DESC:ANTI-PSORIASIS TOPICAL FORMULATION CONTAINING COMBINATION DRUGS AND METHOD OF PREPARATION THEREOF
TECHNICAL FIELD
The present invention relates to microsponge (MS) based gel formulation for multi-drug delivery with dendrimeric peptide(s) as the combination therapy of Psoriasis. The present invention also relates to compositions for enhancing the permeation and deposition of the drug(s) on the skin of mammalian. Further, the present invention specifically relates to microsponge (MS) based anti-psoriasis topical formulation (gel) containing combination drugs (Apremilast (APR) and betamethasone dipropionate (BD)) and dendrimeric peptide for the treatment of skin disease(s) specifically psoriasis and its different types.
REFERENCES
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WO2017216738A1 - Topical compositions of apremilast - Google Patents. (n.d.). Retrieved January 31, 2023, from https://patents.google.com/patent/WO2017216738A1/en
BACKGROUND OF THE INVENTION
Topical administration is the most efficient route for local delivery of therapeutic agents due to its convenience and affordability. The specific challenge of designing a therapeutic system is to achieve an optimal concentration of the drug at the site of action (Roberts et al., 2021; Singh Malik et al., 2016).
Topical drug delivery provides several benefits over other conventional routes by providing localized therapeutic effects, circumventing the first-pass metabolism and enzymatic drug degradation (Navti et al., 2022), and non-invasiveness; (Prausnitz & Langer, 2008). Despite these benefits, skin as a route of drug delivery has some limitations on the physicochemical characteristics of the drug or active agent, such as molecular mass 500–600 Da, solubility in both water and oil, low melting point, octanol–water partition coefficient favoring lipids, low dose, and lack of skin sensitization potential of the drug (Arriagada & Morales, 2019).
Extending topical administration to a wider variety of therapeutic compounds is particularly advantageous in the current context of drug delivery. Numerous methods, including chemical and physical permeation enhancers, have been investigated for this purpose. The chemical permeation enhancers include fatty acids, organic solvents, alcohols, esters, and surfactants. The delivery of chemicals into and across the skin has also been studied by numerous researchers using nanocarrier techniques such as liposomes, polymeric nanoparticles, micelles, dendrimers, and others (Manikkath et al., 2020).
Due to their nanometric size, adaptable surface activity, and ability to conjugate or complex with various agents, dendrimers are a class of hyperbranched synthetic molecules being researched for the delivery of active drugs (Aurelia Chis et al., 2020; Bober et al., 2022; Mendes et al., 2017). Apremilast (APR) a BCS class IV drug has been approved by USFDA in 2014, for the treatment of psoriasis topically in the concentration range of 0.1 – 5%, with different excipients (WO2017216738A1 - Topical Compositions of Apremilast - Google Patents, n.d).
In the treatment of psoriasis, combination therapy is more beneficial rather than a single drug treatment, therefore along with APR, a highly potent topical corticosteroids, betamethasone dipropionate (BD) was selected (M. Lebwohl et al., 2004; M. G. Lebwohl et al., 2021). In psoriasis, it was also observed that the skin begins to shed and there is a lack of skin amino acids (Chen et al., 2021; Hussain et al., 2019); therefore to overcome that we have added a dendrimeric peptide, that contains different amino acids (Hegde et al., 2017; Manikkath et al., 2017; Mutalik et al., 2012, 2014). This not only acts as a permeation enhancer but also acts as a supplement for the amino acids (Avadhani et al., 2017; Shetty et al., 2017). For the effective treatment of psoriasis, sustained release of drugs is beneficial. Therefore to attain this, use of carrier is necessary, and hence, microsponge was used for the same in the present invention.
Microsponges are polymeric delivery systems based on porous microspheres. They are tiny, spherical particles that have a porous surface (Kaity et al., 2010). It is a versatile drug delivery system because of its properties, which include the capacity to suspend or to entrap a variety of compounds and then contained within products like gels (Tripathi et al., 2019), creams, liquids, or powders (Aloorkar et al., 2012). Microsponges are made to improve drug characteristics like enhancing the stability, reduction in side effects, and sustained release behavior (Mahant et al., 2020).
The combination of apremilast and corticosteroid in a topical formulation for the treatment of psoriasis is not in use at present although it appears to be logical combination. In addition, no prior art teaches use of dendrimeric peptide in conjunction with corticosteroids and apremilast. No prior art further teaches use of Microsponges for these drugs. Additionally, patient compliance with the state-of-the-art treatments remains low despite significant economic investment in its treatment.
Based on the foregoing, it is believed that a need exists for an improved topical semisolid/ spray/ liquid formulation containing apremilast and a corticosteroid for improved psoriasis treatment efficacy. Also, a need exists for microsponge (MS) based anti-psoriasis topical formulation containing combination drugs (Apremilast (APR) and betamethasone dipropionate (BD)) and dendrimeric peptide for enhancing the skin permeation and deposition of active agent(s) on skin, as described in greater detail herein.
SUMMARY OF THE INVENTION
The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description.
The foremost objective of the invention is to produce MS based gel formulation using a defined ratio of active ingredient(s), surfactants, polymers, antioxidants, preservatives, emulsifying agents, permeation enhancers etc.
It is yet another object of the present invention to improve the bioavailability of susceptible active agent(s), which undergo first-pass metabolism on oral administration, by delivering the active agent(s) through topical route.
It is an object of the present invention to enhance the topical permeation and/ or deposition of active agent by non-invasive means.
It is an object of the present invention to topical deliver active agent(s) by the use of MS or its formulation which have nil to low irritancy potential on skin.
It is an object of the present invention to enhance the skin availability of poorly soluble and/ or poor permeable active agent(s) when administered topically.
It is an object of the present invention to enhance the skin deposition of active agent(s) by the use of dendrimeric peptide along with other above-mentioned ingredients.
It is an object of the present invention to sustain the release of active agent(s) by the use of microsponge along with other above-mentioned ingredients.
It is further an object of the present invention to deliver the pharmaceutical formulation in the form of carriers, which could augment the efficacy and safety of the encapsulated active agent(s).
