Somatostatin prodrugs

Abstract

The present invention provides prodrugs of somatostatin peptide and peptide analogs that are tissue permeable and oral bioavailable and enable activity of the somatostatin analog at the circulation or target tissue after cleavage of charge-masking lipophilic moieties. Pharmaceutical compositions comprising these prodrugs and their use in therapy and diagnosis are also provided.

Claims

1. A somatostatin prodrug comprising a lipophilic carbamate derivative of a somatostatin peptide or peptide analog, wherein the lipophilic carbamate is having a formula selected from the group consisting of: ##STR00012## wherein R.sup.1 is a primary alkyl having the formula n-C.sub.nH.sub.2n+1, wherein n is in the range of 3 to 15.

2. The somatostatin prodrug of claim 1, wherein the carbamate moiety is formula ##STR00013##

3. The somatostatin prodrug of claim 1, wherein R.sup.1 is n-hexyl.

4. The somatostatin prodrug of claim 1, wherein at least one positively charged group of the somatostatin amino acid sequence is masked with a lipophilic carbamate moiety in the lipophilic carbamate somatostatin derivative.

5. The somatostatin prodrug of claim 1, wherein the somatostatin peptide or peptide analog comprises at least one N-methylated amino acid residue.

6. The somatostatin prodrug of claim 1, wherein the somatostatin peptide or peptide analog comprises 5-15 amino acid residues.

7. The somatostatin prodrug of claim 1, wherein the somatostatin peptide or peptide analog is cyclic.

8. The somatostatin prodrug of claim 1, wherein the somatostatin peptide or peptide analog is selected from the group consisting of octreotide (SEQ ID NO: 1), somatuline (lanreotide, SEQ ID NO: 2), PTR-3173 (Somatoprim, SEQ ID NO: 3), and Pasireotide (SEQ ID NO: 4).

9. The somatostatin prodrug of claim 1, wherein the somatostatin peptide or peptide analog comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6.

10. The somatostatin prodrug of claim 8, wherein the somatostatin peptide or peptide analog is octreotide (SEQ ID NO: 1) coupled with at least one oxycarbonyl moiety.

11. The somatostatin prodrug of claim 10, comprising octreotide coupled to three hexyloxycarbonyl moieties, wherein the somatostatin prodrug has the structure: ##STR00014##

12. The somatostatin prodrug of claim 1, wherein the somatostatin analog is a head-to-tail cyclic N-methylated hexapeptide having the sequence c(PF(NMe)w(NMe)KT(NMe)F), wherein (NMe)w is N-methyl D-Tryptophan and (NMe)F is N-methyl-Phenylalanine (peptide 8, SEQ ID NO: 8).

13. The somatostatin prodrug of claim 1, comprising two hexyloxycarbonyl moieties coupled to peptide 8 (SEQ ID NO: 8) to form the structure: ##STR00015## wherein Hoc represents hexyloxycarbonyl.

14. The somatostatin prodrug of claim 1, wherein the somatostatin analog is a backbone cyclic somatostatin analog, wherein the backbone cyclized somatostatin analog is selected from the group consisting of: PTR 3173 (SEQ ID NO: 3), PTR 3046 (SEQ ID NO: 9), PTR 3205 (SEQ ID NO: 10), PTR 3171 (SEQ ID NO: 13), PTR 3113 (SEQ ID NO: 14), PTR 3123 (SEQ ID NO: 15), PTR 3209 (SEQ ID NO: 16), PTR 3183 (SEQ ID NO: 17), PTR 3185 (SEQ ID NO: 18), PTR 3201 (SEQ ID NO: 19), PTR 3203 (SEQ ID NO: 20), PTR 3197 (SEQ ID NO: 21), PTR 3207 (SEQ ID NO: 22), and PTR 3229 (SEQ ID NO: 23).

15. The somatostatin prodrug of claim 14, comprising three hexyloxycarbonyl moieties coupled to the backbone cyclic peptide Phe-Trp-(NMe)DTrp-(NMe)Lys-Thr-(NMe)Phe to form the formula: ##STR00016## wherein Hoc represents hexyloxycarbonyl.

16. A pharmaceutical composition comprising as an active ingredient a somatostatin prodrug according to claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

17. A method of treating a disease or disorder associated with somatostatin expression or activity, comprising administering to a subject in need thereof, a pharmaceutical composition according to claim 16.

18. The method of claim 17, wherein the disease or disorder is selected from the group consisting of metabolic disease or disorder, endocrine disease or disorder, cancer and angiogenesis.

19. A method of increasing the permeability and bioavailability of a somatostatin peptide or peptide analog, comprising masking at least one terminal amine or side-chain nitrogen atom of the somatostatin peptide or analog to form a lipophilic carbamate somatostatin prodrug, wherein the lipophilic carbamate is having a formula selected from the group consisting of: ##STR00017## wherein R.sup.1 is a primary alkyl having the formula n-C.sub.nH.sub.2n+1 wherein n is in the range of 3 to 15.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 is a bar chart describing the results of permeability transport studies (A to B—apical to basolateral, and B to A—basolateral to apical), of the prodrug octreotide-P (denoted OCT-Hoc-2), in comparison to the control molecules atenolol and metoprolol.

DETAILED DESCRIPTION OF THE INVENTION

(2) In the search for bioavailable somatostatin analogs, lipophilic carbamate prodrug molecules have been produced and showed improved permeability.

(3) The present invention provides methods that modify the active somatostatin peptide structure and enable transcellular absorption pathway from the intestine lumen to the blood circulation. The methods comprise converting charged somatostatin peptides into their lipophilic carbamate prodrugs, thereby, shifting the mechanism of intestinal permeability of the charged drug peptides from para-cellular to trans-cellular pathway of their lipophilic prodrugs. This is done by reducing the polarity and charge of the peptide, and thus improving its membrane permeability and oral bioavailability. The prodrug enhanced intestinal permeability allows its absorption to the blood stream where esterases remove the pro-moiety to regenerate the parent active somatostatin peptide.

(4) The present disclosure is also directed to various synthetic processes for the preparation of somatostatin prodrugs. Said prodrugs are generally characterized by two main chemical features: (a) reduction or omission of electrically charged atoms in the peptide sequence, e.g. through charge masking of charged amino acid residues and terminal amino and carboxylate moieties; and (b) improved lipophilicity provided through introduction of lipophilic groups. A further feature presented by somatostatin-based prodrugs prepared according to some embodiments of the present processes is their lability in the presence of specific enzymes, which transform the somatostatin prodrugs into charged biologically active somatostatin compounds.

(5) A common feature to the processes disclosed herein, is the modification of amino acids and/or amino acid residues to their modified counterparts, which include carbamate(s) of primary alcohols. In some embodiments, amino side chains having amine moieties are transformed into carbamates having —NCO.sub.2R groups; whereas amino side chains having carboxylate moieties are transformed into esters having —CO.sub.2R groups. In some embodiments, since the esters and amines are of primary alcohols, R is primary, i.e. the first group covalently bonded to the carbonyl's α-sp.sup.3 oxygen is a methylene group.

