The technical field of the present invention relates to method of improving the bioavailability of prodrug, targeting absorption via lymphatic system The technical field also relates to solid lipid nanoparticles of prodrug and process of preparation thereof
The dawn of novel drug developing and screening technologies has improved the potency of the molecules, but at the same time has induced greater physicochemical and biological obstacles, such as poor solubility and poor hpophilicity Drug administration through oral route is the most preferred one as it offers numerous advantages but the therapeutic efficacy of an orally administered drug is primarily dependent on the drug's potency and the systemic availability or bioavailability Drug potency is largely under the influence of its chemical or molecular structure and reclines to be an inherent property of an individual molecule Bioavailability is affected by multiple reasons like physicochemical, formulation, and patient related issues As the drug bioavailability is influenced by a number of factors, it can be rmproved by applying formulation strategies involving modification of its physicochemical and to some extent biological response, to obtain highest possible bioavailability and therapeutic benefit However, it is important to understand the behavior of a drug molecule during its (i) processing (n) storage and (in) post administration phase The drug after the oral administration has to successfully overcome a complex gastrointestinal tract (GIT) environment consisting of varying pH, digestive enzymes before it gets absorbed into systemic circulation
To improve the bioavailability of drugs having poor solubility and poor hpophilicity, number of approaches are documented in the prior art These approaches may be broadly classified into two categories including modifications in the chemical structure of the molecule and in the formulation Alteration of the structure of the molecule, preparation of pro-drug and salt formation are amongst the chemical modifications of the drug molecule Use of solubility enhancing agents or absorption enhancers, formation of inclusion complexes and size reduction to micron or even nanometer levels, are amongst formulation development methods to enhance the bioavailability
Prodrug preparation is a well-known approach to improve the bioavailability A prodrug means any pharmaceutically acceptable ester, salt of an ester, or other derivative of an active moiety, which upon administration to a recipient, is capable of readily providing the active moiety enzymatically or non-enzymatically Prodrugs are designed to
improve the solubility and permeability of a molecule, and consist of very weak bonds in their structures These weak bonds help in easy reversal to active parent moiety in the intestinal epithelial cell or blood, but at the same time it makes them susceptible to the actions of digestive enzymes and pH of the gastrointestinal tract, leading to pre-absorption degradation, thus defeating the purpose for which the prodrug was designed The problem is further aggravated when prodrugs show rapid efflux from the intestinal wall, wherein the prodrug is absorbed well, but after entering into the intestinal cells is readily converted into the active moiety, and effluxed back into the intestine instead of entering into the blood circulation, thereby reducing the effective bioavailability Cefpodoxime proxetil is a prototype example of such prodrugs, which has problems of extensive pre-absorption degradation and efflux mechanism The absolute bioavailability of commercially available cefpodoxime proxetil tablets is thereby reduced to only about 50% compared to that of cefpodoxime sodium intravenous infusion Judicious application of formulation technology is required, for achieving better results in such cases
The prior art teaches various formulation approaches targeted mainly to the improvement of solubility, which is thereby expected to improve the bioavailability One such approach relates to the use of hpidic systems for poorly soluble drugs The formation of hpidic systems includes micro or nano emulsions, liposomes, self-emulsifying drug delivery systems, and solid lipid nanoparticles
US 4,880,634 discloses lipid nanopellets for peroral administration of poorly bioavailable drugs It discloses an excipient system containing a drug for peroral administration in the form of an ultrafine aqueous, colloidal suspension of lipid nanopellets comprised of lipids and a surfactant of which the particle diameters of the nanopellets range from 50-1,000 nm, ratio of lipid to surfactant in the lipid nano-pellets ranging from 1 0 1 to 1 2 2, wherein the lipid nano-pellets are present in the suspension in a concentration of from 1-20% by weight The lipid nano-pellets can be provided with pharmacologically active substances, making possible improved biological availability upon peroral administration