These and other objects and features of the present invention will become readily apparent to one skilled in the art from the detailed description given hereafter.
Anti-psoriasis topical formulation containing combination drugs and method of preparation thereof. The present invention relates to microsponge (MS) based anti-psoriasis topical formulation (gel) containing combination drugs (Apremilast (APR) and betamethasone dipropionate (BD)) and dendrimeric peptide for enhancing the skin permeation and deposition of active agent(s) on skin. The microsponges may be prepared suing different polymers or pre-formed Microsponges can be procured from different companies (example: Amcol Household and Personal care, MTI Health & Beauty Solutions, 2870 Forbs Avenue Hoffman Estates, IL, USA).

The microsponge based tropical gel formulation with dual drugs and dendrimeric peptide for the combination therapy of psoriasis comprises an active agent (APR and BD) in the ratio of 1:1, 2:1, 3:1, 10:1, 20:1, a polymer ranging from 0.5-2%, surfactant and emulsifying agent each ranging from 0.05-2% by weight of the composition. The composition may also include other ingredients such as fatty acids, peptides/ proteins, preservatives, antioxidants, permeation enhancers and other excipients. A dendrimeric peptide is used to enhance the permeation and/ or deposition of the active agents into the skin..
The microsponge might either be combined with the active agent(s) in a carrier or incorporated with the active agent(s) in a base that meets pharmaceutical standards. The carrier could also be included in the base that is acceptable to pharmaceutical manufacturers. Before administering the active ingredient, the microsponge could alternatively be applied topically in a base that is acceptable to pharmaceutical manufacturers(s).
The term "active agent" as used in this document refers to any substance, including but not limited to small molecules, drugs, proteins, peptides, nucleic acids (nucleotides, nucleosides, and analogues thereof), prodrugs, and nucleic acids, plant or animal based ingredients/ extracts/ products, which, when administered to a subject (human or non-human animal), alone or in combination with other active or inactive components, may have pharmacological, therapeutic, or disease-treating effects.
The carrier may be chosen from the group of phospholipids, polymers, lipids, carbohydrates, polysaccharides, ionic or non-ionic surfactants, peptides, and/or their derivatives that are natural, semi-synthetic, or synthetic.
It is possible to choose a pharmaceutically acceptable base that is physically and chemically compatible with the aforementioned substances from a range of options, including but not limited to buffer, solution, ointment, cream, paste, gel, jelly, foam, suspension, and lotion. For preparation purposes, the preparation may additionally include other formulation components. When applied to the skin, these preparations will improve the permeation of the active agent(s) through the skin and into the epidermal layer of the skin.
In one embodiment, the dendrimeric peptide (PD) has the amino acid sequence His-Lys-Gly-(Arg)2 (C-N terminus), and the active ingredient is betamethasone dipropionate (BD) and apremilast (APR), an anti-inflammatory and psoriatic drug. The PD along with the active agents is incorporated in the microsponge in the preferred compositions. The carriers are microparticulate in nature (with size ranging from 1 – 20 µm, precisely 5 – 7 µm), which is composed of microscopic sponge-like structure, in particularly desired compositions. A possible weight ratio for PD, APR, and BD is 0.05:0.15:0.05. The formulation may be made in such a way that the preparations can be applied to the skin.
F-moc Solid phase peptide synthesis (SPPS) was used to synthesize the PD. The PD was identified using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy, while the purity was determined using reverse phase-high performance liquid chromatography (RP-HPLC) Using the quasi-emulsion solvent diffusion method (QESD), active compounds (APR and BD) were loaded in a microsponge that contained 0.15:0.05% of each medication. PD and both drugs were introduced into the microsponge through layering technique. Fourier Transform Infrared (FT-IR) Spectroscopy and Differential Scanning Calorimetry (DSC) were used for solid-state characterization, including physical and chemical compatibility investigations between APR and BD and formulation ingredients. No incompatibility between APR and BD and the formulation's excipients was discovered in the FT-IR and DSC investigations.
In order to characterize the PD + APR + BD microsponge (MS-APR+BD+PD) and the APR and BD microsponge without PD, measurements of particle size, SEM, IVRT, and loading efficiency (LE%) were determined. Ex vivo permeation studies and primary skin irritation studies on male Swiss Albino mice skin and in vitro cell viability on HaCaT cells (immortalized human keratinocytes) were performed with APR+BD, MS-APR+BD and MS-APR+BD+PD. Pharmacokinetic study, and pre-clinical efficacy study was performed in comparison with plain APR+BD, MS-APR+BD and MS-APR+BD+PD. Stability studies of MS-APR+BD+PD was also performed. The PD was found to exhibit molecular weight of 515.4 g/mol evident from the MS investigation, single peak purity from the RP-HPLC chromatogram and designated structure from the NMR spectrum. The particle size of the microsponge with the PD and active agent, APR and BD, was found to be in the range of 5 – 7 µm. The entrapment efficiency in the microsponge was found in the range of 70 % to 95 %.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates the chemical structure of the peptide dendrimer (PD) depicting the sequence His-Lys-Gly-(Arg)2 (C-N terminus).
FIG. 1B illustrates a graph showing the mass spectrum of peptide dendrimer (PD) depicting the molecular weight of the peptide dendrimer (PD).
FIG. 2 illustrates the RP-HPLC chromatogram of peptide dendrimer (PD) with a retention time of 4.43 min depicting the purity of the peptide dendrimer (PD).
FIG. 3 illustrates the Differential Scanning Calorimetric thermograms of (a) Microsponge (MS) (b) plain APR (c) Plain BD (d) MS loaded APR+BD (e) PD and (f) MS-APR+BD+PD.
FIG. 4 illustrates the Fourier Transform Infrared spectra of (a) Plain MS, (b) Plain APR, (c) Plain BD, (d) Plain APR+BD and (e) MS loaded APR+BD.
FIG. 5 illustrates SEM images of (a) Plain MS; (b) MS loaded with APR+BD; (c) MS loaded with APR+BD+PD.