(6) The present invention is based in part on the finding that unlike tertiary carbamates, primary carbamates do not transform into their corresponding amines or ammonium ions until after penetrating the intestine membrane and reaching the circulation, the target tissue or the target cell, where specific proteases are present. Without wishing to be bound by any theory or mechanism of action, the commonly used tertiary carbamates (e.g. compound having the tert-butyloxycarbonyl-amino, N—CO.sub.2CMe.sub.3 moiety, N—BOC) undergo O—CMe.sub.3 bond cleavage in gastrointestinal pH. In contrast, primary alkyl carbamates, of the prodrugs of the present invention, are relatively stable until after penetrating through the intestine membrane and reach the circulation, the lymphatic system, and/or the blood stream. Therefore, tertiary carbamates undergo O—CMe.sub.3 bond cleavage before reaching the circulation or target tissue, to form the corresponding carbamic acids (having a —NH—CO.sub.2H group), which undergo spontaneous decarboxylation to form amines. Said amines are then being protonated under gastrointestinal pH to form charged peptides which undergo degradation before reaching the cells. On the other hand, it was surprisingly found that a similar sequence of reactions, occurs with primary carbamates only in the presence of specific esterases, which target and break the O—CH.sub.2 or the carbonyl-OCH.sub.2 bond in the blood stream, lymphatic system, target tissue or inside the target cell.

(7) Example prodrugs according to the present invention have been produced by chemical synthesis and selected based on the results achieved in permeability assay screening. The permeability assays include evaluation of intestinal permeability and metabolic stability in relevant in vitro models. Evaluation of pharmacokinetics following oral administration is tested in vivo in freely moving rats. Comparing the activity of the somatostatin peptide drugs with their prodrugs following oral administration is performed by administration intravenously or orally.

(8) The somatostatin prodrugs of the present invention are used to substitute somatostatin peptides and derivatives in therapeutic application amendable with this drug, for example in treatment of cancer and metabolic diseases. The conversion of the somatostatin treatment from parenteral mode of administration to much more convenient oral intake will significantly increase patient compliance and would be preferred by clinical team.

(9) Somatostatin, both in its natural and synthetic derivative forms is ultimately used as a cyclic peptide and has many derivatives and analogs that are all characterized by having cyclic structure. Cyclization of peptides has been shown to be a useful approach in developing diagnostically and therapeutically useful peptidic and peptidomimetic agents. Cyclization of peptides reduces the conformational freedom of these flexible, linear molecules, and often results in higher receptor binding affinities by reducing unfavorable entropic effects. Because of the more constrained structural framework, these agents are more selective in their affinity to specific receptor cavities. By the same reasoning, structurally constrained cyclic peptides confer greater stability against the action of proteolytic enzymes.

(10) Methods for cyclization can be classified into cyclization by the formation of the amide bond between the N-terminal and the C-terminal amino acid residues, and cyclizations involving the side chains of individual amino acids. The latter method includes the formation of disulfide bridges between two w-thio amino acid residues (cysteine, homocysteine), the formation of lactam bridges between glutamic/aspartic acid and lysine residues, the formation of lactone or thiolactone bridges between amino acid residues containing carboxyl, hydroxyl or mercapto functional groups, the formation of thioether or ether bridges between the amino acids containing hydroxyl or mercapto functional groups and other special methods. Lambert, et al., reviewed variety of peptide cyclization methodologies (J. Chem. Soc. Perkin Trans., 2001, 1:471-484).

(11) Some of the parent somatostatin analogs used to produce the prodrug of the present invention are backbone cyclized peptides. Backbone cyclization is a general method by which conformational constraint is imposed on peptides. In backbone cyclization, atoms in the peptide backbone (N and/or C) are interconnected covalently to form a ring. Backbone cyclized analogs are peptide analogs cyclized via bridging groups attached to the alpha nitrogens or alpha carbonyl of amino acids. In general, the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. During solid phase synthesis of a backbone cyclized peptide the protected building unit is coupled to the N-terminus of the peptide chain or to the peptide resin in a similar procedure to the coupling of other amino acids. After completion of the peptide assembly, the protective group is removed from the building unit's functional group and the cyclization is accomplished by coupling the building unit's functional group and a second functional group selected from a second building unit, a side chain of an amino acid residue of the peptide sequence, and an N-terminal amino acid residue.

(12) As used herein the term “backbone cyclic peptide” or “backbone cyclic analog” refers to a sequence of amino acid residues wherein at least one nitrogen or carbon of the peptide backbone is joined to a moiety selected from another such nitrogen or carbon, to a side chain or to one of the termini of the peptide. According to specific embodiment of the present invention the peptide sequence is of 5 to 15 amino acids that incorporates at least one building unit, said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising an amide, thioether, thioester, disulfide, urea, carbamate, or sulfonamide, wherein at least one building unit is connected via said bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or a terminal amino acid residue. Furthermore, one or more of the peptide bonds of the sequence may be reduced or substituted by a non-peptidic linkage.

(13) A “building unit” (BU) indicates a N.sup.α-ω-functionalized or an C.sup.α-ω-functionalized derivative of amino acids. Use of such building units permits different length and type of linkers and different types of moieties to be attached to the scaffold. This enables flexible design and easiness of production using conventional and modified solid-phase peptide synthesis methods known in the art.

(14) The N.sup.α-ω-functionalized derivative of amino acids preferably have the following structure:

(15) ##STR00007##
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R′ is an amino acid side chain, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting group of G.

(16) According to some embodiments, the building units are ω-functionalized amino acid derivatives wherein X is alkylene; G is a thiol group, an amino group or a carboxyl group; and R′ is the side chain of an amino acid. According to some embodiments R′ is protected with a specific protecting group.

(17) According to some specific embodiments, G is an amino group, a carboxyl group, or a thiol group.

(18) The methodology for producing the building units is described for example in WO95/33765 and WO98/04583 and in U.S. Pat. Nos. 5,770,687 and 5,883,293.

(19) The building units are abbreviated by the three-letter code of the corresponding modified amino acid followed by the type of reactive group (N for amine, C for carboxyl), and an indication of the number of spacing methylene groups. For example, GlyC2 describes a modified Gly residue with a carboxyl reactive group and a two-carbon methylene spacer, and PheN3 designates a modified phenylalanine group with an amino reactive group and a three carbon methylene spacer.

(20) In general, the procedures utilized to construct backbone cyclic molecules and their building units rely on the known principles of peptide synthesis and peptidomimetic synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. Some of the methods used for producing N.sup.αω building units and for their incorporation into peptidic chain are disclosed in U.S. Pat. Nos. 5,811,392; 5,874,529; 5,883,293; 6,051,554; 6,117,974; 6,265,375, 6,355613, 6,407059, 6,512,092 and international applications WO95/33765; WO97/09344; WO98/04583; WO99/31121; WO99/65508; WO00/02898; WO00/65467 and WO02/062819.

(21) The production and activity of some of the backbone cyclic somatostatin peptide analogs, used in the present invention for preparing the prodrugs where disclosed previously, for example in U.S. Pat. No. 5,770,687 and WO99/65508.

