of similar growth performance with less feed consumed. b. Improved FCR that occurs when fish have better growth with similar or higher feed intake. Namely, improved FCR does not necessarily result in an increased growth rate, and vice versa—increased growth rate does not necessarily indicate an improved FCR. According to the first dogma, feed is digested and absorbed in higher efficiency, but growth potential is not altered. In the second dogma, a shift in metabolism towards growth may also be involved. Consistent with these scenarios, the present invention demonstrates different FCR-growth relationships: improved FCR and growth performance, and improved FCR which did not coincide with improved growth but rather with decrease in feed intake. These results suggest that TK antagonists act on both levels of gut physiology and metabolism, but that the effect on metabolism is evidential only at certain regimes.
[0162] The results of the present invention also suggest that treatment with TK antagonists improves fish immune response, mainly in the GI tract, and reduced stress levels resulting in better survival of the fish.
[0163] There are 3 TK receptors: tac1 (high affinity to SP), tac2 (high affinity to NKA) and tac3 (high affinity to NKB). Results presented herein demonstrate that the expression of all TK receptors (tac1, tac2, tac3) are altered in the different parts of the gut and in the skin following AN6 treatment. Without wishing to be bound to any mechanism of action, it is proposed that AN6 effect on fish physiology is mediated by more than one TK receptor and optionally, 2 or all 3 receptors are involved, with expected differences between tissues. The following observations suggest that AN6 effect is mediated primarily by local receptors in the gut: TK receptors mRNA expression (tac1, tac2, tac3) is altered in the different parts of the gut following AN6 treatment, suggesting local effect on gut physiology; AN6 is cleared from blood very quickly (˜4.5 min); there is no effect on gonadal state when administering 10-30 ppm of AN6; effect on growth and FCR observed even before sexual maturation (before 90 dph) and no peptide residual is detected in muscle and blood after treatment.
[0164] The contribution of tachykinins to homeostasis and diseases of the skin is well established (Scholzen et al., Exp Dermatol, 7(2-3):81-961998). The involvement of TK in fish skin pigmentation is addressed in Huali et al. 2018 (The FASEB Journal 32(6):3193-3214). Skin health has great importance for the aquaculture of edible fish (e.g., salmon and trout) as well as for ornamental fish farming. According to some embodiments, the methods and compositions of the present invention are used for improving skin health.
[0165] Fish age is determined in the present invention as days post hatching (dph). For example, in tilapia, early juvenile, characterized by self-feeding and specific coloration is about 7-25 dph; late juvenile characterized by gradual disappearance of specific coloration and by maturation of the gametes, is between 25-95 dph; while adult tilapia fish are usually about 96 dph. In other fish types growth stages are at different dph. Effective treatment age of fish depends on the fish type. Tilapia are treated at age 0 to 4 month and marketed at 8 to 10 months while salmon are marketed at age of 2-3 years and treatment can occur anywhere in until that age.
[0166] The experiments of the present invention suggest that effects on reproduction and growth are not linked and that the compounds of the present invention are safe when administered to fish and do not influence the reproductive system. Histological data on fish gonads indicate that gonads were normally developed in the treated fish and that TK receptor (Tac) expression levels are not altered due to treatment.
[0167] According to some embodiments, gender specific effects may be different in other type of fish.
[0168] According to some specific embodiments, the method of the present invention comprises administering as an active ingredient a compound selected from the group consisting of:
TABLE-US-00003 (SEQ ID NO. 1, AN1) Succ-Asp-Ile-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 2, AN2) Succ-Asp-Phe-N(Me)Val-βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 3, AN3) Succ-Asp-Ser-Phe-N(Me)Val- βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 4, AN4) Succ-Asp-Ile-D-Trp-N(Me)Val- βAla-Leu-Met-NH.sub.2; (SEQ ID NO. 5, AN5) Succ-Asp-D-Trp-N(Me)Val- βAla-Leu-Met-NH.sub.2; and (SEQ ID NO. 6, AN6) Succ-Asp-Ser- D-Trp -N(Me)Val-βAla-Leu-Met-NH.sub.2;
[0169] The peptides of the present invention are preferably synthesized using conventional synthesis techniques known in the art, e.g., by chemical synthesis techniques including peptidomimetic methodologies. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase peptide synthesis procedures are well known in the art. A skilled artesian may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. Synthetic peptides can be purified by preparative high-performance liquid chromatography, and the composition of which can be confirmed via amino acid sequencing. Conjugation of peptidic and permeability moieties may be performed using any methods known in the art, either by solid phase or solution phase chemistry. Some of the preferred compounds of the present invention may conveniently be prepared using solution phase synthesis methods. Other methods known in the art to prepare compounds like those of the present invention can be used and are comprised in the scope of the present invention.
[0170] N-terminal capping or modification according to the present invention denotes alteration of the peptide's sequence by covalently attaching a chemical moiety to the terminal amine resulting in modified charge, activity and/or stability to cleavage by amino peptidases.
[0171] Non-limitative examples of a permeability-enhancing moiety include: hydrophobic moieties such as fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides. According to some embodiments, the hydrophobic moiety is a lipid moiety or an amino acid moiety.
[0172] A permeability-enhancing moiety may be connected to any position in the peptide moiety, directly or through a spacer. According to specific embodiments, the cell-permeability moiety is connected to the amino terminus of the peptide moiety. The optional connective spacer may be of varied lengths and conformations comprising any suitable chemistry including but not limited to amine, amide, carbamate, thioether, oxyether, sulfonamide bond and the like. Non-limiting examples for such spacers include amino acids, sulfone amide derivatives, amino thiol derivatives and amino alcohol derivatives.