US 5,785,976 relates to suspensions of particles of biodegradable lipids solid at room temperature, preferably triglycerides, which can be used as carriers for poorly water-soluble drugs or other bioactive agents, and to suspensions of particles
Eldem et al (Pharm Res 8, 1991, 47-54) describes lipid micropellets prepared by spray-drying and spray-congealing processes The spherical micropellets obtained had smooth surfaces
US 5,250,236 discloses a method to produce solid lipid nanoparticles using microemulsion technique, involving the steps of adding to a molten lipid substance a mixture consisting of water, a surfactant and possibly a co-surfactant to form a microemulsion, dispersing the microemulsion in water, washing with water by diafiltration, and lyophilizing to obtain the final product
US 6,337,092 discloses submicron to micron size particles or microparticles coated with a natural phospholipid in which particles are prepared using a combination of electrostatic and steric stabilizing agents from at least one charged surface modifier and at least one block copolymer respectively The growth in size of the particles, and hence their storage stability, is controlled by such combination of electrostatic and steric stabilizing agents
Lipid nanoparticles are known in the art to improve the bioavailability of few hydrophobic drugs However, we have now discovered that solid lipid nanoparticles could be used as a delivery system to formulate prodrugs having problems of pre-absorption degradation (in the gastrointestinal lumen, or in the enterocytes or epithelial cells), and/or efflux mechanism, and consequently enhancing their bioavailability to surprising levels by targeting absorption via lymphatic system
Hence, in one general aspect there is provided, a method of improving the bioavailability of a prodrug by targeting absorption via lymphatic system
In another general aspect there is provided, a method of improving the bioavailability of prodrug by 1 25 times or more than from conventional oral dosage forms, using solid lipid nanoparticles
In another general aspect there is provided, solid lipid nanoparticles of prodrug comprising prodrug and lipid carrier, and providing bioavailability of 1 25 times or more than from conventional oral dosage forms
In another general aspect there is provided, a process for the preparation of solid lipid nanoparticles of prodrug comprising the steps of dissolving/dispersing the prodrug in mixture of lipid carrier and stabilizer, and further processing into a suitable dosage form
The lymphatic system is a subsystem of the circulatory system Lymphatic system is like the blood circulatory system, wherein the lymph vessels branch through all parts of the body like the arteries and veins, except that the lymphatic system carries a colorless liquid called lymph instead of blood The anatomy and physiology of the lymphatic system varies in different regions of the body The lymphatics of the small intestine are characterized by the presence of a centrally located lacteal in each villi The lacteals join a plexus of lymphatic capillaries and drain, via the mesenteric lymph vessel, into the cisterna chyli Lymph from the cisterna chyli is drained by the thoracic duct, which empties directly into the general circulation at the junction of the left internal jugular and left subclavian veins Gut associated lymphoid tissues comprise tonsils, adenoids (Waldeyer's ring), Peyer's patches, lymphoid aggregates in the appendix and large intestine, lymphoid tissue accumulating with age in the stomach, small lymphoid aggregates in the oesophagus, diffusely distributed lymphoid cells and plasma cells in the lamina propria of the gut Peyer's patches are quite large aggregates of lymphoid tissue found in the small intestine The overlying 'dome' epithelium contains large numbers of intraepithelial lymphocytes Some of the epithelial cells have complex microfolds in their surfaces They are known as M cells and are believed to be important in the transfer of antigens from the gut lumen to Peyer's Patches
Hence, due to its unique anatomy and physiology, potential exists to exploit the lymphatic system as an alternative means of drug delivery Targeting drugs into the lymph has certain advantages These advantages include avoidance of first pass metabolism, direct delivery of drugs to particular regions of the lymphatic circulation e g in the treatment of disease states, and the possibility of regulating the rate of drug delivery into the systemic circulation The major factors affecting apparent lymphatic transport include the route of administration, the design of the drug delivery system, and the physicochemical and metabolic properties of the drug Macromolecules and colloidal particles such as chylomicrons are selectively taken up from the interstitial space via the lymphatics Nanoparticles enter into the lymphatic system through specialized processes called endocytosis Lymphotropic delivery systems, where the
