UNIVERSAL ORAL DELIVERY DEVICE OF INTACT THERAPEUTIC POLYPEPTIDES WITH HIGH BIOAVAILABILITY

Abstract

The invention is related to the fully effective gastro-protected universal oral delivery device of gastro-protected nanoparticles for the transport of intact biologically active polypeptides into the circulatory system. This universal oral delivery device is made of gastro-protected nanoparticles that transport intact therapeutic polypeptides through the gastrointestinal system and it successfully performs the paracellular transepithelial passage of all therapeutic polypeptides from the intestinal lumen into the circulatory system, fully preserving the integrity and biological activity of those therapeutic polypeptides.

Claims

1. A universal oral delivery device of intact therapeutic polypeptides to the circulatory system, the device comprising: a purified recombinant polypeptide, the purified recombinant polypeptide having an amino acid sequence equal to SEQ ID NO. 1 wherein the amino acid in position 560 is a nonpolar amino acid, and a group consisting of at least one or a combination of two or more therapeutic polypeptide.

2. The universal oral delivery device of claim 1, further comprising a group consisting of at least two gastro-protective polymers.

3. A purified recombinant polypeptide, wherein the purified recombinant polypeptide has an amino acid sequence equal to SEQ ID NO 2.

4. The universal oral delivery device of claim 1, wherein at least one therapeutic polypeptide in the group of therapeutic polypeptides are comprised in the group of: recombinant polypeptides, naturally-existing biologically active proteins, fusion proteins, hormones, growth factors, plasma proteins, coagulation factors, toxins and other protein antigens, monoclonal antibodies, replacement enzymes and peptides.

5. The universal oral delivery device of claim 1, wherein at least one therapeutic polypeptide in the group of therapeutic polypeptides is a monoclonal antibody or a fusion polypeptide including but not limited to rituximab, adalimumab, trastuzumab, ranibizumab, pertuzumab, denosumab, cetuximab, bevacizumab, nivolumab, pembrolizumab, eculizumab, ustekinumab, ab, omalizumab, pembrolizumab, and combinations of two or more of these.

6. The universal oral delivery device of claim 1, wherein at least one therapeutic polypeptide in the group of therapeutic polypeptides is a replacement enzyme comprised in the group of: alfa-agalsidase, alglucerase, beta-agalsidase imiglucerase, taliglucerase-alfa, velaglucerase-alfa, laronidase, idulsurfase, elosulfase-alfa, galsulfase, alglucosidase-alfa.

7. The universal oral delivery device of claim 1, wherein at least one therapeutic polypeptide is a recombinant protein comprised in the group of: follicle stimulating hormone, luteinizing hormone, Chorionic gonadotrophic hormone, erythropoietin, filgrastim, molgramostim, soma pin, betaIFN1a, betaIFN1b, IFNalpha2a, IFNalpha2b, interleukin 2, etanercept, ranibizumab, insulin, eptacog alfa, human recombinant Factor VII, human recombinant Factor VIII, human recombinant Factor XII, human recombinant Factor XIII.

8. The universal oral delivery device of claim 1, wherein at least one polypeptide in the group of therapeutic polypeptides is included in therapeutic amounts.

9. The oral delivery device of claim 1, wherein the purified recombinant polypeptide and the group of therapeutic polypeptides are formulated as gastro-protected polypeptide nanoparticles.

10. An isolated nucleic acid comprising a sequence encoding a recombinant polypeptide according to claim 3 or a fragment of that nucleic sequence of at least 18 consecutive bases.

11. An expression vector for an isolated nucleic acid comprising the isolated nucleic acid according to claim 10, wherein the recombinant sequence is operably linked to an expression control sequence.

12. A cultured cell comprising an expression vector as claimed in claim 11.

13. A cultured cell comprising an isolated nucleic acid according to claim 10, wherein the recombinant sequence is operably linked to an expression control sequence.

14. A cultured cell or a progeny of said cell, wherein the cultured cell is transfected with an expression vector for an isolated nucleic acid, the isolated nucleic acid comprising the isolated nucleic acid according to claim 10, and wherein the recombinant sequence is operably linked to an expression control sequence, and the cultured cell expresses the recombinant polypeptide.

15. (canceled)

16. (canceled)

17. A method for synthesizing gastro-protected polypeptide nanoparticles suspension, the gastro-protected polypeptide nanoparticles formulated from a purified recombinant polypeptide having an amino acid sequence equal to SEQ ID NO. 1, wherein the amino acid in position 560 is a nonpolar amino acid, and a group consisting of at least one or a combination of more therapeutic polypeptides, the method comprising solvent-injecting a solution containing a gastro-protective polymer in ethanol into an aqueous solution, the aqueous solution containing the gastro-protected polypeptide nanoparticles, one or more therapeutic polypeptides, and a stabilizing ent.

18. The method of claim 17, wherein the gastro-protected polypeptide nanoparticles are shell-coated.

19. The method of claim 17, wherein the average diameter size of the polypeptide nanoparticles generated is between approximately 250 and 350 nanometers.

20. The method of claim 17, wherein the solvent-injecting of the ethanolic solution into the aqueous solution is done using a narrow tube made of steel and having an internal diameter between approximately 0.5 and 20 millimeters.

21. (canceled)

22. The method of claim 17, wherein the ethanolic solution is injected at a flow rate between approximately 0.5 and 5000 milliliters per minute, or between approximately 2 and 160 millimeters.

23. The method of claim 17, further comprising the step of diluting the gastro-protected polypeptide nanoparticles with the addition of a stabilizing agent.

24. (canceled)

25. (canceled)

26. The method of claim 17, wherein the polypeptide s suspension is freeze-dried.

27. The method of claim 17, wherein the polypeptide nanoparticles suspension is diafiltered and concentrated up to 10 times.

28. (canceled)

29. The method of claim 27, further comprising the step of adding a lyoprotectant agent to the polypeptide nanoparticles suspension before freeze drying.