(22) As used herein “peptide” indicates a sequence of amino acids linked by peptide bonds. Functional derivatives of the peptides of the invention 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. 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 groups (for example those of seryl or threonyl residues) formed by reaction with acyl moieties. Salts of the peptides of the invention contemplated by the invention are organic and inorganic salts.

(23) The compounds herein disclosed may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of double bonds and the like can also be present in the compounds disclosed herein, and all such stable isomers are contemplated in the present invention.

(24) The term “amino acid” refers to compounds, which have an amino group and a carboxylic acid group, preferably in a 1,2-1,3-, or 1,4-substitution pattern on a carbon backbone. α-Amino acids are most preferred, and include the 20 natural amino acids (which are L-amino acids except for glycine) which are found in proteins, the corresponding D-amino acids, the corresponding N-methyl amino acids, side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy-proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, β-cyanolanine), and synthetically derived α-amino acids, such as amino-isobutyric acid, norleucine, norvaline, homocysteine and homoserine. β-Alanine and γ-amino butyric acid are examples of 1,3 and 1,4-amino acids, respectively, and many others are well known to the art.

(25) Some of 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 either 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, the L isomer was used. The D isomers are indicated by “D” or “(D)” before the residue abbreviation or by using the lower case of the amino acid code.

(26) 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. 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.

(27) The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K), Histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(28) The terms “N-methylation” or “NMe” are used herein interchangeably and refer to a form of alkylation wherein a methyl group, CH3, replaces the hydrogen atom of the NH moiety in the backbone amide NHs of residues within the peptides. The terms refer to a peptide having at least one N-methylated amino acid.

(29) Click reactions occur in one pot, are not disturbed by water, generate minimal and inoffensive byproducts, and are “spring-loaded” characterized by a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity (in some cases, with both regio- and stereo-specificity). Click reactions are particularly suitable to the problem of isolating and targeting molecules in complex biological environments. In such environments, products accordingly need to be physiologically stable and any byproducts need to be non-toxic (for in vivo systems).

(30) Prodrugs

(31) The prodrugs disclosed in the present invention are generally characterized by two main chemical features: (a) reduction or omission of electrically charged atoms in the peptide skeleton, e.g. through charge masking of charged amino acid residues and terminal amino and carboxylate moieties; and (b) improved lipophilicity provided through introduction of lipophilic groups. A further feature presented by peptide-based prodrugs prepared according to some embodiments of the present processes is their lability in the presence of cellular enzymes, which transform the prodrugs into charged biologically active peptide drugs.

(32) The term “prodrug” as used herein refers to an inactive or relatively less active form of an active agent that becomes active through one or more metabolic processes in a subject.

(33) The term “masking moiety” as used herein refers to a moiety that reduce the net electric charge of the peptide such as Hexyloxycarbonyl (Hoc).

(34) The term “carbamate” as used herein alone or in combination refers to a chemical group or moiety represented by the general structure —N(CO)O—. Carbamate esters may have alkyl or aryl groups substituted on the oxygen.

(35) A common feature to processes for producing the prodrugs disclosed herein, according to some embodiments, is the modification of amino acids and/or amino acid residues to their modified counterparts, which include an ester(s) and/or carbamate(s) of primary alcohols. In some embodiments and generally, amino side chains having amine moieties are transformed into carbamates having —NHCO.sub.2R moieties; whereas amino side chains having carboxylate moieties are transformed into esters having —CO.sub.2R moieties. In some embodiments, since the esters and amines are of primary alcohols, R is primary, i.e. the first group covalently bonded to the carbonyl's sp.sup.3 oxygen is a methylene group.

(36) It is shown now that unlike tertiary carbamates, primary carbamates do not transform into their corresponding amines or ammonium ions until after penetrating the circulation or target tissue. Without wishing to be bound by any theory or mechanism of action, the commonly used tertiary carbamates (e.g. compound having the tert-nutyloxycarbonyl-amino, NH—CO.sub.2CMe.sub.3 moiety) undergo O—CMe.sub.3 bond cleavage in physiological pH. In contrast, primary alkyl carbamates are relatively stable until after penetrating the circulation or target cells. Therefore, tertiary carbamates undergo O—CMe.sub.3 bond cleavage before reaching the cell (typically in the intestines), to form carbamic acids (NH—CO.sub.2H), which undergo spontaneous decarboxylation to form amines, which are then being protonated to form a charged peptide, which is unable to penetrate the intestinal membrane or undergoes degradation before reaching the circulation or cells. On the other hand, it was surprisingly found that a similar sequence of reactions, occurs with primary carbamates only after penetrating the intestine into blood stream or lymphatic system, where the peptide-based drug is carried as active form to the target tissue. It is hypothesized that the difference stems from the high tendency of tertiary carbamates to form tertiary carbocations under acidic conditions, while primary carbamates tend to cleave in the presence of inter-cellular esterases, which break the O—CH.sub.2 bond in vivo.

(37) In some embodiments, some the processes disclosed herein are distinctive in the stage in which the modification occurs. Whereas in some of the processes a modification is performed on an amino acid prior to its incorporation to the prodrug in a peptide synthesis; in some processes the modification is performed on an amino acid residue during the peptide synthesis; and in some of the processes the modification is preformed after the completion of the peptide synthesis.

(38) 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, if available. 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.

(39) The peptides of the present invention may be produced by any synthetic method known in the art. In some circumstances, a recombinant method can be used to synthesize a somatostatin peptide comprising naturally coded amino acids. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, synthetic peptides are purified by preparative high-performance liquid chromatography. The conjugation of the lipophilic moiety may be performed, according to some embodiments, during synthesis of the peptide or according to other embodiments after the synthesis, cleavage and purification of the peptide.

(40) “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 moiety”, a “permeability enhancing moiety” 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 and transporter peptides.

(41) Pharmacology

(42) The compounds of the present invention can be formulated into various pharmaceutical forms for purposes of administration. Pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant and a mixture thereof, as detailed for example in “Handbook of Pharmaceutical Excipients”; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA.

(43) For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier. Such a carrier can depend on the route of administration, such as oral, rectal, percutaneous or parenteral injection.

(44) A “carrier” as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

(45) For example, in preparing the compositions in oral dosage form, media such as water, glycols, oils, alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions. Alternatively, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents can be used, for example, in powders, pills, capsules or tablets.

