CLIAMS:none ,TagSPECI:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION:
“ORALLY ACTIVE PROTEINS AND PEPTIDES”
2. APPLICANT I
Name : TRANSGENE BIOTEK TLD.
Address : Plot No. 68, 69 & 70, IDA Bollaram, Anrich Industrial Area, Medak
District, Andhra Pradesh 502 325, INDIA

3. PREAMBLE TO THE DESCRIPTION
Orally active proteins and peptides

4. DESCRIPTION:
Technical Field
[001] The present disclosure relates to a field of pharmaceutical formulations and methods for oral delivery of biotherapeutic peptides and proteins to a vertebrate host via receptors present in the gastro intestinal tract. Additionally, the present disclosure relates to processes for preparing suitable formulations for oral delivery of biotherapeutic proteins to a vertebrate host.
Background
[002] With the advancement of recombinant DNA technology and large scale production of recombinant proteins, there is an ever increasing list of bio-pharmaceutical products that are available for administration to patients. Unfortunately, the administration of these molecules must generally be via injections either once, twice or thrice weekly, depending upon the molecule and dose, or, in the case of insulin, up to four times daily. The administration of these molecules via the sub-cutaneous route has many disadvantages, including pain and swelling at the site of injection, highly variable intra and inter-subject variability in dosing, and large fluctuations in the serum profile of the sub-cutaneous administered drugs.
[003] Administration of these proteins via alternative routes is precluded by the very low bioavailability of these molecules when given orally, transdermally, or via pulmonary administration. Despite this, there are obvious benefits to oral delivery of the afore-mentioned molecules, including the ability to maintain a flatter serum profile of the administered protein, an increase in patient comfort and an increase in patient compliance. As such, the oral administration of proteins would be regarded as much more “patient friendly” and clearly more desirable than sub-cutaneous administration.
[004] Even though the oral route of administration may be a highly desirable, the gastro-intestinal tract poses a number of physical and chemical barriers to successful administration of therapeutic agents. The gastro intestinal tract is specifically designed to degrade proteins, and thus orally administered biotherapeutics must withstand acidic meliu of the stomach as well as attack from endogenous proteolytic enzymes, without losing its therapeutic activity. In addition to surviving the harsh milieu of the intestine, the orally administered material must still have some means whereby it can cross the gastrointestinal barrier, enter into the systemic circulation and move to the site of activity. All of this must take place at an appropriate rate to ensure that correct therapeutic dosage is delivered.
[005] An approach has been the use of intestinal transporters to enable the biotherapeutic to cross the intestinal barrier and gain entry into the circulation. One such mechanism has previously been described by Russell-Jones and co-workers (1995, 1996, 2007). These workers have utilized the uptake pathway for the water-soluble vitamin, vitamin B12 (cobalamin or cyanocobalamin). It has been shown that uptake of vitamin B12 is dependent upon binding of the vitamin by gastric intrinsic factor (IF), with subsequent recognition of the IF-VB12 complex by a membrane expressed receptor (IFCR) on the small intestinal epithelial cell (the enterocyte). Following binding of the IF-VB12 complex to the IFCR, the IF-VB12 is internalized, whereupon the IF is degraded and another VB12 binding protein, transcobalamin II binds to the VB12 and transports it across the cell. Russell-Jones and co-workers (2007) have shown that it is possible to link vitamin B12 to peptides, proteins and nanoparticles and use this normal, intrinsic factor-mediated transport system for vitamin B12, to co-transport the attached cargo from the intestine to the circulation following oral feeding. Whilst this transport system has been shown to have commercial utility in the oral delivery of peptides, proteins and nanoparticles, its utility it limited by a number of factors. Firstly, vitamin B12 needs to be chemically modified to provide a suitable site for conjugation to the material to be limited, secondly the uptake capacity of the vitamin B12 transport system is rather low (1 nmole per feed), and thirdly vitamin B12 is relatively large molecule with a molecular weight of 1355.38, when compared with other potential carriers.
[006] Biotin, a 244.31 molecular weight, water soluble vitamin, is an essential vitamin, which has been shown to have good bioavailability following oral feeding to humans. The average daily uptake of biotin is around 15-70 ug (60-300 nmole), which is considerably higher than that of vitamin B12 (1.34 ug, 1 nmole). Single feeding of 1 mg of biotin to human results in serum levels of 34,000 pmol/litre (8.3 ug/litre) 1-3 hours after feeding (Mardach et al, 2002).