30. (canceled)

31. (canceled)

32. (canceled)

33. The method of claim 17, further comprising the step of removing any water miscible organic solvent from the synthesized gastro-protected polypeptide nanoparticles, wherein the removal is done by one of dialysis, ultra-filtration, solvent evaporation at a reduced pressure, N.sub.2 current evaporation or tangential flow filtration.

34. A pharmaceutical composition of gastro-protected polypeptide nanoparticles, the gastro-protected polypeptide nanoparticles formulated from a purified recombinant polypeptide having an amino acid sequence equal to SEQ ID NO. 1, wherein the amino acid in position 560 is a nonpolar amino acid, and a group consisting of at least one or a combination of more therapeutic polypeptides in oral dosage forms as syrups, solutions, ampoules, dispersions, semi-solids, softgels, tablets, capsules, sachets, powders, granules, orally dispersible films.

35. (canceled)

36. A method of oral administration of a pharmaceutical composition to a subject, the method comprising: administering orally to said subject the pharmaceutical composition according to claim 34.

Description

DETAILED DESCRIPTION

[0109] This invention relates to a gastro-protected oral delivery device comprised of therapeutic polypeptides and a purified recombinant polypeptide SERAR and defined later on, the device deliver the therapeutic polypeptides by transepithelial paracellular passage with high bioavailability.

[0110] The gastro-protected oral delivery device successfully transports therapeutic polypeptides in polypeptide nanoparticles through the gastrointestinal system. Furthermore, said device successfully performs the paracellular transepithelial passage of intact therapeutic polpypeptide/s from the intestinal lumen into the circulatory system.

[0111] The transepithelial paracellular passage of therapeutic polypeptides through the intestinal epithelium into the circulatory system is achieved by the presence of the SERAR purified recombinant polypeptide alongside the therapeutic polypeptide.

[0112] The sequence of the SERAR recombinant polypeptide is derived from Seq ID No 1 (FIG. 1). SERAR recombinant polypeptide is specific amino acid sequence described in Seq ID No 2 (FIG. 2) and it comprises a specific amino acid sequence that actively triggers a temporary opening of the intestinal epithelium's intercellular junctions, a linker and a sequence from a maltose-binding protein domain.

[0113] The SERAR purified recombinant polypeptide triggers a temporary opening of the intestinal epithelium's intercellular junctions is derived from the sequence of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), and has been modified in order to eliminate its proteolytic activity. The serratiopeptidase enzyme (Serratia marcescens E15 protease) is an oral anti-inflammatory supplement with EC number 3.4.24.40 having a molecular weight of approximately 52 kDa (kilodaltons). Serratiopeptidase was first isolated from enterobacterium Serratia sp, a microorganism originally isolated in the late 1960s from silk worm Bombyx mori. Serratiopeptidase is present in the silk worm intestine and allows the emerging moth to dissolve its cocoon. It is produced by purification from fermentation of Serratia marcescens or Serratia sp. E 15. The Serratiopeptidase enzyme belongs to the Serralysin group of enzymes, it is a proteolytic enzyme with many favorable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties. Moreover, serratiopeptidase with enteric coating is widely used by oral administration in clinical practice for the treatment of many diseases.

[0114] The purified recombinant polypeptide SERAR actively triggers an innocuous temporary opening of the intestinal epithelium's intercellular junctions in vitro and in vivo. Furthermore, it is fully effective in the transepithelial paracellular passage activity of therapeutic polypeptides in vitro and in vivo, without the proteolytic activity of the wild type serratiopeptidase, and the maltose binding protein (MBP) that grants correct folding of the SERAR polypeptide.

[0115] The crux of the present invention is the gastro-protected oral delivery device of the SERAR recombinant polypeptide and at least one therapeutic polypeptide. The SERAR recombinant polypeptide comprises the specific amino acid sequence of SEQ ID NO. 2 that performs innocuous temporary opening of the intestinal epithelium's intercellular junctions and therapeutic polypeptides pass through the intestinal epithelium by paracellular transepithelial passage in vitro and in vivo.

[0116] In a first embodiment of the present invention a universal oral delivery device, the device delivering intact therapeutic polypeptides into the circulatory system in a subject. The universal oral device is comprised of a purified recombinant polypeptide, including but not limiting to the SERAR purified recombinant polypeptide (SEQ ID NO. 2), and at least one or a combination of more therapeutic polypeptides. In other embodiments of the present invention the universal oral device is gastro-protected and also comprises a group consisting of at least two gastro-protective polymers.

[0117] Embodiments of the present invention may comprise different therapeutic polypeptides, descriptions including but not limited to: [0118] at least one therapeutic polypeptide in the group of therapeutic polypeptides are comprised in the group of: recombinant polypeptides, naturally-existing biologically active proteins, fusion proteins, hormones, growth factors, plasma proteins, coagulation factors, toxins and other protein antigens, monoclonal antibodies, replacement enzymes and peptides. [0119] at least one therapeutic polypeptide in the group of therapeutic polypeptides is a monoclonal antibody comprised in the group rituximab, adalimumab, infliximab, trastuzumab, ranibizumab, pertuzumab, denosumab, cetuximab, bevacizumab, nivolumab, pembrolizumab, eculizumab, ustekinumab, golimumab, omalizumab, pembrolizumab. [0120] at least one therapeutic polypeptide is a recombinant protein comprised in the group of: follicle stimulating hormone, luteinizing hormone, Chorionic gonadotrophic hormone, erythropoietin, filgrastim, molgramostim, somatotropin, betalFN1a, betalFN1b, IFNalpha2a, IFNalpha2b, interleukin 2, etanercept, ranibizumab, insulin, eptacog alfa, human recombinant Factor VII, human recombinant Factor VIII, human recombinant Factor XII, human recombinant Factor XIII, alfa-agalsidase, alglucerase, beta-agalsidase, imiglucerase, taliglucerase-alfa, velaglucerase-alfa, laronidase, idulsurfase, elosulfase-alfa, galsulfase, alglucosidase-alfa and combinations of two or more of these.