(46) The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art e.g. diluents such as lactose (Pharmatose DCL 21), starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agent; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants/antiadherants; chelating agents; vehicles; bulking agents; stabilizers; preservatives; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; chelating agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof. The disintegrants useful in the present invention include but not limited to starch or its derivatives, partially pregelatinized maize starch (Starch 1500°), croscarmellose sodium, sodium starch glycollate, clays, celluloses, alginates, pregelatinized corn starch, crospovidone, gums and the like used either alone or in combination thereof. The lubricants useful in the present invention include but not limited to talc, magnesium stearate, calcium stearate, sodium stearate, stearic acid, hydrogenated vegetable oil, glyceryl behenate, glyceryl behapate, waxes, Stearowet, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate and the like used either alone or in combination thereof. The anti-adherents or glidants useful in the present invention are selected from but not limited to a group comprising talc, corn starch, DL-leucine, sodium lauryl sulfate, and magnesium, calcium and sodium stearates, and the like or mixtures thereof. In another embodiment of the present invention, the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol. In an embodiment of the present invention, the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate and/or tocopherol. In another embodiment, the dosage form of the present invention additionally comprises at least one wetting agent(s) such as a surfactant selected from a group comprising anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, or mixtures thereof. The wetting agents are selected from but not limited to a group comprising oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate and the like, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises at least one complexing agent such as cyclodextrin selected from a group comprising but not limited to alpha-cyclodextrin, beta-cyclodextrin, betahydroxy-cyclodextrin, gamma-cyclodextrin, and hydroxypropyl beta-cyclodextrin, or the like. In yet another embodiment, the dosage form of the present invention additionally comprises of lipid(s) selected from but not limited to glyceryl behenate such as Compritol® ATO888, Compritol® ATO 5, and the like; hydrogenated vegetable oil such as hydrogenated castor oil e.g. Lubritab®; glyceryl palmitostearate such as Precirol® ATO 5 and the like, or mixtures thereof used either alone or in combination thereof. It will be appreciated that any given excipient may serve more than one function in the compositions according to the present invention.

(47) For parenteral compositions, the carrier can comprise sterile water. Other ingredients may be included to aid in solubility. Injectable solutions can be prepared where the carrier includes a saline solution, glucose solution or mixture of both.

(48) Injectable suspensions can also be prepared. In addition, solid preparations that are converted to liquid form shortly before use can be made. For percutaneous administration, the carrier can include a penetration enhancing agent or a wetting agent.

(49) It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier.

(50) Apart from other considerations, the fact that the 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. According to the present invention, novel methods of are being used, in order to synthesize metabolically stable and oral bioavailable somatostatin prodrugs. The preferred route of administration of peptides of the invention is oral administration.

(51) Other routes of administration include but are not limited to intra-articular, intravenous, intramuscular, subcutaneous, intradermal, topical or intrathecal.

(52) Pharmaceutical compositions according to the present invention may also comprise at least one absorption enhancer, such as but not limited to, nanoparticles, piperine, curcumin and resveratrol.

(53) In addition, or alternatively, the compositions and administration methods of the present invention may include agents and formulations that reduce intra-enterocyte metabolism by CYP3A4 and/or reduce P-gp efflux activity, for example, the Pg-p inhibitor verapamil.

(54) Suitable delivery systems for the prodrugs of the present invention, are for example Pro-NanoLipospheres (PNL) and Advanced PNL disclosed in WO2013/208254 [32], and the self-nano-emulsifying drug delivery system (SNEDDS) [33].

(55) 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.

(56) 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.

(57) 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.

(58) 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.

(59) For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

(60) 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.

(61) 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., 2001, Curr. Opin. Chem. Biol. 5, 447). 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.

(62) The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

(63) 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.

(64) 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).

(65) Those skilled in the art of treatment can determine the effective daily amount, optionally based on the known amount of the parent somatostatin compound in use. The precise dosage and frequency of administration depends on the particular compound of the invention being used, as well as the particular condition being treated, the severity of the condition, the age, weight, and general physical condition of the subject being treated, as well as other medication being taken by the subject, as is well known to those skilled in the art. It is also known that the effective daily amount can be lowered or increased depending on the response of the subject or the evaluation of the prescribing physician. Thus, the ranges mentioned above are only guidelines and are not intended to limit the scope of the use of the invention.

(66) The combination of a compound of the invention with another agent used for treatment can be used. Such combination can be used simultaneously, sequentially or separately.

(67) The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

(68) General Procedures

(69) Chromatography

(70) Semi-preparative reversed phase HPLC was performed using Waters instruments: Waters 2545 (Binary Gradient Module), Waters SFO (System Fluidics Organizer), Waters 2996 (Photodiode Array Detector), Waters 2767 (Sample Manager). A C18-column: Reprosil 100 C18, 5 μm, 150×30 mm was used. The Semi-preparative RP-HPLC columns were operated with a flow rate of 40 mL/min with a linear gradient (20 min) of H.sub.2O (0.1% v/v trifluoroacetic acid (TFA)) and acetonitrile (0.1% v/v TFA). Analytical HESI-HPLC-MS (heated electrospray ionization mass spectrometry) was performed on an LCQ Fleet (Thermo Scientific) with a connected UltiMate 3000 UHPLC focused (Dionex) on C18-columns: 51: Hypersil Gold aQ 175 Å, 3 μm, 150×2.1 mm (for 8 or 20 minutes measurements); S2: Accucore C18, 80 Å, 2.6 μm, 50×2.1 mm (for 5 minute measurements) (Thermo Scientific). Linear gradients (5%-95% acetonitrile content) with H.sub.2O (0.1% v/v formic acid) and acetonitrile (0.1% v/v formic acid) as eluents were used.

(71) Permeability Studies

(72) Colorectal adenocarcinoma 2 (Caco-2) cells (ATTC) were grown in 75 cm.sup.2 flasks with approximately 0.5×10.sup.6 cells/flask (Thermo-Fischer) at 37° C. in a 5% CO.sub.2 atmosphere and at relative humidity of 95%. The culture growth medium consisted of DMEM supplemented with 10% heat-inactivated FBS, 1% MEM-NEAA, 2 mM 1-glutamine, 1 mM sodium pyruvate, 50,000 units Penicillin G Sodium and 50 mg Streptomycin Sulfate (Biological Industries). The medium was replaced every other day.

(73) Caco-2 cells (passage 55-60) were seeded at density of 25×10.sup.5 cells/cm.sup.2 on untreated culture inserts of polycarbonate membrane with 0.4 μm pores and surface area of 1.1 cm.sup.2. Culture inserts containing Caco-2 monolayer were placed in 12 mm transwell plates (Corning). Culture medium was replaced every other day. Transepithelial Electrical Resistance (TEER) values were measured by Millicell ERS-2 System (Millipore) a week after seeding up to experiment day (21-23 days) to ensure proliferation and differentiation of the cells. When the cells were fully differentiated and TEER values became stable (200-500 Ω.Math.cm.sup.2). The TEER values were compared to control inserts containing only the medium.

(74) In vitro permeability studies using Caco-2 cells were initiated by replacing the medium from both sides by apical (600 μl) and basolateral (1500 μl) buffers, both warmed to 37° C. The cells were incubated with the buffers solutions for 30 min at 37° C. on a shaker (100 cycles/min). The apical buffer was replaced by apical buffer containing 10 μg/ml of the somatostatin peptide or the somatostatin prodrug. 50 μl samples were taken from the apical side immediately at the beginning of the experiment, resulting in 550 μl apical volume during the experiment. Samples of 200 μl at fixed time points (20, 40, 60, 80, 100, 120 and 150 min) from the basolateral side and replaced with the same volume of fresh basolateral buffer to maintain a constant volume. The experiment included two control compounds, atenolol and metoprolol, as paracellular and transcellular permeability markers respectively.