FIG. 6 illustrates In Vitro release profile of (a) Plain 0.05% of BD + 0.15 % of APR; (b) MS loaded 0.05% of BD + 0.15 % of APR; (c) MS loaded 0.05% of BD + 0.15 % of APR + 0.05% of PD; (d) 0.05 % Betagel (0.05% of Betamethasone Dipropionate).
FIG. 7 illustrates Ex vivo skin permeation profiles of APR and BD from (a) Gel formulation without microsponge; (b) Gel formulation with microsponge; (c) Gel formulation with PD in microsponge; (d)0.05% BD Betagel.
FIG. 8 illustrates comparative bar graph depicting the IC50 values of Apremilast, Betamethasone, APR+BD and APR+BD+PD against HaCaT cell lines by MTT study.
FIG. 9 illustrates overlaid bar graph depicting the IC50 values of APR+BD in 1:1 ratio, MS APR+BD, MS APR+BD+PD against Psoriasis induced HaCaT cell lines by SRB study after the treatment period of 48hrs.
FIG. 10 illustrates overall the given test compounds decreased the % of cells expressing Pro-inflammatory cytokine, TNF-alpha induced by the effect of Psoriasis respectively.
FIG. 11 illustrates Histopathological Images of G-I to G-IX in pre-clinical efficacy studies.
FIG. 12 illustrates Histopathological images of Psoriasis Treatment.
FIG. 13 illustrates the schematic representation of dermatopharmacokinetic (DPK) study to assess topical bioequivalence of apremilast and betamethasone in plain gel formulation. a) absorption phase of both the drugs; b) clearance phase of both the drugs; c) DPK profile of betamethasone dipropionate in plain gel formulation; d) DPK profiles of Apremilast in the plain drug formulation.
FIG. 14 illustrates the schematic representation of dermatopharmacokinetic (DPK) study to assess topical bioequivalence of apremilast and betamethasone in MS based gel formulation a) absorption phase of both the drugs; b) Retention phase of both the drugs; c) DPK profile of Betamethasone dipropionate in MS based gel formulation; d) DPK profile of Apremilast in MS based gel formulation.
FIG. 15 illustrates comparison between Normal, Positive control and Optimized gel treatment.
DETAILED DESCRIPTION
The values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.
The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes all combinations of one or more of the associated listed items.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Invention: Anti-psoriasis topical formulation containing combination drugs along with various excipients and method of preparation thereof. The present invention relates to microsponge (MS) based anti-psoriasis topical formulation (gel) containing combination drugs (Apremilast (APR) and betamethasone dipropionate (BD)) and peptide dendrimer for enhancing the skin permeation and deposition of active agent(s) on skin.
The microsponge based tropical gel formulation with dual drugs and peptide dendrimer for the combination therapy of psoriasis comprises an active agent (APR and BD) in a ratio of 1:1, 2:1, 3:1, 10:1, 20:1, a polymer ranging from 0.5-2%, surfactant and emulsifying agent each ranging from 0.05-2% by weight of the composition. The composition may also include other ingredients such as fatty acids, peptides/ proteins, preservatives, antioxidants, permeation enhancers, etc. A peptide dendrimer is used to enhance the permeation of the active agents through the skin.
The microsponge might either be combined with the active agent(s) in a carrier or incorporated with the active agent(s) in a base that meets pharmaceutical standards. The carrier could also be included in the base that is acceptable to pharmaceutical manufacturers. Before administering the active ingredient, the microsponge could alternatively be applied topically in a base that is acceptable to pharmaceutical manufacturers(s).
The term "active agent" as used in this document refers to any substance, including but not limited to small molecule drugs, proteins, peptides, nucleic acids (nucleotides, nucleosides, and analogues thereof), prodrugs, and nucleic acids, which, when administered to a subject (human or non-human animal), alone or in combination with other active or inactive components, may have pharmacological, therapeutic, or disease-treating effects.
The carrier may be chosen from the group of phospholipids, polymers, lipids, carbohydrates, polysaccharides, ionic or non-ionic surfactants, peptides, and/or their derivatives that are natural, semi-synthetic, or synthetic.
It is possible to choose a pharmaceutically acceptable base that is physically and chemically compatible with the aforementioned substances from a range of options, including but not limited to buffer, solution, ointment, cream, paste, gel, jelly, foam, suspension, spray, emulsion and lotion. For preparation purposes, the preparation may additionally include other formulation components. When applied to the skin, these preparations will improve the permeation of the active agent(s) through the skin and into the epidermal layer of the skin.
In one embodiment, the PD has the amino acid sequence His-Lys-Gly-(Arg)2 (C-N terminus), and the active ingredient is betamethasone dipropionate (BD) and apremilast (APR), an anti-inflammatory and psoriatic drug. The PD along with the active agents is incorporated in the microsponge in the preferred compositions. The carriers are microparticulate in nature (with size ranging from 5 – 7µm), which is composed of microscopic sponge-like structure, in particularly desired compositions. A possible weight ratio for PD, APR, and BD is 0.05:0.15:0.05. The formulation may be made in such a way that the preparations can be applied to the skin.
F-moc Solid phase peptide synthesis (SPPS) was used to synthesize the PD. The PD was identified using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy, while the purity was determined using reverse phase-high performance liquid chromatography (RP-HPLC) Using the quasi-emulsion solvent diffusion method (QESD), active compounds (APR and BD) were loaded in a microsponge that contained 0.15:0.05% of each medication. PD and both drugs were introduced into the microsponge through layering technique. Fourier Transform Infrared (FT-IR) Spectroscopy and Differential Scanning Calorimetry (DSC) were used for solid-state characterization, including physical and chemical compatibility investigations between APR and BD and formulation ingredients. No incompatibility between APR and BD and the formulation's excipients was discovered in the FT-IR and DSC investigations.