[0121] Embodiment of this invention includes a nucleic acid sequence that encodes a SERAR recombinant polypeptide comprising the amino acid sequence of SEQ ID NO. 2 or of a fragment of SEQ ID NO. 2 of 18 bases or larger. Another embodiment of the present invention comprises an expression vector with the nucleic acid sequence of SERAR recombinant polypeptide (Seq. ID No 3). Another embodiment of the present invention includes a cultured cell, wherein the expression vector of SERAR acid nucleic sequence is described above. The nucleic acid sequence Seq ID No. 3 is operably linked to an expression control sequence, where this sequence express the SERAR recombinant polypeptide Seq ID. No 2.

[0122] In other embodiments of the present invention, the gastro-protected oral delivery device is comprised of gastro-protected polypeptide nanoparticles formulated by a combination of therapeutic polypeptides, purified recombinant polypeptide SERAR Seq ID. No 2, a gastro-protective polymer, and a stabilizing polymer.

[0123] Other embodiments of the present invention include methods for preparing polypeptides including but not limited to Seq. ID no 1. In one embodiment, a method for producing recombinant polypeptide SERAR (SEQ ID NO: 2) comprises the steps of cultivating bacteria carrying plasmids with genes coding for the desired SERAR recombinant polypeptide.

[0124] A method for constructing plasmids containing pre-specified genes and controlling DNA sequences, including but not limited to the DNA sequence of the SERAR polypeptide, is given by the so-called recombinant DNA technique.

[0125] It is known in the art that it is possible to obtain, from the cultivated bacteria cells carrying such recombinant plasmid DNA, gene-coded proteins which inherently are characteristic to other organisms than the bacterium used as host cell. In the preparation of recombinant plasmid DNA, a so-called cloning vector (that is, a plasmid which is able to replicate in the host bacterium) is combined with a DNA fragment containing a gene, many genes, DNA sequences or expression cassettes coding for the pre-specified gene polypeptide/s, and/or controlling its expression. The recombinant DNA technique, in its most useful form, is based on the following principle:

[0126] Assume we pre-selected a plasmid vector that is a circular DNA molecule that contains a multiple cloning site with several sites for restriction endonucleases cleavage, other DNA expression control units and at least one selection marker (a DNA fragment coding for resistance to an antibiotic, for example). The vector is treated with one or more restriction enzymes to produce one or several species of a linear molecule.

[0127] On the other hand, we pre-selected a different DNA sample which has been treated with the same endonuclease.

[0128] DNA is cut into pieces in a very specific way by restriction endonucleases including but not limited to HindIII and XhoI. These pieces are joined to each other by DNA ligase in order to obtain molecules composed of the vector to which a foreign DNA fragment has been fused, and which are called recombinant DNA.

[0129] An alternative or complementary method involves one extra step before ligation by DNA ligase, which is the completion of the cohesive ends generated by the restriction endonuclease by using a DNA Polymerase and nucleotides. This performs for ligation of fragments that do not have matching cohesive ends.

[0130] Next, we insert the resulting plasmid into a pre-selected bacterial host cell using either electroporation or calcium chloride transformation. Bacteria that incorporated the plasmid are selected by using the selection marker that was included in the plasmid (for example, resistance to a certain antibiotic, for example, ampicillin, kanamycin). Bacteria that incorporated the plasmid are resistant to the antibiotic and grow in a medium that contains such antibiotic. The larger the number of plasmids per bacterial cell, the highest the resistance to the antibiotic and the higher the chances of such cell of producing the protein of interest in high amounts. Expression of the desired protein coded in the plasmid is granted since the selection marker is included in the same plasmid, adjacent to the DNA fragment that codes for the protein of interest.

[0131] Another embodiment of the present invention includes a method for producing recombinant polypeptide SERAR (SEQ ID NO: 2) comprising a step of cultivating bacteria carrying plasmids with genes coding for the desired SERAR recombinant polypeptide, being such genes cloned in a plasmid sensitive to changes in temperature, in which the plasmid allows both effective plasmid amplification and production of large quantities of the plasmid gene product. The method uses plasmid with a temperature-dependent plasmid copy number pattern and shows a controlled constant plasmid copy number when host bacteria are cultivated at a certain temperature. On the other hand, when the host bacteria carrying the plasmid is grown at a different temperature, a plasmid copy number pattern occurs a higher number of plasmids per cell.

[0132] Hence, the number of copies of such plasmid is low at one temperature, which is an advantage since it decreases the risk that the cloned plasmid or its gene products could disturb the growing of the host bacterium. However, the amount of the plasmid is rapidly increased by a simple temperature shift, whereby simultaneous formation of the cloned plasmid and its gene products are obtained, and the production of plasmid's gene products proceeds rapidly.

[0133] The cultured cell per se is successfully performed using the productivity conventional techniques, including conventional chemically defined nutritive media which, with the productivity for the bacterial species in question, and also, the harvesting of the gene products is performed in accordance with productivity methods to the identity of the gene products of the present invention. An Special and critical parameter of the production process of the gene product is the temperature regulation including at least a period of cultivation to achieve a temperature at which the plasmid shows the plasmid shows an increasing in the copy number. This pattern includes a period of cultivation in which the plasmid is copied in a high number of copies, and gene product of the plasmid are formed in higher amounts.

[0134] The culture of the transformed E. coli cells is grown at 37° C., and ampicillin is added to a concentration (q.s. 100 μg/ml) which it inhibits the growth of cells containing plasmids with normal copy number. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to the cells in order to induce the expression of the gene and the production of the SERAR recombinant polypeptide Seq. ID No 2. The culture of the plasmid-containing cells is then grown at 34° C.