[0173] The term “peptide” or “peptide-based” as used herein is meant to encompass natural (genetically encoded), non-natural and/or chemically modified amino acid residues, each residue being characterized by having an amino and a carboxy terminus, connected one to the other by peptide or non-peptide bonds. The amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. The peptides and peptidomimetics of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
[0174] The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, either the L or D isomers may be used. When a “D”—precedes the amino acid, a D isomer is used.
[0175] Conservative substitution of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions includes replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, affinity to the target protein, metabolic stability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
[0176] The following is an example of classification of the amino acids into six groups, each contains amino acids that are conservative substitutions for one another:
[0177] 1) Alanine (A), Serine (S), Threonine (T);
[0178] 2) Aspartic acid (D), Glutamic acid (E);
[0179] 3) Asparagine (N), Glutamine (Q);
[0180] 4) Arginine (R), Lysine (K);
[0181] 5) Isoleucine (I), Leucine (L), methionine (M), Valine (V); and
[0182] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0183] Other classifications into somehow different groups (for example, aliphatic, polar, non-polar, hydrophilic, hydrophopic etc.) are also known in the art and can be used for conservative amino acid substitutions according to the present invention.
[0184] Also included within the scope of the invention are salts of the peptides, analogs, and chemical derivatives of the peptides of the invention.
[0185] As used herein the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and/or mineralization of calcium minerals.
[0186] A “chemical derivative” as used herein refers to peptides containing one or more chemical moieties not normally a part of the peptide molecule such as esters and amides of free carboxy groups, acyl and alkyl derivatives of free amino groups, phospho esters and ethers of free hydroxy groups. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Preferred chemical derivatives include peptides that have been phosphorylated, C-termini amidated or N-termini acetylated.
[0187] “Functional derivatives” of the peptides of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.
[0188] The term “peptide analog” indicates molecule which has the amino acid sequence according to the invention except for one or more amino acid changes or one or more modification/replacement of an amide bond. Peptide analogs include amino acid substitutions and/or additions with natural or non-natural amino acid residues, and chemical modifications which do not occur in nature. Peptide analogs include peptide mimetics. A peptide mimetic or “peptidomimetic” means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with other covalent bond. A peptidomimetic according to the present invention may optionally comprises at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate “analogs” may be computer assisted. Additional peptide analogs according to the present invention comprise a specific peptide or peptide analog sequence in a reversed order, namely, the amino acids are coupled in the peptide sequence in a reverse order to the amino acids order which appears in the native protein or in a specific peptide or analog identified as active. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of chemical moieties that closely resembles the three-dimensional arrangement of groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site structure, the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.
[0189] A modified amino acid residue is an amino acid residue in which any group or bond was modified by deletion, addition, or replacement with a different group or bond, as long as the functionality of the amino acid residue is preserved or if functionality changed (for example replacement of tyrosine with substituted phenylalanine) as long as the modification did not impair the activity of the peptide containing the modified residue.
[0190] “A peptide conjugate” according to the present invention, denotes a molecule comprising a TK antagonistic peptide to which another moiety, either peptidic or non peptidic, is covalently bound, directly or via a linker. According to some embodiments, a permeability-enhancing moiety, e.g. a fatty acid residue is bound to the peptidic TK antagonist.
[0191] The term “linker” denotes a chemical moiety, a direct chemical bond of any type, or a spacer whose purpose is to link, covalently, a cell-permeability moiety and a peptide or peptidomimetic. The spacer may be used to allow distance between the permeability-enhancing moiety and the peptide.
[0192] “Permeability” refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin layer. A “cell permeability” or a “cell-penetration” moiety refers to any molecule known in the art which is able to facilitate or enhance penetration of molecules through membranes. Non-limitative examples include: hydrophobic moieties such as lipids, fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids, transporter peptides, nanoparticles and liposomes.
[0193] The term “physiologically acceptable carrier” or “diluent” or “excipient” refers to an aqueous or non-aqueous fluid that is well suited for pharmaceutical preparations. Furthermore, the term “a pharmaceutically acceptable carrier or excipient” refers to at least one carrier or excipient and includes mixtures of carriers and or excipients. The term “therapeutic” refers to any pharmaceutical, drug or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury in a patient.
Pharmacology
[0194] Apart from other considerations, the fact that the novel active ingredients of the invention are peptides, peptide analogs or peptidomimetics, dictates that the formulation be suitable for delivery of these types of compounds. Although in general peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes novel methods are being used, in order to design and provide metabolically stable and oral bioavailable peptidomimetic analogs.
[0195] The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally, by inhalation or parenterally, and are specifically formulated for the administration route. The compositions are formulated according to the administration route.
[0196] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
[0197] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
[0198] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
[0199] For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art.
[0200] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
[0201] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0202] For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.
[0203] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art (Pillai et al., Curr. Opin. Chem. Biol. 5, 447, 2001). Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
[0204] Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
[0205] Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).
[0206] The doses for administration of such pharmaceutical compositions range according to some embodiments of the present invention from about 0.1 mg/kg to about 50 mg/kg body weight.
[0207] Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.
[0208] In certain embodiments, peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the peptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Pat. No. 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).