Working of the Invention: In order to characterize the PD + APR + BD microsponge (MS-APR+BD+PD) and the APR and BD microsponge without PD, measurements of particle size, SEM, IVRT, and loading efficiency (LE%) were determined. Ex vivo permeation studies and primary skin irritation studies on male Swiss Albino mice skin and in vitro cell viability on HaCaT cells (immortalized human keratinocytes) were performed with APR+BD, MS-APR+BD and MS-APR+BD+PD. Pharmacokinetic study, and pre-clinical efficacy study was performed in comparison with plain APR+BD, MS-APR+BD and MS-APR+BD+PD. Stability studies of MS-APR+BD+PD was also performed. The PD was found to exhibit molecular weight of 515.4 g/mol evident from the MS investigation, single peak purity from the RP-HPLC chromatogram and designated structure from the NMR spectrum. The particle size of the microsponge with the PD and active agent, APR and BD, was found to be in the range of 5 – 7 µm. The entrapment efficiency in the microsponge was found in the range of 70 % to 95 %.
In the ex vivo skin permeation study, MS-APR+BD+PD was found to achieve higher APR and BD permeation than that from APR and BD microsponge without PD at all time-points up to 24 h. The permeation parameters produced with MS-APR+BD+PD were Q24: 149.89 µg/cm2 of APR and 102.45 µg/cm2 of BD, Jss: 6.54 µg/cm2/h of APR and 4.16 µg/cm2/h of BD and ER: 2.011 of APR and 1.86 of BD compared to passive diffusion of APR and BD.
In vitro cytotoxicity or cell viability assay on HaCaT cells indicated non-toxic potential of MS-APR+BD+PD. Primary skin irritation studies and histopathological evaluation indicated nil to low irritancy potential of MS-APR+BD+PD.
Pharmacokinetic studies revealed that the drug is not going into systemic circulation even after 24 h of application. This showed that the drug is retaining into the epidermal layer, considerably higher skin retention values from MS-APR+BD+PD, compared to plain drug administration.
Stability studies of the microsponge based gel formulation conducted up to 3 months demonstrated that the formulations were stable with respect to viscosity, drug content, pH, consistency, spreadability and phase separation for the tested time period. These findings form the first report on the applicability of MS-APR+BD+PD to bring about enhancement in epidermal and/ or topical delivery of active agent(s).
Test Results: The DSC thermogram of plain MS (Fig. 3a) shows no peak portraying that MS is stable beyond 300 °C, the DSC thermogram of plain APR (Fig. 3 b) depicted a sharp endothermic peak at 157.87 °C (onset: 154.94 °C, endset: 161.55 °C). The DSC thermogram of plain BD (Fig. 3 c) depicted a sharp endothermic peak at 244.70 °C (onset: 241.30 °C; endset: 248.80 °C). The DSC thermograms of MS loaded APR and BD (Fig. 3 d) showed a broad endothermic peaks and reduction in intensity of peak. PD (Fig. 3 e) showed no distinctive peak. MS-APR+BD+PD demonstrated slight shift in the melting transition compared to plain APR and BD which could be due to incorporation of APR and BD into the pores of the microsponge and amorphization of APR and BD at higher temperature. The DSC thermogram of APR and BD suggests crystalline nature of pure APR and BD, while it is available in molecular dispersed state in the formulations. The thermotropic changes in the peak temperature could be due to structural conformation of the additives which could interact with the pores of the microsponge.
The Fourier-transform infrared spectra (FTIR) of APR, BD and APR+BD was recorded over a range of 4000–400 cm-1 to study the principal peaks with FTIR spectrophotometer (Shimadzu Corp.) using the potassium bromide (KBr) disc method The FTIR spectrum of APR (Fig. 4 b) shows the characteristic strong N–H stretching peak at 3360 cm-1. The FTIR spectrum of APR also shows the characteristic peak at 1761 cm-1 due to amide carbonyl (C=O), along with the peaks between 2835 cm-1 and 3084 cm-1 and between 1330 cm-1 and 1392 cm-1 for aliphatic and aromatic benzene ring C–H stretching and S-O stretching respectively. The peak for amide N–H bending was observed at 1523 cm-1 and the peak for C–O stretching was observed at 1139 cm-1. This confirms that the obtained drug is APR. The FTIR spectrum of BD (Fig. 4 c) shows the characteristic strong O–H stretching peak at 3446.79 cm-1. The FTIR spectrum of BD also shows the characteristic peak at 1708 cm-1 due to amide carbonyl (C=O), along with the peak at 1446.61 cm-1 for C–F stretching. The peak for C–O stretching was observed at 1053 cm-1. This confirms that the obtained drug is BD only. The FTIR spectra of A+B (Fig. 4 d and e) shows the characteristic strong N-H stretching peak and O-H stretching peak at 3361.93 cm-1 and 3446.79 cm-1 respectively, which confirms that there is no interaction between both the drugs.
From FIG. 5, SEM images it was confirmed that the prepared microsponge are spherical in shape with numerous of tiny pores.
Different plain and MS based formulations were prepared and all these formulations were compared with marketed formulation i.e., Betagel containing 0.05% of BD. Comparison graph of the formulations with single drug APR, single drug BD and Marketed (Betagel; 0.05% of BD alone) showed that almost 100% drug release was observed from marketed product (OF-10); where are OF-6 (only APR) and OF-7 (only BD) released 33.79% of APR and 67.46% of BD at the end of 12 h (Figure 6 a). Figure 6 b, depicts the drug release of APR and BD from MS gel containing APR + BD without PD. The drug release profiles indicate that the % of APR released at the end of 12 h was increased when combined with BD, in comparison with the MS based gel containing APR alone (APR: 46.25% and BD: 70.76%). MS based gel formulation in combination with APR+BD+PD (OF-9), Figure 6 c, showed 63.46% release of APR and 78.23% of BD at 12th h, which indicates that the drug release is better in OF-9 (containing PD) in comparison with OF-8 (without PD). Figure 6 d shows the release profile of BD from marketed product (Betagel 0.05%). which is almost 100% in 12 h, confirming the results observed in Figure 6a.