[0135] Cells cultured as mentioned above are disrupted and centrifugated to obtain the SERAR inclusion bodies (IBs). These SERAR IBs are washed and resolubilized in lysis buffer and solubilization buffer with 6 M UREA, respectively. Cell pellets are then frozen at −20° C. and then centrifuged at 12000 rpm. The protein of interest is effectively refolded with 20 mM TRIS-HCl sucrose 8% pH 8.0.

[0136] Embodiments of the present invention include an expression vector, which is a plasmid. Said plasmid comprises at least one restriction endonuclease: only one site susceptible to cleavage by the endonuclease. Moreover, said site is such that after insertion of a fragment of foreign DNA at this site, the resulting recombinant DNA replicates autonomously by the temperature-regulated signal of the present invention, increasing the production of the number of identical copies of plasmids regulated by temperature.

[0137] The most suitable restriction endonucleases used in recombinant DNA technology are those giving the so-called “cohesive ends” on both the cloning vector and the DNA fragment, in other words, single-stranded regions at the ends of the molecules with complementary base sequence allowing base pairing to identical sequences. The cloning vector constructed contains a DNA sequence Seq ID No. 3 coding for SERAR recombinant polypeptide Seq. ID No. 2 comprising: a) maltose binding protein sequence and b) the specific amino acid sequence of serratiopeptidase with the modified amino acid in position 560.

[0138] The cloning vector contains genes mediating a so-called marker, useful for identification and/or selection of cells carrying the plasmid. The most useful marker is an antibiotic resistance-related gene, for example, ampicillin resistance, since this is used for an easy counter selection of bacteria that have not received the recombinant plasmid after treatment for transforming a recombinant DNA into a bacterial host.

[0139] Embodiments of the present invention of gastro-protected universal oral delivery device of therapeutic polypeptides into the circulatory system of a human subject. This gastro-protected universal oral delivery device formulated as gastro-protected polypeptide nanoparticles overcome the gastrointestinal tract barriers and are an effective oral delivery device of therapeutic polypeptides into circulatory system using different kinds of oral pharmaceutical compositions (e.g. liquid, solid or a combination of both).

[0140] The purified recombinant polypeptide called SERAR Seq. ID No 2 comprising: a) an artificially mutated amino acid sequence from Serratiopeptidase (Serratia E15 protease) that is involved in the effective transepithelial paracellular passage activity by opening the intestinal epithelium's intercellular junction in vitro and in vivo without proteolytic activity due to the substitution of a glutamic acid in the catalytic site with another amino acid, including but not limited to, a residue of alanine; and b) a stabilizing domain from maltose binding protein (MBP).

[0141] An embodiment of the present invention includes a method for obtaining protein nanoparticles comprising the therapeutic polypeptide and the SERAR polypeptide. Polypeptide nanoparticles are synthesized by nanoprecipitation using a gastro-protective polymer dissolved in an organic solvent injected in an aqueous solution composed by a) at least one therapeutic polypeptide and; b) the SERAR purified recombinant polypeptide Seq ID No 2.

[0142] The protein nanoparticle synthesis method starts from buffer solutions containing the SERAR polypeptide, at least one therapeutic polypeptide, a gastro-protective polymer and a stabilizing hydrophilic polymer. The stabilizing hydrophilic polymer is, but is not limited to, polyvinylpyrrolidone. The resulting solution, composed of an appropriate ratio of SERAR polypeptide between 10 to 30 mg and one therapeutic dose of the therapeutic polypeptide, is stabilized at room temperature.

[0143] A water-miscible organic solvent solution which is, but not limited to, ethanol, is injected from above into the protein buffer solution at a rate of 2 to 160 milliliters per minute (mL/min) under constant stirring. The ethanolic solution is composed by a gastro-protective polymer or copolymer (for example, acrylic acid with methacrylate). The gastro-protective polymer, dissolved in an ethanol solution, is injected to the protein buffer solution producing a decrease in the solubility of the proteins and thus, the formation of protein nanoparticles by nanoprecipitation with efficiency over 99%. Later a solution of a hydrophilic polymer (e.g. polyvinylpyrrolidone) is added in order to stabilize the protein nanoparticles.

[0144] Finally, the ethanolic solvent is eliminated by different methods, including but not limited to, dialysis, ultra-filtration, evaporation at reduced pressure, evaporation with N2 gas injection, or tangential flow filtration.

[0145] According to the present invention, the protein nanoparticles size can range from 50 to 1000 nm, with method variations that can narrow the distribution to an average size population between 250 and 350 nm, are dispersed in an aqueous solution and they act as a vehicle carrying the therapeutic polypeptides (naturally occurring or recombinant proteins, active ingredients of biological origin, including fusion proteins, protein hormones, growth factors, protein vaccines, plasma proteins, coagulation factors, toxins and other protein antigens, monoclonal antibodies, bites and replacement enzymes).

[0146] The therapeutic polypeptides used may be already commercialized and they can be obtained from the purification of human fluids or biotechnological production.

[0147] The synthesized protein nanoparticles may be used directly as liquid dispersion. Alternatively, they may be conserved through a freeze-drying process in the presence or absence of lyoprotectant agents (e.g. sugars, amino acids, polyols, glycerol, or peptides), and then re-dispersed in a liquid medium preserving the proteins nanoparticles shape and size.

[0148] An embodiment of the present invention involves a method for the creation of a the gastro-protected polypeptide nanoparticles wherein these are generated by a gastro-protective shell coating of the polypeptide nanoparticles with a gastro-protective polymer that preserves intact the therapeutic polypeptides from the stomach-intestinal conditions. The correct gastro-protection ratio of polypeptide nanoparticles is a shell coating from 1:3 to 1:9 weight/weight (w/w) gastro-protective polymer.

[0149] An embodiment of the present invention involves a method for the creation of the oral administration to a subject, the gastro-protected oral delivery device of therapeutic polypeptides successfully formulated in gastro-protected polypeptide nanoparticles are formulated in oral pharmaceutical composition. The oral pharmaceutical composition used are liquid oral dosage forms (e.g. solutions, syrups or elixirs) or solid oral dosage forms (e.g. granules, tablets, filled hard or softgel capsules, or extemporal solids).