drug is complexed with a high molecular weight carrier, e g dextran sulphate, have been used in the presence of absorption enhancers to potentiate the selective lymphatic delivery of anticancer drugs
The drugs belonging to class II, III and IV of Biopharmaceutical classification, having solubility and/or permeability problems are the targets for preparing prodrugs Prodrugs are known to improve the bioavailability of poorly soluble drugs by improving their solubility and/or permeability Prodrugs are believed to be absorbed from the intestinal tract after oral administration and hydrolyzed to its parent moiety by enzymatic or non-enzymatic action in the intestinal wall/plasma But in some cases preparation of prodrugs alone does not solve the problem These prodrugs in spite of having improved solubility and permeability shows poor bioavailability because of preabsorption degradation and rapid efflux back into the lumen of intestine One of the approaches to improve the bioavailability of such prodrugs is to target the lymphatic system in which the prodrug is directly entering into the lymph so that there is no preabsorption degradation and efflux mechanism The lymphatic system would also provide certain additional advantages so that the overall bioavailability is enhanced to surprising levels
Cefpodoxime proxetil is one of the prototype example of prodrugs, commercially available oral tablets of which show absolute bioavailability of only 50% compared to that of intravenous infusion Systematic studies were conducted to determine the potential reasons for the poor bioavailability of cefpodoxime proxetil
Solubility and stability studies were conducted at different pH conditions and it was found that cefpodoxime proxetil has pH dependent solubility and stability, both decreasing with increase in pH Though reduced, the solubility at intestinal pH is expected to be sufficient to achieve the desired absorption Therefore stability and not solubility, in the intestine could be one of the probable reasons for the reduced bioavailability
Further studies were conducted to determine the effective permeability coefficients of cefpodoxime and cefpodoxime proxetil using an in vitro rat intestinal "Everted Sac" method Surprisingly the permeability coefficients for both cefpodoxime and cefpodoxime proxetil were found to be similar This was against expectation as cefpodoxime proxetil is supposed to be more lipophilic than cefpodoxime and was
designed to improve permeability and absorption Hence, it appears that permeability coefficients obtained were under the influence of unknown factors like the presence of efflux mechanism, which would reduce the bioavailability further
The role of efflux mechanism in reducing the overall bioavailability was confirmed by the in vitro rat intestinal "Everted Sac" method in which stability of cefpodoxime proxetil in the presence of jejunal segment was studied It was observed that about 90% of the cefpodoxime proxetil present was converted to cefpodoxime acid within fifteen minutes, and within thirty minutes the conversion was nearly complete as represented in Figures 1 and 2 The reason for rapid conversion of cefpodoxime proxetil to cefpodoxime exposed to these epithelial cells was found to be post absorption metabolism of cefpodoxime proxetil to cefpodoxime acid within the enterocytes and its efflux, back into the intestinal lumen
Efflux mechanism was confirmed by intestinal tissue uptake studies using fresh everted jejunal rings The jejunal rings showed presence of high amounts of cefpodoxime proxetil inside the enterocyte in the initial stages as early as less than one minute But the concentrations inside the epithelial cells depleted fast and recurrence of cefpodoxime concentration was observed with time When cefpodoxime proxetil was studied for uptake, its traces were observed in tissue for five minutes only The point illuminated that cefpodoxime proxetil permeates quickly, but is rapidly converted into cefpodoxime, effluxed towards apical side rather than basal side leading to lowered absorption With time, the accumulated cefpodoxime is absorbed but slowly and to a lesser extent When similar study was performed with cefpodoxime, it exhibited a constant intake rate into the tissue giving an upward movement of concentration vs time profile The results of the study are represented in Figures 3 and 4 Such efflux mechanism further reduces the bioavailability in addition to preabsorption degradation