The drug permeation profiles observed with OF-4 (plain gel containing 0.15%:0.05% of APR & BD) over a period of 24 h are shown in Figure 7a. Amount of APR and BD permeated at the end of 24 h are 126.20±0.90 µg cm-2 (Flux: 1.95±0.15 µg cm-2 h-1) and 49.88±0.94 µg cm-2 (Flux: 4.99±0.59 µg cm-2 h-1) respectively. The amount of drug deposited in the skin at the end of 24 h is 7.71±0.14 µg/cm2 for APR and 1.787±0.56 µg/cm2 for BD. The drug permeation profiles observed with OF-8 (MS based gel containing 0.15%:0.05% of APR & BD) over a period of 24 h are shown in Figure 7b. Amount of APR and BD permeated at the end of 24 h are 13.19±0.09 µg cm-2 (Flux: 0.24±0.35 µg cm-2 h-1) and 6.94±0.20 µg cm-2 (Flux: 0.60±0.41 µg cm-2 h-1) respectively. The amount of drug deposited in the skin at the end of 24 h is 134.449±0.455 µg/cm2 for APR and 43.288±0.208 µg/cm2 for BD. The amount of APR and BD deposited in skin from the MS based gel was higher as compared to the plain gel formulation at the end of 24 h. This indicates microsponges improved the drug residence in skin. The high concentration of drug in the skin after application of MS based gel formulation could be explained by the occlusive effect, since MS produced a film on the skin surface, which might have reduced the trans-epidermal water loss and favoured the drug deposition in the skin (de Jalon et al., 2001). Figure 7c, depicts the drug permeation profiles of APR and BD observed with OF-9 (MS based gel containing 0.15%:0.05%:0.05% of APR, BD and PD) for 24 h. Amount of APR and BD permeated at the end of 24 h are 10.44±0.06 cm-2 (Flux: 0.21±0.25 µg cm-2 h-1) and 5.68±0.21µg cm-2 (Flux: 0.45±0.19 µg cm-2 h-1) respectively. The amount of drug deposited in the skin at the end of 24 h is 140.120±0.425 µg/cm2 for APR and 44.388±0.225 µg/cm2 for BD. With the aid of dendrimeric peptide, APR and BD could be deposited within the skin to greater extent compared to plain gel (OF-4) and MS based gel without PD (OF-8). At the same time the amount of drugs permeated across the skin at the end of 24 h was reduced in presence of PD (OF-9) in comparison with OF-4 and OF-8. This indicates the beneficial effect of PD in the optimized formulation. Figure 7d, shows the skin permeation profile of BD from marketed product (Betagel, 0.05%; OF-10). Marketed product showed almost same values for the amount of BD permeated at 6th and 12th h as those observed with OF-4. However, at the end of 24 h, marketed product showed little higher skin permeation than OF-4. The flux value as well as amount of BD deposited at the end of 24 h observed with OF-10 (marketed product) were also comparatively higher than OF-4. These results are in accordance with in vitro drug release studies where the release of BD from Marketed product was higher than that observed with plain gel of APR and BD (Figure 6a).
The observations in Statistical data of MTT cytotoxicity study suggesting us that against HaCaT cell lines, Test Compounds namely Apremilast, Betamethasone, APR+BD and APR+BD+PD showing significant cytotoxic potential properties on dose dependent manner by showing cytotoxic potency with IC50 values of 0.083 µM, 9.35 µM, 1.19 µM and 0.54 µM respectively. The observed results clearly confirmed the significant cytotoxic potency of Apremilast, APR+BD and APR+BD+PD on Human keratinocyte cells.
MS alone did not cause any toxic effect on Psoriasis induced HaCaT cells. The observations in Statistical data of SRB cytotoxicity study suggesting us that against Psoriatic HaCaT cell lines, Test Compound namely APR+BD, MS APR+BD and MS APR+BD+PD showing significant Anti-psoriatic potency with IC50 values of 1.19 µM, 0.85 µM and 0.43 µM respectively. Other compound MS did not show any considerable Anti-psoriatic effect in HaCaT cells. The observed results clearly confirmed the significant anti-psoriatic potency of APR+BD, MS APR+BD and MS APR+BD+PD on Psoriasis induced Human keratinocyte cells.
Histopathological Images of G-I to G-IX in pre-clinical efficacy studies. Nine groups were made, (n=6), namely G-I – positive control (5%IMQ treated); G-II – placebo treated; G-III – negative control (untreated); G-IV – Plain formulation containing 0.05% of BD and 0.1% of APR; G-V – Plain formulation containing 0.05% of BD and 0.15% of APR; G-VI – Plain formulation containing 0.05% of BD and 0.5% of APR; G-VII – Microsponge based formulation containing 0.05% of BD and 0.1% of APR; G-VIII – Microsponge based formulation containing 0.05% of BD and 0.15% of APR; G-IX – Microsponge based formulation containing 0.05% of BD and 0.5% of APR. In Group-I, the epithelium is stratified squamous keratinized consisting of 3 to 8 cell layers. Compared to Normal (G-III), epithelium shows increased thickness of epithelium, increase in keratinization (Hyperkeratinisation). Mild lymphocytic infiltration seen in dermis; congestion of blood vessels was observed. In Group-II, the epithelium is stratified squamous keratinized consisting of 3 to 8 cell layers. Compared to Normal (G-III), epithelium shows increased thickness of epithelium and increased keratinization. Acute and chronic inflammatory infiltration seen in dermis. Compared with G-I, no reduction in epithelial thickness and congestion of blood vessels was observed. In Group-III, the epithelium is thin stratified squamous keratinized consisting of 1 to 2 cell layers. Some amount of keratin is present in stratum corneum. Histological changes were not observed. In Group-IV, the epithelium is stratified squamous keratinized consisting of 3 to 8 cell layers. Compared to G-I, epithelium showed no reduction in thickness of epithelium, lymphocytic infiltration was also observed in dermis, congestion of blood vessels was also observed. As a result, there is no reduction was observed in histological changes. In Group-V, the epithelium is stratified squamous keratinized consisting of 3 to 8 cell layers. Compared to G-I, epithelium showed no reduction in thickness, no reduction in keratinization (Hyperkeratinisation). Mild lymphocytic infiltration and congestion of blood vessels were also observed. In Group -VI, 3 to 7 layers of epidermis was observed. Compared with G-I, there is slight reduction epithelial thickness, congestion of blood vessels and inflammatory infiltration. In Group-VII, 3 to 8 cell layers of epidermal layers. Compared with G-I, no reduction in histological changes seen. In Group-VIII, the epithelium is thin stratified squamous keratinized consisting of 2 to 4 cell layers. Compared with G-I, there is high reduction in epithelial thickness, congestion of blood vessels and inflammatory infiltration were observed. In Group-IX, the epithelium is thin stratified squamous keratinized consisting of 1 to 3 cell layers. Compared with G-I, there is very high reduction in epithelial thickness, congestion of blood vessels and inflammatory infiltration were observed. In comparison to all the groups, G-VIII (microsponge based formulation containing 0.05% of BD and 0.15% of APR) was considered as the best formulation as it showed high reduction in epithelial thickness, congestion of blood vessels and inflammatory infiltration were observed. Although G-IX (microsponge based formulation containing 0.05% of BD and 0.5% of APR) also showed the highest reduction in epithelial thickness, congestion of blood vessels and inflammatory infiltration, but almost 50% animal died due to toxic nature of APR. Therefore, this formulation was not considered for further studies, and G-VIII was considered as an optimized microsponge based gel formulation and separate studies were carried out in comparison with marketed product.