(75) Permeability Coefficient (Papp) for each compound was calculated from the linear plot of drug accumulated versus time, using the following equation:

(76) $\mathrm{Papp}=\frac{\mathrm{dq}\text{/}\mathrm{dt}}{C_0\times A}$

(77) Where dq/dt is steady state appearance rate of the compound on the receiver side, C.sub.0 is the initial concentration of the drug on the donor side, and A is the exposed tissue surface area (1.1 cm.sup.2).

(78) In Vivo Studies

(79) Somatostatin peptides, analogs and prodrugs according to the present invention are evaluated in vivo using methods know in the art for assessing the activity of somatostatin. For example, the in vivo effects on the release of glucagon, insulin and growth hormone may be evaluated according to the method described in WO99/65508.

(80) In a tumor growth model, female nude mice weighing 19-22 g are kept in groups of 5 animals and have free access to drinking water and a pathogen-free rodent diet. Subcutaneous tumors are initiated from cultured AR42J cells. Treatment commences 2-4 days following inoculation of the tumor cells. The tested compounds are administered parenterally or orally. The size of the tumors is determined with a caliper. For statistical calculations Student's t-test is applied.

EXAMPLES

Example 1. Preparation of Octreotide Prodrug

(81) The prodrug three hexyloxycarbonyl-octreotide (Octreotide-P) was synthesized from octreotide using the synthetic pathway shown in Scheme 1:

(82) ##STR00008##

Example 2. Synthesis of Somato8 (Peptide 8) and its Prodrug Somato8-P (Peptide 8-P)

(83) In an effort to develop an improved somatostatin analog, a cyclic N-methylated hexapeptide somatostatin analog denoted “Peptide 8” was selected from a combinatorial library of all possible N-methylated analogs of the potent hexa-cyclic somatostatin analog c(PFwKTF) (SEQ ID NO: 7) [31]. Out of the 30 analogs synthesized, only seven analogs were found to have somatostatin receptor (SSTR) affinity similar to that of the parent peptide, that is, selectivity towards SSTR2 and SSTRS in the nanomolar range. From these seven analogs, one, named “Somato8” (previously “Peptide 8”), having the sequence c(PF(NMe)w(NMe)KT(NMe)F) (SEQ ID NO: 8), that contains three N-methylated amino acid residues, had the most promising PK parameters in vitro (including stability to intestinal enzymes and intestinal permeability). It was further investigated for its bioavailability following oral administration to rats compared to the parent sequence. The calculated absolute oral bioavailability of the multiple N-methylated analog in rats was ˜10% which is nearly five times higher than the parent peptide [27].

(84) Detailed Synthesis of Somato8

(85) Peptide synthesis was carried out using CTC-resin (0.9 mmol/g) following standard Fmoc-strategy. Fmoc-Xaa-OH (1.2 eq.) were attached to the CTC-resin with N,N-diisopropylethylamin (DIEA; 2.5 eq.) in anhydrous dichloromethane (DCM, 0.8 mL/g resin) at room temperature (rt) for 1 h. The remaining trityl-chloride groups were capped by addition of a solution of MeOH (1 mL/g (resin)), DIEA (5:1; v:v) for 15 min. The resin was filtered and washed 5 times with DCM and 3 times with MeOH. The loading capacity was determined by weight after drying the resin under vacuum and ranged from 0.4-0.9 mmol/g.

(86) On-resin Fmoc-Deprotection was performed by treating the Fmoc peptidyl-resin with 20% piperidine in NMP (v/v) for 10 minutes and a second treatment for 5 minutes. The resin was washed 5 times with NMP.

(87) Standard Amino Acid Coupling was performed by adding a solution of Fmoc-Xaa-OH (2 eq.), O-(7-azabenzotriazole-lyl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (HATU) (2 eq.), 1-hydroxy-7-azabenzotriazole (HOAt; 2 eq.), and DIEA (3 eq.) in NMP (1 mL/g resin) to the free amino peptidyl-resin, shaking for 60 min at room temperature and washing 5 times with NMP.

(88) For on-resin N-methylation, the linear Fmoc-deprotected peptide was washed with DCM (3×) incubated with a solution of 2-nitrobenzenesulfonylchloride (o-Ns-Cl, 4 eq.) and 2,4,6-Collidine (10 eq.) in DCM for 20 min at room temperature. The resin was washed with DCM (3×) and tetrahydrofuran (THF) abs. (5×). A solution containing PPh3 (5 eq.) and MeOH abs. (10 eq.) in THF abs. was added to the resin. Diisopropyl azodicarboxylate (DIAD, 5 eq.) in a small amount THF abs. is added stepwise to the resin and the solution was incubated for 15 min and washed with THF (5×) and NMP (5×). For o-Ns deprotection, the o-Ns-peptidyl-resin was stirred in a solution of mercaptoethanol (10 eq.) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 5 eq.) in NMP (1 mL/g resin) for 5 minutes. The deprotection procedure was repeated once more and the resin was washed 5 times with NMP.

(89) For complete cleavage from the resin the linear peptides were treated three times with a solution of DCM and hexafluoroisopropanol (HFIP; 4:1; v:v) at room temperature for half an hour and the solvent evaporated under reduced pressure.

(90) To achieve cyclization, Diphenylphosphoryl Azide (DPPA, 3 eq.) was added to a solution of linear peptide in DMF (1 mM peptide concentration) and NaHCO.sub.3 (5 eq.) at room temperature and the solution was stirred over night or until no linear peptide could be observed by HPLC-MS. The solvent was evaporated to a small volume under reduced pressure and the peptides precipitated in saturated NaCl solution and washed two times in HPLC grade water.

(91) For removal of acid labile side chain protecting group, cyclized peptides were stirred in a solution of TFA, water and TIPS (95:2.5:2.5) at room temperature for one hour or until no more protected peptide could be observed by HPLC-MS and then precipitated in diethylether. The precipitated peptide was collected after centrifugation and decantation and then washed with diethylether and collected two more times.

(92) Orthogonal reductive deprotection of the benzyl-group via hydrogenolysis was performed using a palladium catalyst on activated carbon (10% Pd/C with 50% H.sub.2O as stabilizer, 15 mg/mmol) and hydrogen atmosphere (1 atm. H.sub.2) at room temperature. The completion of the deprotection was monitored by HPLC-MS, the catalyst was removed over diatomaceous earth and the solvent was removed under pressure.

(93) Synthesis of Somato8 Dihexyloxycarbonyl Prodrug “Somato8-P”

(94) Somato8 was dissolved in acetonitrile and DIEA (3 eq.) was added. The solution was cooled in ice/water and under stirring hexyloxycarbonylchloride (Hoc-Cl, 3.1 eq.) was added dropwise. The solution was stirred in the cold for 1 hr. and at room temperature for three hours or until no free peptide could be observed by HPLC-MS. After reaction completion the solvent was evaporated and the product precipitated by hexane.