[0209] Elevated serum half-life can be maintained by the use of sustained-release protein “packaging” systems. Such sustained release systems are well known to those of skill in the art. In one preferred embodiment, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy, 1998, Biotechnol. Prog. 14, 108; Johnson et al., 1996, Nature Med. 2, 795; Herbert et al., 1998, Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the protein in a polymer matrix that can be compounded as a dry formulation with or without other agents.
[0210] In certain embodiments, dosage forms of the compositions of the present invention include, but are not limited to, biodegradable injectable depot systems such as, PLGA based injectable depot systems; non-PLGA based injectable depot systems, and injectable biodegradable gels or dispersions. Each possibility represents a separate embodiment of the invention. The term “biodegradable” as used herein refers to a component which erodes or degrades at its surfaces over time due, at least in part, to contact with substances found in the surrounding tissue fluids, or by cellular action. In particular, the biodegradable component is a polymer such as, but not limited to, lactic acid-based polymers such as polylactides e.g. poly (D,L-lactide) i.e. PLA; glycolic acid-based polymers such as polyglycolides (PGA) e.g. Lactel® from Durect; poly (D,L-lactide-co-glycolide) i.e. PLGA, (Resomer® RG-504, Resomer® RG-502, Resomer® RG-504H, Resomer® RG-502H, Resomer® RG-504S, Resomer® RG-502S, from Boehringer, Lactel® from Durect); polycaprolactones such as Poly(e-caprolactone) i.e. PCL (Lactel® from Durect); polyanhydrides; poly(sebacic acid) SA; poly(ricenolic acid) RA; poly(fumaric acid), FA; poly(fatty acid dimmer), FAD; poly(terephthalic acid), TA; poly(isophthalic acid), IPA; poly(p-{carboxyphenoxy}methane), CPM; poly(p-{carboxyphenoxy} propane), CPP; poly(p-{carboxyphenoxy} hexane) s CPH; polyamines, polyurethanes, polyesteramides, polyorthoesters {CHDM: cis/trans-cyclohexyl dimethanol, HD:1,6-hexanediol. DETOU: (3,9-diethylidene-2,4,8,10-tetraoxaspiro undecane)}; polydioxanones; polyhydroxybutyrates; polyalkylene oxalates; polyamides; polyesteramides; polyurethanes; polyacetals; polyketals; polycarbonates; polyorthocarbonates; polysiloxanes; polyphosphazenes; succinates; hyaluronic acid; poly(malic acid); poly(amino acids); polyhydroxyvalerates; polyalkylene succinates; polyvinylpyrrolidone; polystyrene; synthetic cellulose esters; polyacrylic acids; polybutyric acid; triblock copolymers (PLGA-PEG-PLGA), triblock copolymers (PEG-PLGA-PEG), poly (N-isopropylacrylamide) (PNIPAAm), poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) tri-block copolymers (PEO—PPO-PEO), poly valeric acid; polyethylene glycol; polyhydroxyalkylcellulose; chitin; chitosan; polyorthoesters and copolymers, terpolymers; lipids such as cholesterol, lecithin; poly(glutamic acid-co-ethyl glutamate) and the like, or mixtures thereof.
[0211] In some embodiments, the compositions of the present invention comprise a biodegradable polymer selected from, but not limited to, PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, polyphosphazene and the like. Each possibility represents a separate embodiment.
Fish Feed Compositions
[0212] The feed compositions of the present invention may be part of, or mixed with conventional of specific fish feed. Fish feed is normally specific for the type of fish to be nourished and includes proteins, oils, vitamins and other additives.
[0213] According to some non-limiting examples, the compositions of the present invention are pelletized, solid compositions containing, about 15 to 60 percent protein or protein hydrolysate, about 2 to 15 percent fat, or about 3 to 10 percent crude fiber together with adjuvants, such as minerals, vitamins, and/or trace elements. According to certain embodiments, the feed compositions contain 25-55 percent protein and 15-25 percent lipid (fat). According to some embodiments, the feed compositions further comprise salts, betaine, amino acids, nucleotides and other supplements to improve the osmotic adaptation of young fish to seawater and for better survival. According to further embodiments, the feed composition supplied to the fish is changed during the period of fish growth, for example, the protein content is decreased and the lipid content is increased during the seawater grow-out phase.
[0214] Fish feed composition comprising the antagonists of the present invention are prepared using methods known in the art. For example, pellets, typically 0.5-20 millimeters in diameter, are made from the composition and dried prior to storage and use. The coherence of the pellets may be improved by dissolving a small amount of gelatin in the water. If water-soluble whey powder provides much of the protein content, the pellet surfaces are preferably coated with a little oil or fat to prevent premature disintegration of the pellets upon contact with water. Gelatin-bearing compositions may be foamed in a conventional manner to produce cellular pellets whose overall density is similar to that of water. Such pellets float in water and remain accessible to the fish for a relatively long period. Pellets that sink to the bottom are lost to many fish. The antagonists of the invention may also be mixed with commercial fish feed of conventional composition by uniformly distributing the antagonists in the conventional composition and thereafter making pellets from the mixture obtained. According to some embodiments, feed pellets are coated with the compounds of the invention and used for feeding the fish.
[0215] The feed compositions of the present invention may be prepared by extrusion, a process by which a feed or food material is pushed, using a piston or screw, through an orifice or a die of a given shape and size. Extruders are available in several designs depending on their application, but are generally classified based on the number of screws. The two general types of extruders are single-screw and twin-screw configurations. Single screw extruders operate based on the pressure requirement of the die, slip at the barrel wall and the extent to which the screw is filled, whereas twin screw extruders operate based on the direction the two screws rotate and the extent of intermeshing between the two screws. There are two main extrusion process: dry process, wherein the heat from friction of materials is used to force the materials to pass through the die holes; and wet process, similar to the dry process but with addition of water and vapor up to 20-30% moisture level. Any extruder and extruding method may be used for preparing feed compositions according to the present invention.