Histopathological images of Psoriasis Treatment: Seven groups were made, (n=6), namely G-A – positive control (5%IMQ treated); G-B – placebo; G-C – Standard; G-D – Optimized formulation OF-3 (gel containing only BD); G-E – Optimized formulation OF-4 (containing APR+BD without MS); G-F – Optimized formulation OF-8 (MS based gel formulation containing APR+BD); G-G – Optimized formulation OF-9 (MS based gel formulation containing APR+BD+PD). In Group-A, the tissue section showed thick epidermis. The epithelium consisting of 3 to 6 cell layers with the thickness of 270.10±4.55 µm. Increase in keratin of stratum corneum was observed. Infiltration of acute and chronic inflammatory cells were seen in all areas of lower dermis with abundance of neutrophils. Group-B also showed epidermis similar to G-A. The epithelium consists of 3 to 6 cell layers with the epidermal thickness of 263.80 ± 3.011 µm. Very high amount of keratin was present in stratum corneum. Infiltration of acute and chronic inflammatory cells were seen in all layers. Lower dermis shows abundance of neutrophils. In Group-C, the tissue section showed slightly thinner epidermis as compared to the group G-A and G-B. The epithelium is slightly thin consisting of 3 to 4 cell layers with the thickness of 123.60 ± 5.113 µm. Slight decrease in keratin of stratum corneum was observed. Compared with G-A, there is reduction in inflammatory infiltration. Lesser amount of neutrophilic infiltration in lower dermis was observed. In Group-D almost same results were observed as that of G-C. In Group-E, the tissue section showed thinner epidermis as compared to G-A, G-B, G-C and G-D epidermis. The epithelium consists of 2 to 3 cell layers with the thickness of 106.40 ± 2.324 µm. No increase in keratin of stratum corneum. Few lymphocytes were seen in all areas Compared with G-A. There was reduction in acute inflammatory infiltration. Very few neutrophilic infiltrations in lower dermis were observed. Similarly, G-F also showed same results as that of G-E with slight decrease in epidermal thickness 102.50 ± 1.661 µm, while Group-G, showed the epithelium consists of 1 to 2 cell layers with the thickness of 73.02 ± 6.061 µm. No acute and chronic inflammatory cells were seen in all areas. No increase in keratin of stratum corneum. No neutrophilic infiltration was observed in lower dermal areas.
Visual Observation values are expressed as mean ±SE; the optimized formulation is significant as compared to formalin (p<0.05); Inflammation (inf) Erythema scale: 0 – None; 1 – slight; 2 – well defined; 3 – moderate; 4 – scar formation. Edema scale: 0 – None; 1 – slight; 2 – well defined; 3 – moderate; 4 – severe. Histopathological scale: +slight; ++moderate; +++severe. Formalin produced severe erythema and edema (standard irritant), histopathological examination of skin indicated that the optimized formation doesn’t cause any erythema and edema to the skin, while formalin produced discontinuity and thinning of epidermis at some extent caused hyperplasia. Figure 15 and Table 1, illustrated that the optimized formulation is safe to use in the treatment of psoriasis.

Groups Visual Observation Histopathological Evaluation
Erythema Edema Inflammation Edema Remarks
Normal 0.00 ±0.00 0.00 ±0.00 - - -
Positive Control (0.8% Formain) 3.44 ±0.22 3.58 ±0.24 +++ +++ Discontinuity in epidermis, Epidermal hyperplasia, Ulceration
Optimized Formulation 1.20 ±0.16 1.13 ±0.20 + + -
TABLE-1: Results of skin irritation studies
This invention relates to the development of a microsponge based gel formulation or composition containing apremilast and betamethasone dipropionate in combination with peptide dendrimer using a quasi-emulsion diffusion solvent method. The following is a complete procedure for the formulation of the present invention that anyone versed in the art may understand.
Example 1: In this example, the polymer used was methyl methacrylate and the active ingredient refers to apremilast and betamethasone dipropionate, an anti-psoriatic and anti-inflammatory agent. The gel-based formulation was made by weight using the following composition
Carbopol 943 is present in an amount ranging from 0.05-1% by weight of the composition
Tween 80 is present in an amount ranging from 0.05-1% by weight of the composition
PEG-400 is present in an amount ranging from 0.05-1.5% by weight of the composition
DMSO is present in an amount ranging from 0.5-2% by weight of the composition
Sodium metabisulphite is present in an amount ranging from 0.01-0.05% by weight of the composition
Methyl paraben is present in an amount ranging from 0.01-0.05% by weight of the composition
Propyl paraben is present in an amount ranging from 0.01-0.05% by weight of the composition
Triethanolamine is present in an amount ranging from 0.01-0.05% by weight of the composition
Example 2:
In this example, the polymer used was methyl methacrylate and the active ingredient refers to apremilast and betamethasone dipropionate, an anti-psoriatic and anti-inflammatory agent. The gel-based formulation was made by weight using the following composition
Carbopol 943 is present in an amount ranging from 1-2% by weight of the composition
Tween 80 is present in an amount ranging from 1-2% by weight of the composition
PEG-400 is present in an amount ranging from 1-2% by weight of the composition
DMSO is present in an amount ranging from 2-5% by weight of the composition
Sodium metabisulphite is present in an amount ranging from 0.1-0.2% by weight of the composition
Methyl paraben is present in an amount ranging from 0.1-0.2% by weight of the composition
Propyl paraben is present in an amount ranging from 0.01-0.05% by weight of the composition
Triethanolamine is present in an amount ranging from 1-3% by weight of the composition
Example 3:
In this example the ratio of apremilast, betamethasone dipropionate in the plain gel composition or formulation is 1:1, 2:1, 3:1, 10:1, and 20:1
Example 4:
In this example the ratio of apremilast, betamethasone dipropionate in the MS based gel composition or formulation is 1:1, 2:1, 3:1, 10:1, and 20:1
Example 5:
In this example the ratio of apremilast, betamethasone dipropionate and peptide dendrimer in MS based gel composition or formulation is 3:1:1.