Conversion of prodrugs to active moieties, prior to absorption into the systemic circulation will remain a hurdle in all types of conventional oral dosage forms Hence, a formulation needs to be prepared for prodrugs such as cefpodoxime proxetil, which can bypass and provide sufficient protection to the prodrug from the intestinal contents as well as from the enterocytic enzymes Solid lipid nanoparticles, which undergo lymphatic absorption, would be a better choice in such cases Solid lipid nanoparticles instead of going into blood are taken into lymph by means of a special persorption
process called endocytosis, by which small droplets or solid particles pass through the intestinal wall and there upon are routed into the lymph flow As the prodrug is not entering into the enterocytes in which it may be degraded, secondly as direct contact with gastrointestinal environment is avoided, there are less chances of preabsorption-degradation Moreover when the prodrug is absorbed through lymphatic system, there would not be any first pass effect, if any, and hence bioavailability would additionally be enhanced
Solid lipid nanoparticles of prodrugs of the present invention may improve the bioavailability by 1 25 times or more, in particular 1 5 times or more than from conventional oral dosage forms Less amount of prodrug would be required to achieve the plasma levels of the drug compared to conventional dosage forms Hence solid lipid nanoparticles would be cost-effective and have reduced chances of dose-dumping leading to toxicity problems
Solid lipid nanoparticles consist of prodrug conjugated with a lipid carrier and formulated into spherical solid particles in nano range, particularly in the range of 100 to 500nm Solid lipid nanoparticles combine advantages of polymeric nanoparticles, fat emulsion and liposomes but simultaneously avoid some of their disadvantages Size, nature of the lipid carrier, zeta potential, method of fabrication, presence of nutrients are among the factors affecting the performance of solid lipid nanoparticles Hence, in a simplest version solid lipid nanoparticles of the present invention comprises prodrug, and a lipid carrier
Solid lipid nanoparticles may be used for prodrugs having poor bioavailability due to problems of preabsorption degradation and/or efflux mechanism Examples of the prodrugs include cefpodoxime proxetil, cefetamet pivoxil, cefditoren pivoxil, cefuroxime axetil, valacyclovir, valganciclovier, azidothymidine, capecitabine, famciclovir, nabumelone, pivampicillin, innotecan, terfenadine, enalapril, ramipril, dipivefnn, omeprazole, sulfasalazine, olsalazme, methanamie, bambuterol, allopunnol, gemcitabine, fludarabine, cladnbme, simvastatin, tegafur, fosphenytoin and viramidine The amount of prodrug may vary from about 5% w/w to about 90% w/w, in particular from about 10% w/w to about 40% w/w of the solid lipid nanoparticles
Examples of lipid carrier includes one or more of mono-, di- and tri-glycendes of saturated, straight-chain fatty acids with 12 to 30 carbon atoms such as launc acid, mynstic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignocenc acid, cerotinic acid, melissic acid, esters of polyhydnc alcohols such as ethylene glycol, propylene glycol, mannitol, sorbitol, saturated fatty acids with 12 to 22 carbon atoms such as lauryl alcohol, mynstyl alcohol, cetyl alcohol, stearyl alcohol, arachidyl alcohol, behenyl alcohol, saturated wax alcohols with 24-30 carbon atoms such as hgnoceryl alcohol, ceryl alcohol, cerotyl alcohol, myncyl alcohol, and the like Other examples include animal or plant phospholipids such as lecithin and their hydrogenated forms, polypeptides such as gelatins along with their modified forms, waxes such as carnauba wax, bees wax, and the like The lipids employed preferably exhibit a melting point in the range of from about 30° to about 100° C The ratio of prodrug and lipid carrier in solid lipid nanoparticles may vary from about 1 0 5 to about 1 10 The amount of lipid carrier may vary from about 5% w/w to about 90% w/w, in particular from about 20% w/w to about 70% w/w of the solid lipid nanoparticles
Besides this, solid lipid nanoparticles of the present invention may further comprise one or more of other pharmaceutical^ acceptable additives such as stabilizer, co-solvents, cryoprotectants, preservatives, and the like