[0216] Although the present invention has been described with respect to various specific embodiments thereof in order to illustrate it, such specifically disclosed embodiments should not be considered limiting. Many other specific embodiments will occur to those skilled in the art based upon applicants' disclosure herein, and applicants propose to be bound only by the spirit and scope of their invention as defined in the appended claims.
EXAMPLES
[0217] The following examples demonstrate the production and in-vivo activity of TK antagonists in improving fish FCR and health and reducing overall mortality.
[0218] Three separate experiments (#1, #2 and #3) on fish are described in the following examples. Experiment #1 studied the effects of supplementing fish feed with two different doses of AN6 peptide in two phases, from 10 dph for 90 days and from 207 dph for 50 days. Experiment #2 examined the effects of supplementing fish feed with either 5 or 10 mg AN6/kg different doses of AN6 from 60 dph for 90 days. Experiment #3 examined the effects of supplementing fish feed starting from 10 or 90 dph with either 5, 10, or 30 mg AN6/kg for various lengths of time. Results were analyzed for a number of growth-related parameters.
Example 1—Feed Coating
[0219] MeM (powder, manufactured by Bern Aqua), Nanolis P1, P2 (pellet sizes of 0.8-1 mm, 1-1.5 mm, respectively, manufactured by Ocialis) and Tilaphi 1, 2 & 3 (pellet sizes of 1-1.5 mm, 2 mm, 3-5.5 mm, respectively, manufactured by Ocialis) were used as basic regular feed. The basic feed was coated according to the following general procedure: 9 ml or 3 ml of Tris-HCl solution (50 mM, pH=8.8) were diluted to 60 ml with distilled H.sub.2O. For AN6-containing feed preparation, desired amounts of AN6 peptide (a TK antagonist having the sequence Succ-Asp-Ser-D-Trp—N(Me)Val-βAla-Leu-Met-NH.sub.2, SEQ ID NO. 6) were dissolved in the Tris-HCl solution. Then, each of the various solutions was mixed with 3 Kg of basic feed pellets using an electric mixer. The peptide-feed mixtures where then dried either in oven at 40° C. for 24 hours or in drying drum for few hours to form coated pellets. The feed compositions, containing different amount of peptide coating are summarized in Table 1. The total amount of each feed was 3 Kg.
TABLE-US-00004 TABLE 1 Compositions of feed coating and peptide doses for treatment groups of experiments #1 and #2 Treatment Feed Final Peptide Dose Group type Coating composition (mg AN6/Kg feed) A Phase 1 Feed A 9 ml of 50 mM Tris-HCl 0 (control) 51 ml distilled H2O B Phase 1 Feed C 3 ml of 50 mM Tris-HCl 10 57 ml distilled H2O 30 mg AN6 A Phase 2 Feed A 9 ml of 50 mM Tris-HCl 0 (control) 51 ml distilled H2O B Phase 2 Feed D 9 ml of 50 mM Tris-HCl 30 51 ml distilled H2O 90 mg AN6 C Feed A 9 ml of 50 mM Tris-HCl 0 (control) 51 ml distilled H2O D Feed B 3 ml of 50 mM Tris-HCl 5 57 ml distilled H2O 15 mg AN6 E Feed C 3 ml of 50 mM Tris-HCl 10 57 ml distilled H2O 30 mg AN6
Example 2. Oral Tachykinin Antagonist Treatment Improves Growth of Adult Fish
[0220] Tilapia fish (n=3600) have been raised from age 10 to 106 days post-hatch (dph) in indoor tanks kept under artificial lightening (12 h light; 12 h dark), and then transferred to cages in ponds for 160 days (total 8-9 months). The fish were fed to apparent satiation 2-4 times a day, for about 90 days (10-106 dph, Phase 1), either with regular feed (Feed A, Group A, n=600 in 3 repeats) or with feed coated with AN6 peptide solution in a ratio of 10 mg AN6/Kg feed (10 ppm, Feed C, Group B, n=600 in 3 repeats). In the second phase, fish in group B were fed for 50 days (207-257 dph, Phase 2) with feed coated with AN6 peptide solution, in a ratio of 30 mg AN6/Kg feed (30 ppm, Feed D, Group B). Between days 107 and 206 the fish received control feed (feed A). The experimental conditions are summarized in Table 2.
TABLE-US-00005 TABLE 2 Summary of experimental conditions and treatments (experiment #1). Initial Final Tanks/ density density Age Cages Tanks (Kg (Kg (dph) size (L) location Fish/m.sup.3) Fish/m.sup.3) Treatment 10-83 400 Indoors 0.075 25 Feed A/Feed C 83-106 200 Indoors 13 130 Feed A/Feed C 106-195 10400 Outdoors 2.5 9.6 Feed A 195-263 41600 Outdoors 2.3 4.3 Feed A/Feed D
[0221] During the experiment period, the average fish weight was measured, and the daily growth rate was calculated by dividing the average weight gained in each measuring period by the number of days since the last measurement. The results showed that there were no differences in fish average weight and daily growth rate during the first 200 days of experiment (
[0222] In order to eliminate the effect of initial body weight on average growth rate, the weight gained between each two measuring points was normalized according to the initial body weight, showing the same trend (
Example 3: Oral Tachykinin Antagonist Treatment Improves Growth of Adult Fish
[0223] Tilapia fish (n=1440) have been raised from day 60 to day 119 post-hatch in indoor tanks and then transferred to outdoor tanks (120-150 dph) followed by cages in ponds (150-243 dph). The fish were fed to apparent satiation 2-4 times a day, for 90 days (60-150 dph) either with regular feed (Feed A, Group C, n=60 in 8 repeats); feed coated with AN6 peptide solution in ratio of 5 mg AN6/Kg feed (5 ppm, Feed B, Group D, n=60 in 8 repeats); or feed coated with AN6 peptide solution in ratio of 10 mg AN6/Kg feed (Feed C, Group E, n=60 in 8 repeats). The experimental conditions are summarized in Table 3.