Example 6:
In this example, the procedure for the characterization of the MS based composition or formulation containing apremilast and betamethasone dipropionate as the active agent is described.
Fourier transform infrared spectroscopy (FTIR): The KBr pellet technique was used to determine IR spectra using an FTIR spectrophotometer (Shimadzu FTIR -8300, Kyoto, Japan). APR, BD, physical mixture (containing APR, BD, Carbopol, sodium metabisulphite, methyl and propyl paraben) and PD composition were dried under a sodium lamp and then grinded with KBr in an agate mortar. The dry materials were then compressed into pellets under 5 tons of pressure for 5 minutes and scanned at 500 to 4000 cm-1.
Differential Scanning Calorimetry (DSC): The DSC APR, BD, physical mixture (containing APR, BD, Carbopol, sodium metabisulphite, methyl and propyl paraben) and PD composition were performed using DSC (Shimadzu-TA-60 WS, Kyoto Japan). The samples were placed in an aluminum pan and heated at a rate of 10 °C/ min from 25 °C to 350 °C under continuous nitrogen flow. An empty aluminum pan was used as the reference for the analysis. Each sample had its heat flow measured as a function of temperature. For every sample, heat flow was measured as a function of temperature.
Scanning Electron Microscopy (SEM): Scanning electron microscope (SEM) was used to examine the surface morphology of MS, MS loaded with drugs and PD. The instrument used was EVO MA18 with Oxford EDS(X-act), Zeiss, Germany. Pelletized samples were mounted on aluminum stubs using double-sided gold tape. To improve the conductivity of the samples, they were placed in a vacuum at 10 Torr. An electronic beam with a 20 kV acceleration potential was used to scan the samples. The obtained images were collected.
Example 7:
In this example, the in vitro drug release study of Plain gel formulation and MS loaded gel formulation is described. The in vitro drug release test for the formulations were conducted by using Franz diffusion vertical type apparatus. The receptor medium was phosphate buffer pH 5.8 + 20% methanol to replicate the pH of skin. The receptor compartment of 5mL volume with donor compartment bearing an area of 0.78 cm2 was used. The receptor medium was maintained at room temperature and stirred magnetically at 500 rpm. Samples were analyzed by HPLC. The fresh buffer was immediately replenished into the receptor compartment after each sampling (15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. Percent cumulative drug release was plotted as a function of time.
Example 8:
In this example, drug permeation and deposition study were performed on the excised swiss albino mice skin using Franz diffusion cell (Mutalik et al., 2014). Epidermal side of the skin was exposed to ambient condition, while dermal side was kept facing the receptor solution. Receptor compartment containing 5 mL phosphate buffer pH 5.8+20% methanol (Amrutiya et al., 2009) was thermostated at 37±0.5°C and stirred at 750 rpm. Skin was saturated with diffusion medium for 1 h before the application of sample. A 100 mg of was applied on the donor compartment. For determination of drug deposited in the skin, the diffusion cell was dismantled after a period of 24 h. The skin was carefully removed, and drug present on the skin surface was cleaned with diluent (1:1 ratio of ACN : MilliQ water). APR and BD were extracted from skin using a modification of the procedure described by (Echevarría et al., 2003). Briefly, the skin was cut into small pieces and homogenized with 5 mL diluent by tissue homogenizer. The homogenized sample was subjected to ultrasonication for 10 min for complete extraction of drug. This extract was centrifuged at 10,000 rpm for 10 min. The supernatant was collected and filtered through a 0.2-µm syringe filter and analyzed by developed HPLC method.
Example 9:
In this example, in vitro cytotoxicity studies by MTT assay was performed by taking 200 µL cells suspension was seeded in a 96 – well plate at required cell density (20,000 cells/ well), without the test agent. The cells were allowed to grow for about 24 h to reach 70-80% confluence. After 24 h the spent media was removed and test agent was added. The plates were then incubated for 48 h at 37 °C in a 5% CO2 atmosphere. After completion of incubation, the plates were taken out from the incubator. MTT reagent was added after removing spent media to make the final concentration of 0.5 mg/mL of total volume. The plates were wrapped with aluminum foil to avoid exposure of light. The plates were then again kept in incubator of 3 h. After completion of 3 h, the MTT reagent was removed and 100 µL of DMSO (solubilization solution) was added. To enhance dissolution this solution was gently shaken in a gyratory shaker. The solution was also occasionally pipetting up and down to completely dissolve the MTT frozen crystals in dense cultures. The absorbance was read on a spectrophotometer or an ELISA reader at 570 nm wavelength. Then % cell viability was calculated by the following formula:
% Cell Viability= (Absorbance of treated cells)/(Absorbance of untreated cells) x100
Example 10:
In this example, the expression of TNF-? was determined in psoriatic induced HaCaT cell lines with the aid of FACS method by Flow cytometry, in this method, human keratinocytes were used to develop the reliable model of psoriasis in vitro. The cells (5×105 cells/mL) were cultured in 6 well plate using calcium free Keratinocyte Medium II, containing 10% FBS and incubated for 24 h in CO2 incubator. Further cells were incubated with 2 mM calcium chloride (CaCl2) for 6 h. After treatment with CaCl2, Imiquimod (IMQ) at a concentration of 100 µM was added to induce psoriasis like inflammatory condition in differentiated HaCaT cells. After induction of in vitro model of psoriasis an appropriate concentration of the test agents was added to differentiated cells.