[007] It is generally believed that the mechanism of uptake of biotin from the intestine is due to the sodium dependent multivitamin transporter (SMVT), which has affinity for biotin, pantothenic acid and lipoate, and which has been identified in rabbit small intestine, rat small intestine and colon and Caco-2 cells (Chatterjee et al, 1999). A Na+ dependent transport of 3H-biotin was shown in Caco-2 transwell cultures (Ng and Brochardt, 1993). Ortiz etal (1998) described the functional existence of a specialized carrier -mediated, Na+-dependent system (the SMVT) for biotin uptake in colonic epithelial cells.
[008] The possible use of the SMVT system described above, as an uptake mechanism for biotin-targeted peptides, proteins or nanoparticles in a similar fashion to that described for vitamin B12 conjugates is precluded by the need to maintain a free carboxyl group on the biotin, pantothenic acid or lipoate molecules, which is lost during conjugation to the drug to be delivered orally. Furthermore, the SMVT utilizes a “pore-like” structure, which is only suitable for transport of very small molecules (less than 1,000 MW), and hence the SMVT has very limited utility for the transport of pharmaceuticals.
[009] An alternative cellular uptake system for biotin, which is different from the SMVT has been described on several cancer cell lines. This system is capable of uptake of the free vitamin as well as biotin-polymer and biotin-nanoparticle conjugates (Russell-Jones et al, 2003; 2004; McTavish et al, 2003). This uptake system is highly expressed on many aggressive tumour cell types and is separable from SMVT, as the later requires a free carboxyl group on the biotin.
[0010] Whilst SMVT has been shown to be responsible for some uptake of biotin in the small intestine, the presence of an alternative cellular uptake system for biotin has, until recently, not been known. Thus, Balamurugan and co-workers (2003) could find no evidence of uptake of biotin via the high affinity Na+-independent biotin carrier in CaCo-2 cells or HepG2 cells grown as cell monolayers.
[0011] In order to address the long felt need of such a solution, the present disclosure discloses methods and formulations for using the transport system for the intestinal uptake and transport of biotin-conjugated peptides, proteins, polymers, and nanoparticles.

Summary
[0012] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements or delineate the scope. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
[0013] Exemplary objective of the present subject matter is to prepare pharmaceutical formulations and methods for oral delivery of biotherapeutic peptides and proteins to a vertebrate host via biotin specific receptors present in the gastro intestinal tract.
[0014] Another exemplary objective of the present subject matter is to produce complexes of biotin or its analogues with the pharmaceutical biotherapeutics, without altering its therapeutic activity, to be delivered orally and utilizing the biotin transport system for effective oral delivery of such complexes.
[0015] Another exemplary objective of the present subject matter is to use biotin which is relatively smaller in size as compared to other essential vitamin like vitamin B12 and has a relatively high uptake capacity.
[0016] Another exemplary objective of the present subject matter is to use biocytin which is a natural derivative of biotin having a free carboxyl group and amino group for conjugation.
[0017] Another exemplary objective of the present subject matter is to provide a new set of oral biotherapeutics to which a biotin molecule, or analogue thereof, has been conjugated, thereby facilitating oral delivery of biotherapeutic which do not have their own receptor in the gastrointestinal tract.
[0018] Another exemplary objective of the present subject matter is to provide a pharmaceutical composition which comprises a drug-biotin conjugate of the present subject matter encapsulated with a pharmaceutically acceptable excipient. The excipient disclosed in the present disclosure is pH sensitive and protects the API from the harsh acidic milieu of the stomach containing various proteolytic enzymes. Once into the duodenum and into an increased pH environment, the encapsulation falls apart releasing the API to interact with its receptor. On the other hand, biotin conjugated to the API interacts with biotin receptor present on the surface of enterocytes.
[0019] Another exemplary objective of the present subject matter is to provide a pharmaceutical composition whose transport across the intestinal barrier and bioactivity is independent of particle size. The formulation releases the API in the alkaline pH of the intestine making the API accessible to its receptors.
Brief description of drawings
[0020] Other objects and advantages of the present disclosure will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:
[0021] FIG. 1 illustrates an in vitro assay for enterocyte cell permeability for predicting in vivo absorption of the targeted drugs across the gut.
[0022] FIG. 2 is a graph illustrating the movement of biotin-insulin formulation across polarized and differentiated Caco2 monolayer.
[0023] FIG. 3A is a graph illustrating the internalization of Biotin-Q dots into CHO cells.
[0024] FIG. 3B is a graph illustrating the internalization of Biotin-Q dots into Caco2 cells.
[0025] FIG. 4 is a diagram representing the presence and location of insulin and biotin receptors in various regions of the gut.
[0026] FIG. 5 is a graph illustrates the hypoglycemic effect of therapeutic proteins in a diabetic rat.
[0027] FIG. 6 is a bar diagram describing the distribution of insulin in major organs of a diabetic rat when biotin-insulin formulation is administered orally.