TABLE-US-00006 TABLE 3 Summary of experimental conditions and treatments (experiment #2). Initial Final Tanks/ density density Age Cages Tanks (Kg (Kg (dph) size (L) location Fish/m.sup.3) Fish/m.sup.3) Treatment 60-119 200 Indoors 3 30 Feed A/Feed B/ Feed C 120-150 1000 Outdoors 6 13 Feed A/Feed B/ Feed C 150-250 2600 Outdoors 5 8 Feed A
During the experimental period, the average fish weight was measured, and the daily growth rate was calculated by dividing the average weight gained in each measuring period by the number of days since the last measurement. The results showed that from age 95 to 165 dph AN6 peptide treatment (10 mg/Kg feed, Group E) resulted in steady and relatively low increase in fish average weight and growth rate (˜+3%). However, from age 200 dph, a considerable jump in fish performance and FCR was observed, reaching a maximal level at the last measurement point (243 dph, +8.4% average weight, +18% weight gain, +10% total biomass;
[0224] The observed effect of AN6 treatment (10 mg/Kg feed) on growth rate, long after peptide treatment (age 150 dph), may point on long-term or delayed physiological changes following AN6 peptide treatment.
Example 4: Oral Tachykinin Antagonist Treatment Improves Fish Survival
[0225] During the experiments described above, dead fish were removed from tanks before each feeding, and then counted and weighed. Dead fish were not replaced after the beginning of the experiment. In the cage systems, in which dead fish are undetectable, actual mortality was estimated by the difference between initial and final count.
[0226] During the experiment described in example 2 (experiment #1), the fish were exposed to high stocking densities in indoor tanks (13-130 kg/m.sup.3, 83-106 dph, Table 2,
[0227] Pond water differ from tank water in some parameters including the presence of cellular organisms such as algae, small arthropod and bacteria that are normally absent in the relatively clean tank water. While algae and small arthropod are added directly to the fish diet, bacteria and virus can challenge fish immune system.
[0228] The importance of functional immune system in improving fish survival following transfer was suggested regarding the migration of wild salmonid smolts from their nursery lake to the north pacific ocean (Jeffries et al. Mol Ecol. 2014, 23(23), 5803-5815). This study showed that differences in immune gene expression can predict the ability of smolts to survive viral pathogens and elevated stress levels during the freshwater migration.
[0229] The high mortality observed in the pond may therefore be attributed to exposure to pathogens inside the pond, followed by death due to compromised immune system, and/or elevated stress levels during migration. It is proposed that treatment with the TK antagonist AN6 improves fish immune response, mainly in the GI tract, and reduced stress levels resulting in better survival in the pond (
Example 5: Oral Tachykinin Antagonist Treatment Improves Growth Tilapia Fish (Experiment #3)
[0230] The following fish feed was used in this experiment: MeM (powder, manufactured by Bern Aqua), Nanolis P1, P2 (pellet sizes of 1 mm, 1.2-1.5 mm, respectively, manufactured by Ocialis) and Tilaphi 2, 3 & 4 (pellet sizes of 2 mm, 3-4 mm & 5.5 mm manufactured by Ocialis). The feed content is in described in Table 4.
TABLE-US-00007 TABLE 4 Composition of feed coating per 3 kg of feed (for use in experiment #3) Final Peptide Dose Feed type Coating composition (mg AN6/Kg feed) Feed E (Control diet) 30 ml distilled H2O 0 Feed F (5 ppm AN6) 1.25 ml of 50 mM Tris-HCl 5 28.75 ml distilled H2O 15 mg AN6 Feed G (10 ppm AN6) 2.5 ml of 50 mM Tris-HCl 10 27.5 ml distilled H2O 30 mg AN6 Feed H (30 ppm AN6) 7.5 ml of 50 mM Tris-HCl 30 22.5 ml distilled H2O 90 mg AN6
[0231] Feed was coated using the following general procedure: Different amounts of the peptide AN6 (SEQ ID NO: 6) were prepared by dissolving the peptide in Tris-HCl solution (50 mM, pH=8.8) in ratio of 15 mg peptide to 1.25 ml Tris-HCl solution. The Tris-HCl-Peptide solution was then diluted to final volume of 30 ml with distilled H.sub.2O as indicated in Table 4). Then, each of the various solutions was mixed with 3 Kg of the basic feed pellets (depending on fish size) using an electric mixer. The peptide-feed mixtures where then dried either in oven at 40° C. for 24 hours or in drying drum (at room temperature) for few hours to form coated pellets.
[0232] Tilapia fish (n=4800) were raised from day 10 (0.05 g) post-hatch (dph) in indoor tanks and then transferred to outdoor cages in ponds (40-279 dph). Each cage/tank is an experimental unit. The fish were fed to apparent satiation 2-4 times a day with feed appropriate to their age and treatment as described in Table 5.