a. Positive control – IMQ with 100 µM
b. Negative control – Untreated cells
c. IMQ with 100uM+ Apremilast 0.020 µM
d. IMQ with 100uM+ Apremilast 0.041 µM
e. IMQ with 100uM+ A+B+P- 0.135 µM
f. IMQ with 100uM+ A+B+P- 0.27 µM
After addition of the test agents mentioned above, the cells were incubated for 48 h at 37 °C in a 5% CO2 atmosphere. At the end of the treatment, the cells were harvested directly into 12 x 75 mm polystyrene tubes. The tubes were then centrifuged for five min at 300 x g at 25 °C. After centrifugation, the supernatant was decanted carefully and cells were washed with PBS. The cells were then fixed with 1 mL cold 70% ethanol. Ethanol was added drop wise to cell pellet while vortexing to ensure fixation of all cells and for minimizing clumping. The cells were then incubated for 30 min in -20 °C freezer. To form the pellet the cells were centrifuged at higher speed for 5 min as compared to the live cells, then the supernatant was aspirated carefully without losing the pellet. Note that ethanol-fixed cells require higher centrifugal speeds to form pellet as compared to unfixed cells since they become more buoyant upon fixation; after fixation the cells were washed twice with PBS. 10 µL of TNF - ? antibodies were added, mixed thoroughly and incubated for 30 min in the dark at room temperature (20 to 25 °C). 500 µL of DPBS was added, mixed thoroughly and analysed by FACS – Cell quest pro software.
It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.
,CLAIMS:I/WE CLAIM
1. Anti-psoriasis topical formulation, consisting of:
an anti-psoriasis topical formulation (microsponges based) containing drug(s) (combination of Apremilast (APR) and betamethasone dipropionate (BD), and any other active agent which enhances the therapeutic effect or Apremilast alone), with or without dendrimeric peptide(s), for enhancing the skin permeation and deposition of active agent(s) on skin wherein the microsponges based tropical gel formulation with dual drugs or Apremilast alone and dendrimeric peptide for the combination therapy of psoriasis comprises active agents (APR and BD) in a ratio ranging from 0.01:9.99 to 9.99:0.01 for APR and BD respectively or Apremilast alone in the range of 0.001% to 10%, polymer(s) ranging from 0.1-5%, surfactant and emulsifying agent each ranging from 0.01-5% by weight of the composition wherein the composition contains fatty acids, peptides/ proteins, preservatives, antioxidants, permeation enhancers, other additives, etc wherein dendrimeric peptide(s) ranging from 0.01 – 20% is used to enhance the permeation and deposition of the active agents onto the skin.
2. The formulation as claimed in claim 1 wherein the microsponges (either prepared or procured by commercial source) are combined with the active agent(s) in a carrier or incorporated with the active agent(s) in a base that meets pharmaceutical standards wherein the carrier can also be included in the base.
3. The formulation as claimed in claim 1 wherein the active agent(s) comprise of a class of phosphodiesterase inhibitors and the active second agent belongs to a class of corticosteroids and other agent(s) which enhances the efficacy of the combination for the treatment of psoriasis and or reduces the toxicity.
4. The formulation as claimed in claim 1 wherein the active agent(s) comprise small molecule drug, protein, peptide, amino acid, nucleic acid (nucleotides, nucleosides, and analogues thereof), prodrug, which provide pharmacological action upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components.
5. The formulation as claimed in claim 1 wherein the active agent is drug combination comprises of Apremilast and Betamethasone dipropionate or single drug (Apremilast).
6. The formulation as claimed in claim 5 wherein the Aprimelast and Betamethasone are ranging from 0.001% w/w to 10% w/w and are present in the ratio ranging from 0.01:9.99 to 9.99:0.01 or single drug Apremilast in the range of 0.001% to 10%.
7. The formulation of claim 5 wherein the combination drugs are added with/ into a carrier which may be selected from the class of microsponges, microspheres, nanosponge, liposomes, nanoparticles, micelles, emulsions, suspensions, dendrimer, natural, synthetic or semisynthetic phospholipids, polymers, lipids, carbohydrates, polysaccharides, ionic or nonionic surfactants, peptides, and/or other derivatives.
8. The formulation as claimed in claim 1 wherein the microsponge composition consisting of one or more porous materials that are loaded with one or more active agents, either inside the pores, on the surface, or both inside the pores and on the surface.
9. The formulation as claimed in claim 1 wherein the microporous material is prepared using natural and/or semi-synthetic and/or synthetic polymers, which may be combined with other excipients used in the preparation.
10. The formulation as claimed in claim 1 wherein the dendrimeric peptide or poly(amino acid) or individual amino acid(s) are loaded in the microsponge either along with the active agent(s) in a carrier or associated with the active agent(s) in a pharmaceutically acceptable base or administered as pre-treatment or post-treatment on the skin in a pharmaceutically acceptable base.
11. The formulation as claimed in claim 10 wherein the dendrimeric peptide may be composed of glycine, lysine, arginine, proline, histidine, methionine, leucin or other combination of amino acids thereof or the composition as explained in claims 1-9 may contain individual amino acid(s) wherein the % w/w of dendrimeric peptide or poly (amino acid) or individual amino acid(s) may range between 0.001 – 20% w/w.
12. The formulation as claimed in claim 1 wherein the microsponge with drug combination or single drug and dendrimeric peptide or poly(amino acid) or amino acid(s) are added in a pharmaceutically acceptable base encompasses but is not limited to buffer, solution, ointment, cream, paste, gel, jelly, foam, suspension, spray, emulsion, lotion, and may contain one or more skin protective agent(s), permeation enhancer(s) and/ or one or more formulation component(s) and/ or additives.