[0150] An embodiment of the present invention involves a method for the generation of a syrup pharmaceutical composition. According to this method, the protein nanoparticle dispersion (containing the encapsulated therapeutic polypeptide/s) is homogenized within the sucrose syrup containing, or not, preservative agents. Next, the preparation is conditioned in the proper container and is dosed by volume. The syrup formulation is appropriate when the dose of therapeutic protein is bigger than 500 mg/dose and ease of swallowing is an issue for pediatric, elder, hospitalized patients.

[0151] Another embodiment of the present invention involves a method for the manufacture of an oral solid pharmaceutical composition. Firstly, this oral solid pharmaceutical composition is based on the gastro-protected oral delivery device of therapeutic polypeptides Layering. This layering consisting of the first coating with the gastro-protected oral delivery device of therapeutic polypeptides over sugar micro/spheres to obtain the vehicle system. Secondly, an Enteric Coating with a gastro-protective polymer to preserve the vehicle system from the digestion of said composition in a stomach-intestine of a subject, and thirdly, the double-coated microspheres are dosage and loaded within gelatin capsules.

[0152] The coating processes are performed on the sugar microspheres with an average size between 300 and 1000 micrometers (μm) and/or standard spheres from 0.5 to 2 millimeters. The coating processes consist of a fluid bed coater with a bottom-up spraying system (Würster) and constant aerodynamic convection at up to 40° C. for drying and protection of the polypeptides involved in the process. The Pharm-a-spheres® (Evonik Industries AG) brand represents an example of sucrose micro/spheres that may be used for this step. This manufacturing method is appropriate for those therapeutic proteins administered at very low doses (in the order of micrograms, or μg in symbols) and the fluid bed coating process control the loading of protein nanoparticles over the sugar microspheres. This method uses a fluid bed coater which is loaded with the sucrose micro/spheres and these are thermostabilized at up to 40° C. The coating suspension is then pumped (using, for example, a peristaltic pump).

[0153] The first coating suspension (the gastro-protected oral delivery device of therapeutic polypeptides layering suspension) is composed by a) the gastro-protected polypeptide nanoparticles dispersion containing the therapeutic polypeptide/s, b) an adhesion polymer (for example 4-7% w/v polyvinylpyrrolydone or hydroxypropyl cellulose) (here w/v refers to weight over volume in grams and milliliters, e.g., 1% w/v 1 gram of sugar in 100 mL of water), c) talc or colloidal silica (c.a. 3% w/v) for the prevention of the electrostatic and auto-adhesion of micro/spheres and d) distilled water as solvent. The gastro-protected polypeptide nanoparticles layered micro/spheres are dried for 10 min. Later, the gastro-protected polypeptide nanoparticles coated micro/spheres are subjected with a second gastro-protective coating with a gastro-protective polymer.

[0154] The choice of the polymer (30-40% w/w of total solids) depends on the kind of controlled delivery or the site of releasing where the therapeutic protein is aimed to be absorbed. For example, the Eudragit® polymers commercialized by Evonik Industries AG, such as L 100-5S or L 30 D-55 are used for delivery at duodenum, the L 100 or L 12.5 for delivery at jejunum, the S 100, S 12.5 or FS 30 D are used for delivery at ileum and colon.

[0155] The second gastro-protected coating suspension also contains a) a plasticizer (1-5% w/v, for example triethyl citrate, trioctyl citrate, triehexyl citrate and acetylated monoglycerides) for diminishing the glass transition temperature of the gastro-protective polymer, b) talc (6-7% w/v), c) colored or transparent lacquer and c) distilled water as solvent. The micro/spheres are dried again for 10 min. Both coating suspensions (gastro-protected polypeptide nanoparticles layering and gastro-protective coating) have c.a. 5-15% w/v solid concentration giving a favorable viscosity for the spraying application.

[0156] The final microspheres with the double coating, gastro-protected polypeptide nanoparticles layered and the gastro-protective coating are dosed and filled in capsules for oral administration to a subject. The dose of the therapeutic polypeptide determines the microsphere size, the therapeutic polypeptide concentration in the gastro-protected polypeptide nanoparticles layering suspension and the capsule size.

[0157] Next, experiments are reproduced where different embodiments of the present invention take place. The first three examples include the SERAR recombinant polypeptide obtention. The following examples show the use of the SERAR as the universal oral delivery. The gastro-protected polypeptides nanoparticles dispersion obtained is administered directly as an oral liquid pharmaceutical composition and/or lyophilized for liquid or solid oral pharmaceutical compositions (e.g. extemporal suspensions, syrups, liquid-filled hard capsules, soft capsules, coated microspheres filled capsules, and granulates).

EXAMPLES

[0158] The following examples are included for explanatory purposes only and no limit to the result of the invention. The examples demonstrate generic processes.

Example 1

Expression Vector Obtention and Clone Selection of SERAR

[0159] Target DNA sequence (SEQ ID No 3, introduced in FIG. 3) coding for SERAR recombinant polypeptide (SEQ ID No 2) is successfully synthesized. The synthesized sequence is cloned into vector pET-22b(+), containing a ampicillin-resistance gene, by using two restriction endonucleases (HindIII and XhoI) with unique sites within the plasmid for protein expression in E. coli.

[0160] For evaluation of the expression the E. coli strain BL21(DE3) is transformed with the recombinant plasmid and cultured in plates with ampicillin. A single colony from such plate is inoculated into chemically defined medium containing ampicillin; culture is incubated at 37° C. at 200 rpm and then induced 4 hours with IPTG. SDS-PAGE and capillary gel electrophoresis is used to monitor the expression amount of the SERAR recombinant polypeptide successfully obtained.