TABLE-US-00008 TABLE 5 Feed type and feeding frequency (experiment #3) Stages Ages Pellet sizes Frequency Feeding time 1 10 DOA-20 DOA 300-500 MeM 4 8:00; 10:30; 13:00; 15:00 2 20 DOA-30 DOA 500-800 MeM 4 8:00; 10:30; 13:00; 15:00 3 30 DOA-50 DOA Nanolis 1 (1 mm) 4 8:00; 10:30; 13:00; 15:00 4 50 DOA-80 DOA Nanolis 2 (1.2-1.5 mm) 4 8:00; 10:30; 13:00; 15:00 5 80 DOA-120 DOA Tilaphi 2 (2.0 mm) 2 8:00; 15:00 6 120 DOA-150 DOA Tilaphi 3 (3-4 mm) 2 8:00; 15:00 7 150 DOA-180 DOA Tilaphi 3 (5.5 mm) 2 8:00; 15:00 8 180 DOA-270 DOA Tilaphi 4 2 8:00; 15:00
[0233] The trial included total 48 cages representing 8 treatments, with 6 repeats each. Each cage contained about 100 fish. Treatments are below: [0234] Treatment 1 (Control): The control diet (Feed E) without the addition of the peptide (AN6). Fed to fish throughout the entire growth-trial period (for 279 days); [0235] Treatment 2 (5 ppm from 10 dph for 120 days): The control diet with the addition of 5 mg peptide/kg of diet (Feed F). Fish were fed the peptide enriched diet for 120 days (4 months), from 10 days of age (D0—the first day of the experiment) to 130 days of age (D120), and the control diet during the last 6 months (159 days) (Feed E); [0236] Treatment 3 (10 ppm from 10 dph for 60 days): The control diet with the addition of 10 mg peptide/kg of diet (Feed G). Fish were fed the peptide enriched diet for 60 days (2 months) from 10 days of age (D0) to 70 days of age (D60), and the control diet during the last 8 months (219 days) (Feed E); [0237] Treatment 4 (10 ppm from 10 dph for 90 days): The control diet with the addition of 10 mg peptide/kg of diet (Feed G). Fish were fed the peptide enriched diet for 90 days (3 months) from 10 days of age (D0) to 100 days of age (D90) and the control diet for the last 7 months (189 days) (Feed E); [0238] Treatment 5 (10 ppm from 10 dph for 120 days): The control diet with the addition of 10 mg peptide/kg of diet (Feed G). Fish were fed the peptide enriched diet for 120 days (4 months) from 10 days of age (D0) to 130 days of age (D120) and the control diet for the last 6 months (159 days) (Feed E); [0239] Treatment 6 (10 ppm from 100 dph for 30 days): The control diet with the addition of 10 mg peptide/kg of diet (Feed G). Fish were fed the peptide enriched diet for 30 days (1 month) from 100 days of age (D90) to 130 days of age (D120) and the control diet for the first three months (90 days from D0 to D90) and the last 6 months (159 days) (Feed E); [0240] Treatment 7 (30 ppm from 10 dph for 60 days): The control diet with the addition of 30 mg peptide/kg of diet (Feed H). Fish were fed the peptide enriched diet for 60 days (2 months) from 10 days of age (D0) to 70 days of age (D60), and the control diet during the last 8 months (219 days) (Feed E); [0241] Treatment 8 (30 ppm from 10 dph for 90 days): The control diet with the addition of 30 mg peptide/kg of diet (Feed H). Fish were fed the peptide enriched diet for 90 days (3 months) from 10 days of age (D0) to 100 days of age (D90) and the control diet for the last 7 months (189 days) (Feed E).
[0242] During the experimental period, fish weight and length were measured once a month. Individual fish sex was determined at days 96 and 279 of the experiment. Average fish weight per cage were calculated for each measurement period (
TABLE-US-00009 TABLE 6 Effect of TK antagonist on average survival, weight, weight gain and FCR in last month of experiment (D 236 to D 279) Average Total Average Average Average Treatment density weight/ weight/ weight gain/ FCR/ group (body/cage) cage (g) fish (g) fish (g) cage 2 0.45% 6.71% 6.41% p = 0.0006 6.18% −2.14% 3 6.91% 10.27% 3.44% p = 0.0776 9.19% −4.28% 4 3.34% 7.37% 3.89% p = 0.0222 1.69% −2.14% 5 −1.78% 1.28% 3.29% 7.59% −0.53% 6 −1.55% −0.53% 0.97% 2.11% 0.00% 7 −4.89% −4.56% 1.37% 1.43% 0.00% 8 −0.67% 6.23% 7.11% p = 0.0001 13.57% −2.14%
All values were calculated as % change relative to control.
[0243] Differential analysis of male vs female performances revealed that both males and females are affected by AN6 treatment, having improved average weight compared to their respective controls (Tables 7A and B).