Detail description
[0028] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[0029] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[0030] According to a non limiting exemplary embodiment of the present subject matter, a pharmaceutical formulation and a process for oral delivery of pharmaceutically active compounds which may include but not limited to proteins and peptides like insulin or its analogues via biotin receptors present in the gastrointestinal tract of the vertebrate host is disclosed. According to exemplary aspect of the present disclosure, the pharmaceutical formulation includes the pharmaceutically active compound which is conjugated to targeting moiety may include but not limited to biotin or its analogues and the like to form a peptide-biotin complex.
[0031] According to a non limiting exemplary embodiment of the present subject matter, the peptide-biotin complex reacts with each other through a cross-linking agent and a spacer compound so that the biotin does not interfere with the peptides binding to its own receptor.
[0032] In accordance with a non limiting exemplary aspect of the present disclosure, the peptide-biotin complex is encapsulated by a polymer which is stable in the harsh environment of the gastrointestinal tract. The capsule opens up in the alkaline pH of the gut thereby releasing peptide-biotin conjugate, in which the peptide or biotin can bind to its intestinal receptor of the vertebrate host.
[0033] According to a non limiting exemplary embodiment of the present subject matter, the peptide-biotin conjugate is transported by carrier-mediated transcyotsis across the intestinal barrier using peptide and biotin receptors. Thus there are two potential receptor systems that partake in absorption of peptide thereby increasing the total bioavailability of peptide in the systemic circulation.
[0034] In accordance with a non limiting exemplary aspect of the present disclosure, the peptide-biotin conjugate is preferentially taken up by the lymphatic system for example in case of insulin or its analogues, resulting in a sustained release in systemic circulation and a prolonged therapeutic effect. The uptake of peptide-biotin conjugate by the lymphatic system in the intestinal space which avoids first pass metabolism of liver and results in increasing total bioavailability of the drug in the systemic circulation.
[0035] Referring to FIG. 1 is a diagram 100 illustrating an in vitro enterocyte cell permeability assay for predicting in vivo absorption of the targeted drugs across the gut. According to a non limiting exemplary embodiment of the present subject matter, colon adenocarcinoma Caco2 cells are cultured on the semi permeable membranes for ~18 days where they differentiated to form a Caco2 monolayer which is morphologically and biochemically similar to the intestinal enterocyte monolayer having distinct apical and basolateral membranes. The transepithlial electrical resistance (TEER) between apical and basolateral chambers indicates the tightness of the cells and reaches a maxima in around 15 days preventing any paracellular movement of the drug in between the cells. The targeted drug is added on the apical chamber and its appearance in the basolateral chamber indicates its potential to cross the intestinal barrier.
[0036] Examples of typical biotherapeutics for delivery according to the present disclosure include but not limited to hormones and bioactive peptides such as vasopressin, oxytocin, insulin, testosterone, interferons, somatotrophin, somatostatin, Erythropoietin, Colony Stimulating Factors (GCSF, GM-CSF, CSF), PMSG, HCG, inhibin, PAI-1, PAI-2, therapeutic agents such as neomycin, salbutamol, pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine, zylazine, ketamine HCl, GABA, iron dextran, nucleotide analogues or ribozyme.