Generally, the solid lipid nanoparticles comprise a stabilizer for minimizing electrostatic forces leading to aggregation when dispersed in the contents of the gastrointestinal tract, and thereby affect reproducibility Examples of stabilizer include one or more of any polymer and/or surfactant, which can prevent aggregation of solid lipid nanoparticles and thus stabilizes the composition Specific examples of polymer include polyvinyl alcohol, polyethylene glycol, polyvmylpyrollidone, and the like Surfactant may either be ionic or non-ionic in nature Examples include naturally occurring as well as synthetic phospholipids, their hydrogenated derivatives and mixtures thereof, sphingolipids and glycosphmgohpids, physiological bile salts such as sodium cholate, sodium dehydrocholate, sodium deoxycholate, sodium glycocholate and sodium taurocholate, saturated and unsaturated fatty acids or fatty alcohols, ethoxylated fatty acids or fatty alcohols and their esters and ethers, alkylaryl-polyether alcohols such as tyloxapol, esters and ethers of sugars or sugar alcohols with fatty acids or fatty alcohols, acetylated or ethoxylated mono- and diglycendes, synthetic biodegradable polymers like block co-polymers of polyoxyethylene and polyoxypropyleneoxide,
sulfosuccinic acid esters, polyoxyethylenesorbitane esters ethoxylated sorbitanesters or sorbitanethers, amino acids, polypeptides and proteins such as gelatin and albumin, lecithin, egg lecithin, polyoxyethylene fatty alcohol ethers, polyoxyethylene stearic acid esters and corresponding mixed condensates of polyoxyethylene ethers and polyoxypropylene ethers (e g , Pluronic®), ethoxylated saturated glycendes, (e g , Labrafil®) and partial fatty acid glycendes and polyglycides (e g , Gelucire®)
Examples of co-solvent include one or more volatile solvent such_as organic solvents including chloroform, methanol, ethanol, and the like
Cryoprotectants are used to prevent the composition from damage during freezing Examples include sugars such as sucrose, glucose, trehalose, glycols (alcohols containing at least two hydroxyl groups) such as ethylene glycol, propylene glycol and glycerol, and the like
Examples of preservatives include benzyl alcohol, pheny|ethyl alcohol, phenoxyethanol, sodium benzoate, methyl paraben, propyl paraben, butyl paraben, propyl gallate, demercaprol, butylhydroxyanisole, butylhydroxytoluene, palmityl ascorbate, sodium pyrosulfite, tocopherol and/or its esters, and the like
Solid lipid nanoparticles may be prepared using conventional techniques known in the art such as high pressure homogenization (hot homogenization or cold homogenization), microemulsification-solidification, solvent emulsification/evaporation method, and the like
In one of the embodiments, solid lipid nanoparticles of prodrug may be prepared a process of high-pressure hot homgenization comprising the steps of
(a) melting lipid and dissolving/dispersing the prodrug in the lipid,
(b) dispersing the prodrug loaded lipid in a hot aqueous surfactant mixture,
(c) pre-mixing to form a coarse pre-emulsion,
(d) homogenizing at high pressure to form hot oil in water nanoemulsion, and
(e) solidification of nanoemulsion to get solid lipid nanoparticles, by cooling down to room temperature
In another embodiment, solid lipid nanoparticles of prodrug may be prepared by a process of high-pressure cold homgenization comprising the steps of
(a) melting lipid and dissolving/dispersing the prodrug in the lipid,
(b) solidifying the drug loaded lipid in liquid nitrogen or dry ice,
(c) suitably grinding the solid,
(d) dispersing in aqueous solution/dispersion of surfactant in an organic solvent,
(e) mixing to form a coarse pre-emulsion,
(f) homogenizing at high pressure to get solid lipid nanoparticles, at room temperature or lower
In another embodiment, solid lipid nanoparticles of prodrug may be prepared by process of solvent emulsification/evaporation method comprising the steps of
(a) dissolving prodrug and lipid in organic solvent,
(b) mixing with stabilizer,
(c) homogenizing and evaporating the solvent to get solid lipid nanoparticles
Alternatively, solid lipid nanoparticles may also be prepared by microemulsification-solidification or multiple microemulsification-sohdification techniques
Solid lipid nanoparticles prepared above may be combined with one or more pharmaceutical^ inert excipient, if desired, and processed into conventional oral dosage forms such as suspension, tablets, capsules, pellets, powder in sachets, and the like
The performance of solid lipid nanoparticles may be analyzed by evaluating certain parameters listed below
(Table Removed)
The invention is further illustrated by the following examples but they should not be construed as limiting the scope of the invention any way
Examples

(Table Removed)
Procedure
1 Cefpodoxime proxetil and tnbehenin were dissolved in chloroform to form a solution
2 Solution of step 1 was added dropwise to aqueous solution containing polyvinyl alcohol maintained at 40-50° C, with continuous stirring
3 The homogenized mix of step 2 was purified by ultracentnfugation, and the filtrate collected