TABLE-US-00010 TABLE 7A Effect of TK antagonist on average female weight and length (experiment #3) D 96 D 279 Female Fish Weight/fish (g) Length/fish (cm) Weight/fish (g) Length/fish (cm) Treatment Mean Std Err Mean Std Err Mean Std Err Mean Std Err 1 (Control) 82.22 1.44 15.79 0.09 601.9 8.91 30.13 0.16 2 87.35 1.24 15.85 0.08 651.83 10.43 30.76 0.15 3 87.67 1.52 16.03 0.09 629.02 9.95 30.5 0.15 4 86.4 1.53 15.96 0.09 631.17 9.45 30.48 0.14 5 82.96 1.54 15.95 0.09 611.23 12.45 30.33 0.19 6 87.28 1.41 16.04 0.09 617.44 9.62 30.45 0.15 7 82.52 1.44 15.73 0.08 628.2 10.42 30.53 0.14 8 89.02 1.55 16.17 0.09 649.16 11.37 30.65 0.16 Δ Mean % (Treatment vs. control) 2 6.24% p = 0.0090 0.38% 8.30% p = 0.0004 2.09% p = 0.0027 3 6.63% p = 0.0079 1.52% p = 0.0433 4.51% 1.23% 4 5.08% p = 0.0418 1.08% 4.86% p = 0.0418 1.16% 5 0.90% 1.01% 1.55% 0.66% 6 6.15% p = 0.0136 1.58% p = 0.0391 2.58% 1.06% 7 0.36% −0.38% 4.37% 1.33% 8 8.27% p = 0.0010 2.41% p = 0.0016 7.85% p = 0.0010 1.73%
TABLE-US-00011 TABLE 7B Effect of TK antagonist on average male weight and length (experiment #3) D 96 D 279 Male Fish Weight/fish (g) Length/fish (cm) Weight/fish (g) Length/fish (cm) Treatment Mean Std Err Mean Std Err Mean Std Err Mean Std Err 1 (Control) 105.5 1.29 17.22 0.07 865.31 10.21 34.38 0.13 2 111.8 1.44 17.22 0.08 916.05 11.92 34.37 0.15 3 106.51 1.26 17.19 0.07 877.38 11.21 34.14 0.13 4 111.87 1.38 17.26 0.07 881.92 10.87 34.15 0.12 5 100.46 1.12 17.08 0.06 859.1 10.16 34.21 0.12 6 105.78 1.33 17.07 0.07 863.26 11.6 34.19 0.13 7 109.68 1.54 17.78 0.62 840.39 9.84 34.04 0.13 8 106.64 1.26 17.21 0.07 912.05 11.13 34.56 0.12 Δ Mean % (Treatment vs. control) 2 5.97% p = 0.0011 0.00% 5.86% p = 0.0013 −0.03% 3 0.96% −0.17% 1.39% −0.70% 4 6.04% p = 0.0006 0.23% 1.92% −0.67% 5 −4.78% p = 0.0060 −0.81% −0.72% −0.49% 6 0.27% −0.87% −0.24% −0.55% 7 3.96% p = 0.0289 3.25% −2.88% −0.99% 8 1.08% −0.06% 5.40% p = 0.0028 0.52%
[0244] Fish size distributions reveals a higher proportion of large fish (>1000 g) and a lower proportion of small fish (<476 g) in AN6 treated groups (Treatments 2, 3, or 8) (Table 8).
TABLE-US-00012 TABLE 8 Effect of TK antagonist on total number of large and small fish in each treatment Big fish 279 dph >1000 g Small fish 279 dph >476 g Treatment Fish# Big fish # % big fish Fish # Small fish # % small fish Control 449 46 10.2% 449 41 9.1% 2 451 75 16.6% 451 21 4.7% 3 480 79 16.5% 480 23 4.8% 4 463 68 14.7% 463 22 4.8% 5 441 53 12.0% 441 38 8.6% 6 442 57 12.9% 442 29 6.6% 7 427 46 10.8% 427 23 5.4% 8 446 91 20.4% 446 25 5.6% Δ Treatment - Control (%) 2 6.4% p = 0.0397 −4.5% p = 0.0139 3 6.2% p = 0.0459 −4.3% p = 0.0164 4 4.4% −4.4% p = 0.0187 5 1.8% −0.5% 6 2.7% −2.6% 7 0.5% −3.7% p = 0.0267 8 10.2% p = 0.0019 −3.5% p = 0.0395
[0245] The observed effect of AN6 treatment (5 and 10 mg/Kg feed) on average weight and growth rate, is significant 159 to 189 days after peptide treatment ended, may point on long-term or delayed physiological changes following AN6 peptide treatment.
Example 6: Oral Tachykinin Antagonist Treatment Improves Feed Conversion Rate (FCR) in Adult Fish
[0246] In order to determine the efficiency of feed conversion into fish substance in experiments (#1, #2, #3), FCR values which reflect the amount of feed needed to grow a kilogram fish, were calculated for each period and for the entire fish growth period. In order to determine the amount of consumed feed, the amount of feed provided was measured daily for each tank and the leftovers were collected, weighted and subtracted from the total amount of feed. Dead fish were removed every day before each feeding and then counted and weighed for accurate calculation of feed intake and FCR. It is to be noted that high mortality can still influence FCR values since feed eaten by fish that have later died cannot be subtracted from the total feed consumed and on the other hand periodic total fish weight gained is underestimated (dead fish are not included). Three FCR values were calculated according to the following formulas:
[0247] The calculated FCR values clearly indicate that all AN6 peptide treatments significantly improves FCR in adult fish (
[0248] The physiology of improved FCR can be broken into several successive steps: Feed Intake.fwdarw.Digestion.fwdarw.Absorption.fwdarw.Conversion. While digestion and absorption relate to gut physiology, conversion occurs mainly in the soma and is determined by the availability of metabolites and energy in growth processes on the expanse of other energy consuming processes (metabolism).