[0037] Further examples of active substances include polypeptides such as, insulin, somatostatin, somatostatin derivatives, growth hormones, prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone(TRH), thyroid stimulating hormone (TS), luteinizing hormone (LH), follical stimulating hormone (FS), vasopressin, vasopressin derivatives, oxytocin, calcitonin, parathyroid hormone, glucagon, gastrin, secretin, pancrozymin, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin (HCG), enkephalin, enkaphalin derivatives, endorphin, kyotorphin, interferons (?, ?, ?), interleukins (I, II and III), tufstin, thymopoietin, thymosin, thymostimulin, thymic humoral factor (TFH), serum thymic factor (FTS), and its derivatives, tumor necrosis factor (TNF), colony stimulating factor (CSF), motilin, dinorphim, bombesin, neurotensin, cerulein, bradykinin, urokinase, asparaginase, kallikrein, substance P and its antagonists, nerve growth factor, blood coagulation factors VIII and IX, lysozyme chloride, polymyxin B, colistin, gramicidin, bacitracin, protein synthesis stimulating peptides, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), platelet-derived growth (PDGF), growth hormone, growth hormone factor (GRF, somatocrinin), bone morphogenic factor (BMP), epidermal growth factor (EGF), Fab and Fc portions of mAb’s etc.
[0038] Accordingly, this invention provides a new set of biotherapeutics to which a biotin molecule, or analogue thereof, has been conjugated. These biotin -drug conjugates are suitable for oral delivery to a vertebrate host whereby as they can utilize the aforementioned biotin receptor system for uptake binding and uptake of the pharmaceutical from the intestine into the circulation of the vertebrate host.
[0039] Referring to FIG. 2 is a graph 200 describing the movement of insulin and biotin-insulin API across differentiated Caco2 monolayer. In accordance with a non limiting exemplary aspect of the present disclosure, the API’s dissolved in buffer solution are added on the apical chamber and the amount of API appearing in the basolateral chamber is monitored by Enzyme-linked immunosorbent assay (ELISA). The amount of biotin-insulin formulation appearance in the basolateral chamber increased from 0 to 4 hours indicates the potential of biotin-insulin formulation to bind to biotin receptors on the Caco2 cells and the movement of the biotin-insulin formulation across the monolayer. This indicates that binding capacity of biotin to the insulin has the potential to take it across the intestinal barrier. Here, since unmodified insulin also appears to cross the monolayer, it is difficult to infer whether movement is through biotin receptors only.
[0040] Referring to FIG. 3a is a graph 300a illustrating the internalization of Biotin-Q dots into CHO cells. Biotin Q dots represent biotin conjugated to a drug moiety and since the size of the Q dots is similar to a small drug, internalization of these particles into the cells cannot be via SMVT. The only way Biotin Q dots can cross the barrier is through carrier mediated endocytosis utilizing biotin receptors. Internalization of Biotin-Q dots into these cells indicates the presence of the biotin receptor and the ability of the biotin to take a drug sized molecule into the cells. These cells represent 1 hour and 4 hour internalization of Biotin-Q dots, in which the internalization of Biotin-Q dots is increased with the increased time interval. When non conjugated biotin is added to the cells before Biotin-Q dots, internalization is inhibited indicating that biotin mediated internalization of Q-dots is specifically using biotin receptors and not through any other pathway. Similarly FIG. 3b is a graph illustrating the internalization of Biotin-Q dots into Caco2 cells. These cells are intestinal cell lines and internalization of Biotin-Q dots into these cells indicate the presence of the biotin receptor and the ability of the biotin to take a drug sized molecule into the cells.
[0041] Suitable analogues of biotin according to the disclosure may include but not limited to biotin, iminobiotin, Biocytin hydrazide, Biotin hydrazide, biocytin, 5-(Biotinamido)pentylamine, Sulfo-NHS(n-Hydroxysuccinimidyl)-Biotin, Sulfo-HNS-hexanyl-biotin (Sulfo-NHS-LD-Biotin), NHS-Biotin, Pentafluorophenyl-biotin, Pentafluorophenyl-polyethylenoxide-biotin, NHS-biotin Trifluoroacetamide, NHS-Iminobiotin trifluoroacetamide, Maleimido-polyethylenoxide biotin, Maleimido-polyethylenoxide iminobiotin, chloroacetylated biotin, Phosphonoacetatyl-1’N-biotin, biocytin
[0042] Suitable extended spacers for the conjugation of the pharmaceutical to biotin according to the disclosure may include but not limited to disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BSS), ethylene glycolbis(succinimidylsuccinate) (EGS), ethylene glycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS), p-amino-phenylacetic acid, dithiobis(succinimidylpropionate) (DSP), 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (Sulfo-DST), bis[2-(succinimidyloxycarbonyloxy)-ethylene]sulfone (BSOCOES), bis[2- (sulfosuccinimidooxycarbonyloxy)-ethylene]sulfone (Sulfo-BSOCOES), dimethyl adipimidate.2 HCl (DMA), dimethyl pimelimidate.2 HCl (DMP), dimethyl suberimidate.2 HCl (DMS), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophyl)butyrate (SMPB).