4 The filtrate of step 3 was freeze-dned to get the solid lipid nanoparticles
Solid lipid nanoparticles prepared above had more or a less spherical and had a mean particle size in the range of 100-500 nm The loading of cefpodoxime proxetil on lipid was about 5-20% Further, the solid lipid nanoparticles as per the compositions of example 1-3 were stored at 40° C and cefpodoxime proxetil was analyzed at regular intervals over a period of 2 weeks using a validated HPLC method the cefpodoxime proxetil content was found to be at least 95%
In vivo performance of solid lipid nanoparticles (T) was evaluated with respect to the Cepodem™ 100 (Marketed By Ranbaxy Lab Ltd ) tablets (R) in male Sprague-Dawely rats (weight 250-275 gms) at a dose of 10 mg per kg (equivalent to cefpodoxime acid) body weight, under fasted conditions (with free access to water) All experiments and procedures on animals were conducted in accordance with protocols approved by the Animal Institutional Ethical Committee Each formulation at the specified dose was administered orally, with the help of oral gauge needle along with saline Blood samples (0 4-0 5 ml) were collected at 0 5, 1, 1 5, 2, 3, 4, 6, 8, 12, and 24 hours Samples were centnfuged to separate plasma and drug content analyzed using a validated HPLC method Pharmacokinetic parameters Cmax (Maximum plasma concentration), Tmax
(Time to attain maximum plasma concentration), AUCot (Area under the plasma concentration vs time curve from 0 hours to the time of last sample collected) and AUCo α (Area under the plasma concentration vs time curve from 0 hours to infinity) were calculated from the data obtained The data was analyzed using both MS-Excel™ and using non-linear regression software- PC-NONLIN™ The results of the study are represented in the table below

(Table Removed)
The above results clearly indicate the importance of solid lipid nanoparticles in improving the oral bioavailability of prodrugs

WE CLAIM:
1. A method of improving the bioavailability of prodrug by 1.25 times or more than from conventional oral dosage forms, using solid lipid nanoparticles.
2. The method of improving the bioavailability of prodrug according to claim 1 wherein poor bioavailability is due to preabsorption degradation and/or efflux mechanism.
3. Solid lipid nanoparticles of prodrug comprising prodrug and lipid carrier; and providing bioavailability of 1.25 times or more than from conventional oral dosage forms.
4. The solid lipid nanoparticles according to claim 3 wherein prodrug is selected from the group consisting of cefpodoxime proxetil, cefetamet pivoxil, cefditoren pivoxil, cefuroxime axetil, valacyclovir, valganciclovier, azidothymidine, capecitabine, famciclovir, nabumelone, pivampicillin, irinotecan, terfenadine, enalapril, ramipril, dipivefrin, omeprazole, sulfasalazine, olsalazine, methanamie, bambuterol, allopurinol, gemcitabine, fludarabine, cladribine, simvastatin, tegafur, fosphenytoin and viramidine.
5. The solid lipid nanoparticles according to claim 4 wherein prodrug comprises from about 5% w/w to about 90% w/w of the solid lipid nanoparticles.
6. The solid lipid nanoparticles according to claim 3 wherein lipid carrier is selected from the group consisting of more of mono-, di- and tri-glycerides of saturated, straight-chain fatty acids with 12 to 30 carbon atoms, esters with polyhydric alcohols, saturated wax alcohols with 24-30 carbon atoms, animal or plant phospholipids such as lecithin and their hydrogenated forms, and polypeptides.
7. The solid lipid nanoparticles according to claim 6 wherein lipid carrier comprises from about 5% w/w to about 90% w/w of the solid lipid nanoparticles.
8. The solid lipid nanoparticles according to any of the preceding claims wherein solid lipid nanoparticles may further comprise one or more of pharmaceutically acceptable excipients selected from the group consisting of stabilizer, co-solvents, cryoprotectants, and preservatives.
9. The solid lipid nanoparticles according to any of the preceding claims prepared by a process comprising the steps of dissolving/dispersing the prodrug in mixture of lipid carrier and stabilizer; and processing into a suitable dosage form.
10. The solid lipid nanoparticles according to claim 9 wherein dissolving/dispersing of prodrug is carried out by high-pressure homogenization, microemulsification-solidification, multiple microemulsification-solidification, or solvent emulsification/ evaporation techniques.
11. The solid lipid nanoparticles according to claim 9 wherein the dosage form is selected from the group consisting of suspension, tablets, capsules, pellets, powder in sachets.
12. A method of improving the bioavailability of prodrug as described and illustrated in the examples herein.