Example 2

Obtention of SERAR Purified Recombinant Polypeptide

[0161] Production of the purified recombinant polypeptide SERAR (SEQ ID NO: 2) comprises the cultivation of bacteria carrying plasmids with genes coding for the desired recombinant protein. The method for producing the recombinant polypeptide SERAR comprises growth of bacteria carrying a plasmid for recombinant expression of such polypeptide, induced with IPTG which must be added to the culture medium. The bacteria master cell bank (MCB) is manufactured by amplification of the bacteria containing the expression plasmid, and vials of such bank are used as seed for preparation of the inoculum.

[0162] Presence of expression plasmid in the bacterium is confirmed by isolation and identification via sequencing.

Inoculum:

[0163] 10 Erlenmeyer flasks containing 100 mL of LB medium with ampicillin 100 μg/mL are inoculated with 90 μl from one vial of the MCB each. All flasks are incubated at 34° C. for 15.5 h with continuous orbital shaking at 250 rpm.

[0164] After this incubation, samples from each flask are taken and OD600 nm is measured to each sample to determine bacterial concentration. Microscopic observation is also performed to discard contamination.

[0165] OD values above 3.0 are obtained and the content of all Erlenmeyer's are pooled to a final volume of 1000 mL and OD=3.41.

Fed-Batch Cultivation in Bioreactor:

[0166] A bioreactor with a working volume of 15 L is used, and 10 L of chemically defined medium added to the vase. The following parameters are set: 400-800 rpm agitation, higher than 30% pO.sub.2, 1-2 vvm aeration, pH 7.00 and 34° C. temperature.

[0167] Initial values after inoculation are: OD 0.38, 1 vvm, 400 rpm, pH 7.03 and pO.sub.2 84%.

[0168] Agitation and aeration are adjusted according to the O.sub.2 consumption during the culture in order to maintain the desired parameters.

[0169] After 3 h of culture the OD is 4.52 and glucose concentration is 1 g/L. Induction is started in this moment by addition of yeast extract and 50 mL of IPTG 1 M.

[0170] Glucose 40% feed is initiated at 3.5 h from the start to maintain pO.sub.2 above 30% in a cascade mode, with a set up concentration of glucose of 1 g/L.

[0171] The total induction period is 4 h, and samples are collected every hour. Maximum OD 14.08 is reached at 3 h post induction. Final OD after 7.5 h of process is 13.86, with a maximum velocity of growth during the exponential phase of the culture. Total wet biomass or wet pellet obtained from the 10 L of working volume is 180 g. Total glucose added during the fed-batch is 112 g.

Harvest and Clarification:

[0172] Total culture volume (10 L) is centrifuged at 17500 g and 4° C. for 15 min. The pellet obtained (180 g) contains mostly intact bacteria, and is stored at −70° C. until further use.

Bacterial Rupture and Separation of Inclusion Bodies:

[0173] The total amount of wet pellet obtained in harvest (180 g) is resuspended in 2 L of Buffer Tris 20 mM, NaCl 200 mM, EDTA 1 mM, pH 7.4 until total dissolution. OD of such solution is 66.7.

[0174] The total volume of resuspended pellet is subjected to 4 serial homogenization cycles. Product obtained is centrifuged at 9500 g at 4° C. for 45 min, and supernatant is separated for purification of SERAR in soluble fraction (2 L).

[0175] Pellets containing inclusion bodies (72 g) are washed with Buffer Tris 20 mM, NaCl 200 mM, EDTA 1 mM, pH 7.4, and centrifuged at 9500 g at 4° C. for 45 min. Two further washing steps with Tris 20 mM Urea 4M are executed, and one final wash step with Buffer Tris 20 mM, NaCl 200 mM, EDTA 1 mM, pH 7.4 is performed. Supernatant is discarded and pellets (12 g) are stored at −70° C. for further processing.

[0176] Total distribution of the recombinant polypeptide SERAR is 80% in IBs and 20% in the soluble fraction determined by densitometric SDS-PAGE and capillary gel electrophoresis.

[0177] Higher than 98% of the protein content of the pellets is recovered as SERAR recombinant polypeptide.

Solubilization of Inclusion Bodies (IB) by Denaturation:

[0178] IBs containing 8.5 g of total protein are fully solubilized with 6 M urea at a concentration of 20 g of pellet per liter of buffer, followed by magnetic stirring at 900 rpm for 30 min.

[0179] This solubilized IBs is stored at −20° C.

Refolding of SERAR from Inclusion Bodies:

[0180] The dissolved IBs in 6 M urea solution from the previous step are centrifuged at 4000 g at 4-8° C. for 20 min. The recovered supernatant contains SERAR recombinant polypeptide and the pellet is discarded.

[0181] The supernatant is dropped at a constant rate of 50 mL/min into a vessel containing refolding buffer TrisHCl 20 mM sucrose 8% pH 7.4 with mild magnetic stirring.

[0182] The supernatant is finally diluted 10 times with the refolding buffer, and pH is adjusted to 6.0 with acetic acid.

[0183] This mixture is maintained for 48 h at 4-8° C. with mild magnetic stirring.

[0184] After 48 h the refolding volume is diafiltered against 10 volumes of Tris HCl 20 mM pH 7.4 using TFF with a molecular cut off of 10 kDa.

[0185] Concentration is also adjusted to 4-6 g/L and purity obtained is higher than 90%.

SERAR Recombinant Polypeptide Purified and Refolded from Inclusion Bodies:

[0186] SERAR recombinant polypeptide solution from the refolding step is subjected to chromatography with Capto DEAE resin.

[0187] Protein solution containing the SERAR polypeptide already folded at a concentration of 4.3 g/L in a Tris 20 mM pH7.4 buffer is loaded in the column at a concentration of 30 mg of protein per mL of resin.

[0188] Resin has been previously sanitized with NaOH 0.1 N and equilibrated in Tris 20 mM pH 8.0

[0189] Sample is loaded at a 150 cm/h linear flow followed by 3 column volumes (CVs) of wash buffer composed of Tris 20 mM NaCl 50 mM pH 8.0.