(95) The structures of Somato8 (SEQ ID NO: 8) and its prodrug are illustrated in Scheme 2:

(96) ##STR00009##

Example 3. Backbone Cyclic Somatostatin Analogs and their Prodrugs

(97) In an attempt to identify novel somatostatin analogs, libraries of backbone cyclic peptides have been previously prepared with compounds having identical or highly similar sequences to the somatostatin pharmacophoric sequences. Four libraries, each containing 96 compounds, were synthesized and screened for their binding affinities to somatostatin receptors. Following the screening process, several candidates were further investigated for their metabolic stability and pharmacodynamic profile compared to SRIF and to octreotide. Some of the compounds are PTR-3046 (SEQ ID NO: 9) [28], PTR-3205 (SEQ ID NO: 10) [29] and PTR-3173 (SEQ ID NO: 3) [30] depicted in Scheme 3:

(98) ##STR00010##

(99) All backbone cyclic analogs were found to be stable against enzymatic degradation in serum and renal homogenate. However, their biological activity and selectivity varied toward the somatostatin receptors: while PTR-3046 was found to be selective toward the SSTR5 (IC50 in the nanomolar range), PTR-3205 was found to be selective towards SSTR2 and PTR-3173 was selective towards the SSTR2, SSTR4 and SSTR5. These analogs were also evaluated for their in vivo efficacy compared to octreotide. PTR-3173 was found to be 1000-fold more potent in the in vivo inhibition of GH than that of glucagon, with no effect on insulin secretion at physiological concentrations (GH:insulin potency ratio >10,000). This was the first description of a long-acting somatostatin analog possessing complete in vivo selectivity between GH and insulin inhibition. PTR-3046 inhibits bombesin- and caerulein-induced amylase and lipase release from the pancreas without inhibiting GH or glucagon release. PTR-3173 has been reported to bind uniquely to SSTR2, SSTR4 and SSTR5 in vitro with outstanding in vivo selectivity in GH inhibition [30]. All backbone cyclic analogs were found to be stable against enzymatic degradation in serum and renal homogenate.

(100) The active N-methylated sequence (NMe)w-(NMe)K-T-(NMe)F-(SEQ ID NO: 11) was incorporated into the framework of the backbone cyclic analog PTR 3173 to form the somatostatin analog Somato3M (SEQ ID NO: 12), and its three hexyloxycbarbonyl prodrug, namely Somato3M-P (Scheme 4) was prepared in the same manner as Octreotide-P:

(101) ##STR00011##

(102) Each combination of bridge type and length imposes certain pharmacodynamics selectivity towards the somatostatin receptor subtypes. In addition, the N-methylation at different sites may elevate intestinal permeability.

Example 4. Intestinal Permeability and Oral Bioavailability Studies

(103) In-vitro permeability studies model are essential for development of peptides as therapeutic agents, as they allow good prediction for in-vivo oral absorption of compounds [22]. The Caco-2 model is a widely used tool in the academia and pharmaceutical industry to evaluate and predict compounds' permeability mechanism. The Caco-2 system consists of human colon cancer cells that multiply and grow to create a monolayer that emulate the human small intestinal mucosa [23].

(104) Transport studies were performed through the Caco-2 monolayer mounted in an Ussing-type chamber set-up with continuous trans-epithelial electrical resistance (TEER) measurements to assure TEER between 800 and 1200 Ω*cm.sup.2. HBSS supplemented with 10 mM IVIES and adjusted to pH 6.5 were used as transport medium in the donor compartment and pH 7.4 in the acceptor compartment. The donor solution contained the test compound. The effective permeability coefficients (Papp) were calculated from concentration-time profiles of each of the tested compounds in the acceptor chamber [24]. In every assay, the compounds were compared to the standards atenolol and metoprolol which represent para-cellular and trans-cellular permeability mechanisms respectably [25].

(105) Permeability mechanism of compounds is studied by evaluating the Papp of a compound from the apical to the basolateral (A-to-B) membrane and its Papp from the basolateral to the apical membrane (B-to-A). The A-to-B assay simulates passive and transporter-mediated permeability. The B-to-A assay is essential complementary experiment indicative of the activity of P-gp. The ratio of the A-to-B and B-to A Papps (efflux ratio) is calculated to determine the permeability mechanism. A significant difference between the permeability coefficients in the two directions (efflux ratio of 1.5-2 or above), is a strong indication of active transport or efflux system involvement [26].

(106) It is important to note that the involvement of efflux system is actual indication that the prodrug is permeate through the enterocytes membrane and afterwards removed from these cells by the efflux system. To further study the efflux system involved in the permeability mechanism, a Caco-2 study in the presence of verapamil (100 mM), a known P-gp inhibitor is performed.

Example 5: In Vitro Intestinal Permeability Studies with the Octreotide Prodrug (OCT-Hoc-2) in the Caco-2 Model

(107) Among the cell-based models, the most popular is the well-established Caco-2 cell culture model, originating from human colorectal adenocarcinoma cells. Caco-2 cells spontaneously differentiate into an epithelial monolayer similar in structure to that of the intestinal epithelium, including functional properties of mature enterocytes. These include formation of microvilli, tight-junctional complexes and expression of various transporters, efflux systems and brush-border enzymes. The cells are grown on semi-permeable membrane supports, which enhances their polarization and maximizes their similarity to the intestinal epithelial membrane while allowing transport studies across it.

(108) Growth and Maintenance of the Cells

(109) Caco-2 cells were obtained from ATCC (Manassas, Va., USA) and grown in 75 cm2 flasks with approximately 0.75×106 cells/flask at 37° C. in a 5% CO2 atmosphere and at a relative humidity of 95%. The culture growth medium consisted of Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 2 mM sodium pyruvate, 2 mM Penicillin-Streptomycin solution and 2 mM L-glutamine. The medium was replaced three times weekly. For the transport studies, cells in a passage range of 53-60 were seeded at a density of 25×105 cells/cm2 on pretreated culture inserts of a polycarbonate membrane with 0.4 μm pores and a surface area of 1.1 cm2 and then placed in 12-well transwell plates, 12 mm, Costar™. The culture medium was changed every other day. Transport studies were performed 21-22 days after seeding, allowing the cells proper proliferation, differentiation and development of their proper morphology.

(110) Experimental Protocol

(111) Transport studies (apical to basolateral, A to B) were initiated by removing the medium from both sides of the monolayer and replacing it with 600 μL of apical buffer (0.025M D-glucose monohydrate, 0.02M MES biological Buffer, 1.25 mM calcium chloride and 0.5 mM magnesium chloride in Hanks Balanced Salt Solution, filtered and titrated to pH 6.5 with NaOH) and 1500 μL of basolateral buffer (0.025M D-glucose monohydrate, 0.02M HEPES biological Buffer, 1.25 mM calcium chloride, and 0.5 mM magnesium chloride in Hanks Balanced Salt Solution, filtered and titrated to pH 7.4 with NaOH), both preheated to 37° C. The cells were incubated for 30 min at 37° C. with shaking (100 cycles/min). After the incubation period, the buffers were removed and replaced with 1500 μL of basolateral buffer on the basolateral side. Test solutions containing tested drug or prodrug (10 μg/mL) in apical buffer were preheated to 37° C. and added (600 μL) to the apical side of the monolayer. Samples (50 μL) were immediately taken from the apical side at the beginning of the experiment, leaving a 550 μL apical volume during the experiment. For the period of the experiment, the cells were kept at 37° C. with shaking. At predetermined times (20, 40, 60, 80, 100, 120 and 150 min), 200 μL samples were taken from the basolateral side and replaced with the same volume of fresh basolateral buffer to maintain a constant volume and sink conditions. For the basolateral to apical study (B to A), the test solution of the drug or prodrug was placed in the basolateral chamber, followed by immediate sampling from the basolateral side and continued sampling from the apical side at predetermined times, similarly to the A-to B protocol. Samples were kept frozen at a temperature of −20° C. pending analysis by HPLC-MS.