[0043] These biotin -drug conjugates are suitable for oral delivery to a vertebrate host whereby as they can utilize the aforementioned biotin receptor system for uptake binding and uptake of the pharmaceutical from the intestine into the circulation of the vertebrate host.
[0044] Such protective excipients include but are not restricted to carboxymethyl dextran, carrageenan, inulin, carboxymethy cellulose, starch and derivatives thereof; chondroitin sulfate, styrene-maleic anhydride copolymer, divinylether-maleic anhydride copolymer, poly-glutamic acid, polyaspartic acid, polylactic acid.
[0045] Referring to FIG. 4 is a diagram 400 representing the presence and location of insulin (red) and biotin (green) receptors in various regions of the gut. As is clear from the images insulin receptors are located in the duodenum and ileum whereas biotin receptors are present all along the length of the gastrointestinal tract including jejunum. According to the present disclosure, insulin formulation is transported across the intestinal barrier using a combination of both the receptors which increases the length of the absorptive surface. Further, it is these receptors which are responsible for the movement of the biotin-insulin formulation across the Caco2 cells described in Fig 2.
[0046] Referring to FIG. 5 is a graph 500 illustrating the hypoglycemic effect of oral insulin formulation in a diabetic rat study. Here, as a control, a set of diabetic rat were injected with the insulin represented by solid blue colored line showing sharp reduction in blood glucose levels. Further the hypoglycemic effect was rapid and short lived. In another set, diabetic rats were injected with an insulin conjugated to biotin represented by a solid red colored line. Here again, there was observed a hypoglycemic effect indicating that biotin conjugation did not alter its hypoglyceic activity. However, the reduction in blood glucose was not as rapid as unmodified insulin and the effect lasted up to 4 hours after administration. In another set, diabetic rats were orally administered with a biotin-insulin formulation represented by a solid green colored line. Oral administration of insulin formulation resulted in sustained and prolonged hypoglycemic effect for up to 8 hours. An explanation for this sustained activity could be the utilization of both insulin and biotin receptors which are present throughout the gut thereby increasing the absorptive length for the formulation. The levels of blood plasma insulin when biotin insulin was injected is represented by dotted red colored line and when biotin insulin formulation was orally administered represented by a dotted green colored line.
[0047] Referring to FIG. 6 is a bar diagram describing the distribution of insulin in major organs of diabetic rats when biotin-insulin formulation was administered orally. These include spleen, kidney, liver and heart. The levels of insulin are observed to be highest in the spleen while compared to liver and the other organs. This observation indicates that the insulin after crossing the intestinal barrier is taken up preferentially by the lymphatic circulation. This also explains the lower serum levels of insulin in diabetic rats that were orally administered with biotin insulin formulations.
[0048] Also, those skilled in the art can appreciate from the foregoing description that the present disclosure can be implemented in the variety of forms. Therefore, while the embodiments of this disclosure have been described in connection with particular examples thereof, the true scope of the embodiments of the disclosure should not be so limited since other modifications will be apparent to the skilled practitioner upon a study of the drawings and following claims.

5. CLAIMS
6. DATE AND SIGNATURE:

Dated this 17th June 2013,
Authorized Signatory’s Name: Dr. Kollipara Koteswara Rao
Authorized Signatory’s Signature:

7. ABSTRACT
Exemplary aspects of the present invention disclosure relates to a novel pharmaceutical formulation and a process for oral delivery of biotherapeutic peptides and proteins like insulin conjugated with biotin to form a complex and encapsulated by a polymer for binding to its own as well as biotin receptors of the gastrointestinal tract of the vertebrate host is disclosed. The encapsulation prevents proteolytic degradation of the biotherapeutic in the GI tract and releases the API in the duodenum as a result of increase in pH. Released API conjugated to biotin is thus presented to its own receptor as well as biotin receptor which facilitate its movement across the intestinal barrier into systemic circulation in a sustained and prolonged rate. Utilization of potentially two receptor systems increases the bioavailability of the biotherapeutic protein. The process further comprises the uptake of the pharmaceutical formulation by the lymphatic system of biotherapeutics like insulin resulting in a prolonged therapeutic effect.