[0190] Elution is performed at the same linear flow, with Elution buffer (Tris 20 mM NaCl 1 M pH 8.0) ranging from 0 to 100% of Elution buffer in 10 CVs.

[0191] The recombinant polypeptide SERAR starts eluting with 0.4 M NaCl up to 0.55 M NaCl.

[0192] Purity of SERAR recombinant polypeptide in such eluate is 98% and the yield of the process is higher than 89%.

Example 3

[0193] Transepithelial Paracellular Passage of FSH with SERAR Study:

Cell Culture:

[0194] Caco-2 cell line HTB-37 (ATCC, Rockville, Md.), derived from human colon cells, and is used for all experiments. Cells are maintained in Dulbecco's Modified Eagles Medium (DMEM, American Type Culture Collection (ATCC), Rockville, Md.) supplemented with 67 IU/mL of penicillin, 67 μg/L of streptomycin, and 100 mL/L of fetal bovine serum. Monolayers are grown on Bio FIL-24 well pore 1 μm translucent PET membrane filter supports according to supplier instructions. At the end of the growth period, the integrity of the cell monolayer is confirmed by transepithelial electrical resistance (TEER) measurements (Millicell-ERS Voltohm meter, Millipore, Billerica, Mass.).

TEER Measures the Opening of Intercellular Junctions with Different Concentrations of SERAR:

[0195] Upper filter supports containing viable Caco-2 monolayers are transferred into a 24-well cell culture plate and 1000 μL of media is dispensed into each basolateral compartment. Solutions containing the recombinant FSH 11.000 mUI/ml (Gonal F®) and different doses of SERAR polypeptide solution (0.42, 1, 3, 6, 9, 12, 15 mg) are applied to the apical compartment and TEER readings are taken at each time 0 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h.

Transepithelial Paracellular Passage Study:

[0196] Firstly, purified SERAR recombinant polypeptide and FSH are dissolved in Dulbecco's Modified Eagles Medium (DMEM, American Type Culture Collection (ATCC), Rockville, Md.).

[0197] rFSH and different concentrations of SERAR Solutions are added to the apical side of Caco-2 monolayers. Samples are taken from the basolateral compartment at time: 0 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h and Transepithelial Paracellular Passage is quantified by the amount of rFSH transported across the barrier in the basolateral well through the time by IMMULITE. Positive control experiments are performed with addition of rFSH+EDTA 2.5 mM on the apical section of the cells.

Results

SERAR Recombinant Polypeptide Opens the Intercellular Junctions and Reduces the TEER of the Caco-2 Monolayer:

[0198] Using TEER as a surrogate marker for FSH permeability, the study of all doses of the SERAR purified recombinant polypeptide is studied. The use of TEER as a measurement for permeability has several advantages, including convenience and a lack of dependence on the size of the solute, thereby ensuring the generality of results.

[0199] SERAR recombinant polypeptide opens the intercellular junctions and reduces the TEER of the Caco-2 monolayer after 20 min of exposure to SERAR with a reversible mechanism. SERAR exhibited maximal reduces the TEER of the Caco-2 monolayer at the dose of 15 mg in FIG. 5.

SERAR Performs the Transepithelial Paracellular Passage of FSH:

[0200] SERAR exhibited pronounced reduces the TEER of the Caco-2 monolayer at the dose of 6 mg at different times during the study in FIG. 6A.

[0201] The purified recombinant polypeptide SERAR is effective transepithelial paracellular passage device validated by methods used in this example. The device increases the transepithelial paracellular passage of the therapeutic polypeptides molecules, such us the glycoprotein Follicle Stimulating Hormone (FSH) of 30 kDa, more than 1000 times. These values are better than the maximum attainable permeability achieved, indeed better than a positive control EDTA 2.5 mM. This serves as an example of SERAR recombinant polypeptide successfully perform the transport of polypeptide macromolecules across intestinal epithelial cells.

Example 4

Successful of SERAR Amount

[0202] Formulations composed by SERAR and FSH as therapeutic protein, with increasing amounts of SERAR are compared for their ability to facilitate absorption of FSH by transepithelial paracellular passage in methods and compositions of the present invention. SERAR and FSH are co-formulated as described in the above Examples. Increasing amounts of SERAR polypeptide is added to formulations with fixed amount of FSH and those formulations are tested in a Caco-2 monolayer in vitro test. The most effective SERAR polypeptide amount is used in subsequent studies FIG. 7.

Example 5

Successful of Protease Inhibitor Amount

[0203] Aprotinin protease inhibitor is studied to preserve the SERAR recombinant polypeptide and the therapeutic polypeptide following oral administration in methods and compositions of the present invention. SERAR and the therapeutic polypeptide are co-formulated as described in the above Examples, except that aprotinin is added as a gastroenteric protease inhibitor. The amounts of this protease inhibitor is also varied, to determine the successful amount of gastro-protective protease inhibitor. The most effective protease inhibitor amount is used in Subsequent Examples.

Example 6

Successful of Therapeutic Polypeptide FSH Concentration

[0204] Formulation are compared for their ability to facilitate absorption of FSH following oral administration in methods and compositions of the present invention. SERAR and FSH are co-formulated as described in the above Examples. The most effective SERAR/FSH is used in Subsequent experiments.