(112) During the three-week period of differentiation of the cells, the transepithelial electrical resistance (TEER) of the cells was measured continuously using the Millicell2 ERS Epithelial Volt-Ohm meter and STX01 electrode (Millipore corporation, Billerica, Mass.), to evaluate the proper development of the monolayer. For Caco-2 cells, at 21-22 days post seeding, the cells reach their full differentiation and generation of the monolayer, reaching stable TEER values of 300-500 Ω×cm.sup.2. Inserts with deviational values were not used.

(113) Atenolol, a commonly used marker for paracellular permeability in Caco-2 cells, was used in combination with metoprolol, a commonly used marker for transcellular permeability.

(114) Similarly, to the A-to-B studies of the peptide permeability, 600 μl of apical buffer containing 10 μg/ml of atenolol and metoprolol each were added to the apical side of the monolayer and a sample (50 μl) was immediately withdrawn from the apical side. Further samples of 200 μl were taken at predetermined times up to 150 min, and similar volumes of blank buffer were added to the basolateral side to maintain constant volume and sink conditions during the experiment. The samples were analyzed for atenolol and metoprolol content by means of HPLC-MS, followed by calculation of atenolol and metoprolol Papp.

(115) Data Analysis

(116) The samples obtained from the Caco-2 permeability experiments were analyzed for peptide, atenolol and metoprolol content using the HPLC-MS system. The permeability coefficient (Papp) of each peptide was calculated from the linear plot of drug accumulated vs. time, using the following equation: Papp=dQ/dt C0×A where dQ/dt is steady state appearance rate of the drug on the receiver side, C0 is the initial concentration of the drug on the donor side, and A is the exposed tissue surface area, 1.1 cm2 in the specified experiments.

(117) As can be seen in FIG. 1, the octreotide prodrug OCT-Hoc-2 permeate in the A to B direction through enterocyte monolayer, with permeability coefficient values less than atenolol (0.03 cm/s×10.sup.6), the marker for paracellular permeability.

(118) It is known that the intestinal absorption of Octreotide is poor. As previously shown in Caco2 studies, the mechanism of absorption of octreotide is via the paracellular pathway and the formulative efforts to elevate its permeability had only minor success. The octreotide prodrug of the present invention is the first one that demonstrated enhanced intestinal permeability via the transcellular pathway.

(119) The permeability mechanism of the pro-drug was also studied by evaluating the P.sub.app of the compounds from the basolateral to the apical membrane (BA). The P.sub.app BA of the Octreotide prodrug (11.95 cm/s×10.sup.6) were significantly higher than the P.sub.app AB(0.03 cm/s×10.sup.6)(P<0.05, indicating efflux system involvement. The use of specific delivery systems and/or absorption enhancers designed for inhibiting p-gp activity is therefore preferred. Such delivery system is for example, an advanced lipid based Self-Emulsifying Drug Delivery System termed Advanced Pro-NanoLiposphere (PNL) pre-concentrate.

Example 6: Metabolic Stability Studies

(120) Generally, the purpose of metabolic stability studies is to evaluate the compounds rate of elimination in the presence of hostile environments: a rat plasma or extractions of the gut wall. In these environments, compounds are prone to enzymatic degradation, as there are high concentrations of peptidases, esterases, lipases and other peptides that metabolize xenobiotics to building units for synthesizing essential structures in the body [27, 28].

(121) Specifically, in our case, the purposes of the metabolic stability studies are (1) to prove that the prodrug is digested by esterases to furnish the drug and (2) to demonstrate that the somatostatin peptides and their prodrugs are stable to digestion in the intestine.

(122) The enzymatic reactions are performed as follows: 2 mM stock solutions of the tested compounds are diluted with serum or purified brush border membrane vesicles (BBMVs) solution to a final concentration of 0.5 mM. During incubation at 37° C. samples are taken for a period of 90 minutes. The enzymatic reaction is stopped by adding 1:1 v/v of ice cold acetonitrile and centrifuge (4000 g, 10 min) before analysis. Preparation of BBMVs: The BBMVs is prepared from combined duodenum, jejunum, and upper ileum (male Wistar rats) by a Ca++ precipitation method. Purification of the BBMVs is assayed using GGT, LAP and alkaline phosphatase as membrane enzyme markers

(123) The peptides and prodrugs are subjected to rat plasma and followed their degradation. Rat plasma is known to be rich with esterases. Next, peptides and prodrugs are subjected to extractions of the gut wall (brush border membrane vesicles, BBMV) and followed their rate of degradation. The BBMV assay determines the peptides stability in the presence of digestive enzymes in the brush border membrane of the intestine especially peptidases.

(124) Selected peptides and prodrugs are subjected to additional in vitro assay to evaluate the involvement of liver metabolism, through the Pooled Human Liver Microsome assay. Liver microsomes are subcellular particles derived from the endoplasmic reticulum of hepatic cells. These microsomes are a rich source of drug metabolizing enzymes, including cytochrome P-450. Microsome pools from various sources are useful in the study of xenobiotic metabolism and drug interactions.

Example 7: Pharmacokinetic Study

(125) The pharmacokinetic in-vivo study allows a further evaluation of the prodrug concept in the whole animal. The PK studies are performed for example in conscious Wistar male rats. An indwelling cannula is implanted in the jugular vein 24 hours before the PK experiment to allow full recovery of the animals from the surgical procedure. Animals (n=4) receive either an IV bolus dose or oral dose of the investigated compound. Blood samples (with heparin, 15 U/ml) are collected at several time points for up to 6 hours post administration and are assayed by HPLC-MS method. Non-compartmental pharmacokinetic analysis is performed using WinNonlin software.