[0249] In the relationship between FCR and growth, two main dogmas are possible: a. Improved FCR value which is a manifestation of similar growth performance with less feed consumed. b. Improved FCR that occurs when fish have better growth with similar or higher feed intake. Namely, improved FCR does not necessarily result in an increased growth rate, and vice versa—increased growth rate does not necessarily indicate an improved FCR. According to the first dogma, feed is digested and absorbed in higher efficiency, but growth potential is not altered. In the second dogma, a shift in metabolism towards growth may also be involved. Consistent with these scenarios, the current experiments showed different FCR-growth relationship. Treatment with 10 and 30 mg AN6/Kg feed resulted in both improved FCR and growth performance (experiment #1). On the other hand, treatment with 5, 10 mg and 30 mg AN6/Kg feed in other cases resulted in improved FCR which did not coincide with improved growth but rather with decrease in feed intake (experiments #2 and #3). These results suggest that TK antagonist act on gut physiology and metabolism in separate pathways. The differential activation of these pathways may correlate with treatment age and peptide dose per Kg fish at the time of treatment.
[0250] At the end of the experiment described in example 3 (experiment #2), 6 fish from each cage/tank (3 females and 3 males, n=24 per treatment group) were sacrificed and whole intestine was removed and weighted. A difference in the intestine size was observed in male fish treated with 10 mg AN6/Kg feed and the ratio between intestine weight and fish weight (ISI) was significantly higher in male treated with 10 mg AN6/Kg feed (+21%,
Example 7: Safety of Treatment with AN6
[0251] At the end of each of the experiments described in examples 2, 3 and 5 (#1, #2, #3), a sample of fish from each treatment group was sacrificed and dissected for measurements of internal parts as indicated in Table 9.
TABLE-US-00013 TABLE 9 Sacrificed samples from experiments Average Number fish of fish Total Cage number/ sampled/ fish Experiment Treatment number cage cage sampled 1 2 6 360 5 Males & 60 5 Females 2 3 24 53 3 Males & 144 3 Females 3 8 48 73 3 144
[0252] No significant effect of AN6 treatment on gonad size and histology was observed in any of the experiments. Similarly, no effect of treatment with AN6 was observed on kidney, liver and fat weight and rates in any of the experiments.
[0253] It was also demonstrated that the half-life time of AN6 in blood is very short and that non residual peptide was observed in muscle and plasma following treatment, indicating the safety of the compound.
[0254] Fillet rate (calculated as the ratio between the maximal boneless longitudinal fish muscle slice, from head to tail, and total fish mass), was significantly higher in males and females treated with 10 ppm AN6 (experiments 2 & 3, +3.38% female fillet ratio, +5.6% male fillet ratio).
Example 8: In Vitro Inhibition of Tilapia and Salmon NKB Receptor Expressed in Naïve COS7 Cells
[0255] Naïve COS7 cells (fibroblast-like cell lines derived from monkey kidney tissue) were transfected with DNA of Tilapia Tac3Ra (t1NKB receptor a, cloned in pcDNA3.1). Exogenous receptor activity is determined by dynamic reads of Fluorescent emission from Ca++ indicator reporter system (Fluo4AM, Molecular Probes). Fluo-4 molecule and their derivatives all exhibit large increase in fluorescence emission intensity upon Ca++ binding. Time-laps reading of florescence intensity initiates ˜12 sec after activator is added to the cells, in 6 sec intervals for 2 minutes (readmax˜30-40 sec). Alternatively, Naïve COS7 cells were co-transfected with DNA of Salmon Tac3R (NKB receptor, slNKBr Cloned in pcDNA3.1) and Luciferase reporter under the regulation of SRE promoter. Nuclear SRE promoter activation is part of the PKA signaling pathway that is typical for NKB receptor activation. In this case, salmon NKB receptor activity is determined by reading of Luciferase fluorescence emission in plate reader 6 hours after activation.
[0256] For background subtraction, average reads in the last minutes is subtracted from reads in the first minutes which includes peak Ca++ fluorescence activity (Afluorescence). Read over time are smoothen by Polynomial Fit Degree=4 (JMP®, SAS). Peak value (Apeak fluorescence), usually achieved 12-24 seconds after reading initiation, is used as the result value for each concentration. For verifying ligand specific receptor activation, in each experimental setup cell activity following DMSO treatment is also determined (null). Receptor activation state in different ligand concentrations is expressed as a single peak value (Apeak fluorescence) divided by the read of maximal receptor activity concentration ((Apeak/Amax=1). EC50 is determined as AN6/Senktide concentration required for half maximal activity. IC50 is determined as AN6 concentration required for half maximal inhibition. Peptide concentration of EC50 and IC50 are predicted by Logistic 4P Roadbard Fit or Logistic 4P Fit (JMP®, SAS).
[0257] In order to determine inhibition capacity of the peptides, cells were treated with increasing concentrations of the peptide AN6, AN2, AN3, AN4 or AN5 (SEQ ID Nos: 6, 2, 3, 4 and 5 respectively), and tilapia NKBr or salmon NKBr were simultaneously activated by Senktide (Cayman), a potent, synthetic and specific NKB activator or by Salmon NKB (s1NKB) ligand, respectively (
[0258] The peptides' inhibition affinity (IC50), determined as the concentration required to reduce by half the average florescence emission (from Ca++ or luciferase reporter systems), induced by agonist in constant concentration.
[0259] It is demonstrated that all tested TK antagonists inhibit binding of salmon and tilapia NKB receptor agonist.