Example 7

Manufacture of Polypeptide Nanoparticles Using Purified Recombinant Polypeptide SERAR and Therapeutic Polypeptide Follicle Stimulating Hormone

[0205] 75 IU of FSH and 25 mg of correctly purified recombinant polypeptide SERAR are dissolved in a final volume of 12.5 mL of polyvinylpyrrolidone (PVP) 0.15% w/v under constant magnetic stirring at 500 rpm. This protein solution is further mixed for 20 min. In the polypeptide nanoparticles synthesis, an appropriate volume of ethanolic solution of gastroprotective methacrylic acid copolymer 0.15% w/v is injected into the protein solution at 3 mL/min using a narrow tube (0.5 mm inner diameter) under constant magnetic stirring at 500 rpm. The SERAR-FSH nanoparticles suspension is kept under magnetic stirring at 500 rpm for another 20 min. Then, one volume of PVP 0.15% w/v is added under magnetic stirring. The nanoparticle suspension, with an average particle diameter between 100-350 nm, is concentrated 5 times followed by a diafiltration using 10 volumes of Tris buffer solution pH 7.4. Both processes are performed by tangential flow filtration using a 300 kDa polyethersulfone membrane (Pellicon 3 Merck-Millipore). The mean particle size remains stable during the concentration and diafiltration. Finally, the protein nanoparticle suspension is lyophilized for 72 h with or without the addition of any lyoprotectant. The mean particle size and polydispersity index remain stable after storing at room temperature and reconstitution with saline solution. The gastro-protected polypeptide nanoparticle synthesis yield (>99%), encapsulation efficiency (>99%) and mean particle size (100-350 nm) are performed as quality control of the reconstituted formulation.

Example 8

Extemporaneous Preparation of Gastro-Protected Polypeptide Nanoparticles of Cetuximab

[0206] First, 250 mg of cetuximab and 2.5 g of purified recombinant polypeptide SERAR are dissolved in a final volume of 1.4 L of polyvinylpyrrolidone (PVP) 0.15% under constant magnetic stirring at 500 rpm. This protein solution is mixed for 20 min. For the polypeptide nanoparticle synthesis (yield >99%), an appropriate volume of ethanolic solution of Eudragit L-100 0.15% is injected to the protein solution at 3 mL/min using a narrow tube (0.5 mm inner diameter) under stirring at 500 rpm. The SERAR-Cetuximab nanoparticle suspension is kept under magnetic stirring at 500 rpm for 20 min and later. Then, one volume of PVP 0.15% is added under magnetic stirring. The nanoparticle suspension, with an average particle diameter between 200-500 nm, is concentrated 5 times followed by a diafiltration using 10 volumes of Tris buffer solution pH 7.4. Both processes are performed by tangential flow filtration using a 300 kDa polyethersulfone membrane (Pellicon 3 Merck-Millipore). The mean particle size remains stable during the concentration and diafiltration. Finally, the cetuximab nanoparticle dispersion is lyophilized for 72 h with the addition of mannitol as lyoprotectant. The extemporaneous suspension powder composed by SERAR-Cetuximab nanoparticles remains stable—in terms of mean particle size, polydispersity index and encapsulation efficiency—after storing at room temperature and reconstitution with saline solution.

[0207] The gastro-protected polypeptide nanoparticle synthesis yield (>99%), encapsulation efficiency (>99%) and mean particle size (100-350 nm) are performed as quality control of the reconstituted formulation.

Example 9

[0208] Microspheres Coated with Gastro-Protected Polypeptide Nanoparticles of Follicle Stimulating Hormone

[0209] First, 75 IU (or 150 IU) of Follicle Stimulating Hormone (FSH) and 25.0 mg of purified recombinant polypeptide SERAR are dissolved in a final volume of 10 mL of polyvinylpyrrolidone (PVP) 0.15% under constant stirring at 500 rpm. This polypeptide solution is mixed for 20 min. For the polypeptide nanoparticles synthesis (yield >99%), an appropriate volume of ethanolic solution of Eudragit L-100 0.15% is injected to the protein solution at 2 to 160 mL/min using a narrow nozzle under stirring at 500 rpm. The FSH protein nanoparticle dispersion is kept under stirring at 500 rpm for 20 min and later. One volume of PVP 0.15%, used as stabilizing dilution, is added under magnetic agitation at 500 rpm. The mixture is kept at room temperature for 30 min. The average size of polypeptide nanoparticles are between 250 and 500 nm were obtained. Finally, the polypeptide nanoparticle dispersion is freeze-dried for 48 h with or without the addition of any kind of lyoprotectant (e.g. mannitol).

[0210] Gastro-protected FSH polypeptide nanoparticle layering and enteric coating processes in sugar microspheres are carried out with a fluid bed coater Mini Glatt (Glatt®). 50 g of sucrose microspheres (size 710 μm) are weight and put into the Mini Glatt and dried at 35° C. by air current at 40° C. The instrument parameters are:

[0211] Coating mode: Würster. Silicon tubes measures: 2 mm (internal diameter) and 4 mm (external diameter).

[0212] Sprayer diameter: 0.8 mm.

[0213] Inlet air pressure: 0.2-0.4 bar.

[0214] Atomization pressure: 1.5-2-5 bar.

[0215] Bed temperature: 35-40° C.

[0216] Peristaltic pump (Flocon®): 5 rpm.

[0217] Protein nanoparticle layering suspension percentage composition

TABLE-US-00002 Ingredient Commercial name Percentage (%) Therapeutic protein Freeze dried FSH protein 0.19 nanoparticles PVP K-29/32 PLASDONE ™ K-29/32 6.54 Talc Van Rossum 3.27 Distilled water — 90 Total 100

[0218] Solid content: 10%.

[0219] Once finished the gastro-protected polypeptide nanoparticle layering process the microspheres are dried by air current for 10 min. Then the enteric coating suspension is applied.

Enteric Coating Percentage Composition

[0220]

TABLE-US-00003 Ingredient Commercial name Percentage (%) EUDRAGIT ® L 30 D-55 EUDRAGIT ® L 30 D-55 41.65 Triethyl citrato (TEC) Citrofol ® AI 1.25 Talc Van Rossum 6.25 Distilled water — 50.85 Total 100

[0221] Solid content: 10%.

[0222] After the gastro-protective coating, the microspheres are dried again under air current for 10 min. Once the fluid bed coating process is finished, #4 capsules are filled with 130 mg coated microspheres.