(126) While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

REFERENCES

(127) 1. Ovadia O, Greenberg S, Laufer B, Gilon C, Hoffman A, Kessler H. Improvement of drug-like properties of peptides: the somatostatin paradigm. Expert Opin. Drug Discov. Informa UK Ltd London, UK; 2010; 5:655-71. 2. Grozinsky-Glasberg S, Shimon I, Korbonits M, Grossman A B. Somatostatin analogues in the control of neuroendocrine tumours: efficacy and mechanisms. Endocr. Relat. Cancer. BioScientifica; 2008; 15:701-20. 3. Patel Y C, Murthy K K, Escher E E, Banville D, Spiess J, Srikant C B. Mechanism of Action of Somatostatin: An Overview of Receptor Function and Studies of the Molecular Characterization and Purification of Somatostatin Receptor Proteins Model of Somatostatin Action. 4. Lahlou H, Guillermet J, Hortala M, Vernejoul F, Pyronnet S, Bousquet C, et al. Molecular Signaling of Somatostatin Receptors. Ann. N. Y. Acad. Sci. Blackwell Publishing Ltd; 2004; 1014:121-31. 5. Reichlin S. Somatostatin. N. Engl. J. Med. 1983; 309:1495-501. 6. Mezey É, Hunyady B, Mitra S, Hayes E, Liu Q, Schaeffer J, et al. Cell Specific Expression of the SST2A and SST5 Somatostatin Receptors in the Rat Anterior Pituitary. Endocrinology. Oxford University Press; 1998; 139:414-9. 7. Kumar U, Sasi R, Suresh S, Patel A, Thangaraju M, Metrakos P, et al. Subtype-selective expression of the five somatostatin receptors (hSSTR1-5) in human pancreatic islet cells: a quantitative double-label immunohistochemical analysis. Diabetes. 1999; 48. 8. Mitra S W, Mezey É, Hunyady B, Chamberlain L, Hayes E, Foor F, et al. Colocalization of Somatostatin Receptor sst5 and Insulin in Rat Pancreatic β-Cells.sup.1. Endocrinology. 1999; 140:3790-6. 9. Schally A V. Oncological applications of somatostatin analogues. Cancer Res. American

(128) Association for Cancer Research Inc.; 1988; 48:6977-85. 10. Scarpignato C, Pelosini I. Somatostatin analogs for cancer treatment and diagnosis: an overview. Chemotherapy. 2001; 47 Suppl 2:1-29. 11. Pollak M N, Schally A V. Mechanisms of Antineoplastic Action of Somatostatin Analogs. Exp. Biol. Med. SAGE PublicationsSage U K: London, England; 1998; 217:143-52. 12. Bousquet C, Puente E, Buscail L, Vaysse N, Susini C. Antiproliferative effect of somatostatin and analogs. Chemotherapy. Karger Publishers; 2001; 47 Suppl 2:30-9. 13. Oberg K. Future aspects of somatostatin-receptor-mediated therapy. Neuroendocrinology. Karger Publishers; 2004; 80 Suppl 1:57-61. 14. Heron I, Thomas F, Dero M, Gancel A, Ruiz J M, Schatz B, et al. Pharmacokinetics and efficacy of a long-acting formulation of the new somatostatin analog BIM 23014 in patients with acromegaly. J. Clin. Endocrinol. Metab. 1993; 76:721-7. 15. Hruby V, Balse P. Conformational and Topographical Considerations in Designing Agonist Peptidomimetics from Peptide Leads. Curr. Med. Chem. 2000; 7:945-70. 16. Weide T, Modlinger A, Kessler H. Spatial Screening for the Identification of the Bioactive Conformation of Integrin Ligands. doi.org. Springer Berlin Heidelberg; 2006; 1-50. 17. Lamberts S W J, van der Lely A-J, de Herder W W, Hofland L J. Octreotide. Wood A J J, editor. N. Engl. J. Med. Massachusetts Medical Society; 1996; 334:246-54. 18. Bauer W, Briner U, Doepfner W, Haller R, Huguenin R, Marbach P, et al. SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action. Life Sci. 1982; 31:1133-40. 19. Gilon C, Halle D, Chorev M, Selincer Z, Byk G. Backbone cyclization: A new method for conferring conformational constraint on peptides. Biopolymers. Wiley Subscription Services, Inc., A Wiley Company; 1991; 31:745-50. 20. Assaf Friedler ‡ Nehama Zakai §, Orit Karni §, Yehoshua C. Broder §, Lea Baraz I, Moshe Kotler I, et al. Backbone Cyclic Peptide, Which Mimics the Nuclear Localization Signal of Human Immunodeficiency Virus Type 1 Matrix Protein, Inhibits Nuclear Import and Virus

(129) Production in Nondividing Cells†. American Chemical Society; 1998; 21. Ovadia O, Linde Y, Haskell-Luevano C, Dirain M L, Sheynis T, Jelinek R, et al. The effect of backbone cyclization on PK/PD properties of bioactive peptide-peptoid hybrids: the melanocortin agonist paradigm. Bioorg. Med. Chem. 2010; 18:580-9. 22. Chatterjee J, Gilon C, Hoffman A, Kessler H. N-Methylation of Peptides: A New Perspective in Medicinal Chemistry. Acc. Chem. Res. 2008; 41:1331-42. 23. Linde Y, Ovadia O, Safrai E, Xiang Z, Portillo F P, Shalev D E, et al. Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides. Biopolymers. NIH Public Access; 2008; 90:671-82. 24. Han H K, Amidon G L. Targeted prodrug design to optimize drug delivery. AAPS PharmSci. Springer; 2000; 2:E6. 25. Jornada D, dos Santos Fernandes G, Chiba D, de Melo T, dos Santos J, Chung M. The Prodrug Approach: A Successful Tool for Improving Drug Solubility. Molecules.

(130) Multidisciplinary Digital Publishing Institute; 2015; 21:42. 26. Simplicio A L, Clancy J M, Gilmer J F. Prodrugs for Amines. Molecules. Molecular Diversity Preservation International; 2008; 13:519-47. 27. Biron E, Chatterjee J, Ovadia O, Langenegger D, Brueggen J, Hoyer D, et al. Improving Oral Bioavailability of Peptides by Multiple N-Methylation: Somatostatin Analogues**. 28. Chaim Gilon ‡, Martin Huenges §, Barbara Mathä §, Gary Gellerman ⊥, Vered Hornik ⊥, Michel Afargan ⊥, et al. A Backbone-Cyclic, Receptor 5-Selective Somatostatin Analogue: Synthesis, Bioactivity, and Nuclear Magnetic Resonance Conformational Analysis†. American Chemical Society; 1998; 29. Falb E, Salitra Y, Yechezkel T, Bracha M, Litman P, Olender R, et al. A bicyclic and hsst2 selective somatostatin analogue: design, synthesis, conformational analysis and binding. Bioorg. Med. Chem. 2001; 9:3255-64. 30. Afargan M, Janson E T, Gelerman G, Rosenfeld R, Ziv O, Karpov O, et al. Novel Long-Acting Somatostatin Analog with Endocrine Selectivity: Potent Suppression of Growth Hormone But Not of Insulin. Endocrinology. 2001; 142:477-86. 31. Veber D F, Freidlinger R M, Perlow D S, et al. Nature 1981; 292(5818):55-8, 32. Cherniakov, Domb A J, Hoffman A. Self-nano-emulsifying drug delivery systems: an update of the biopharmaceutical aspects. Expert Opin Drug Deliv. 2015 July; 12(7):1121-33. 33. Klinger et al., Enhancing oral bioavailability of cyclic RGD hexa-peptides by the lipophilic prodrug charge masking approach: redirection of peptide intestinal permeability from paracrine to transcellular pathway.