DESC:Technical field of Invention:
The invention relates to stable fixed dose combination (FDC) of four anti-tuberculosis (TB) drugs viz., Rifampicin (RIF), Isoniazid (INH), Pyrazinamide (PZA) and Ethambutol Dihydrochloride (EDH). The invention further relates to the method for preparation of stable fixed dose combination and advantage of cocrystals in stabilizing the FDC compared to the FDC of pure drugs.

Background of the Invention:
Tuberculosis (TB) remains one of the largest health problems in the world today, and ranks as the second leading cause of death from a single infectious disease after HIV. According to WHO an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320,000 deaths among HIV-positive people) in 2012 (World Health Organization (WHO) report 2013, http://www.who.int/tb/publications/factsheet_global.pdf). The numbers of TB deaths are unacceptably large given that many are preventable. WHO has recommended fixed dose combinations (FDCs) against TB over six to nine months treatment for better patient compliance and lesser chance of developing drug resistance.FDC drug regimens consists of Rifampicin (RIF), Isoniazid (INH), Pyrazinamide (PZA), and Ethambutol dihydrochloride (EDH). All the drugs in the FDC work by different mechanisms of action towards tuberculosis, in which rifampicin is one of the effective anti-tuberculosis drugs which binds to the ß–subunit of bacterial DNA dependent RNA polymerase, resulting in the blocking of the initiation chain formation for RNA synthesis and growth inhibition of the susceptible organisms (L. C. D. Toid, V. Pillay, M. P. Danckwerts, Respiratory research. 2006, 7, 118–135). Isoniazid is a prodrug activated by KatG, exhibiting its activity by inhibiting the synthesis of mycolic acid in the bacterial cell wall (J. Suarez, K. Ranguelova, A. A. Jarzecki, J. Manzerova, V. Krymov, X. Zhao, S. Yu, L. Metlitsky, G. J. Gerfen, R. S. Magliozzo, J. Biol. Chem. 2009, 284, 7017–7029).Pyrazinamide prodrug gets converted to pyrazanoic acid by pyrazinamidase enzyme and pyrazanoic acid lowers the pH of the surroundings of TB bacteria, resulting in reduced growth of the organisms (Y. Zhang, D. Mitchison, Int. J. Tuberc. Lung Dis., 2003, 7, 6–21). Ethambutol is bacteriostatic against actively growing TB bacilli and it works by obstructing the formation of the cell wall (R. G. Shepherd, C. Baughn, M. L. Cantrall, B. Goodstein, J. P. Thomas, R. G. Wilkinson, Annals of the New York Academy of Sciences. 1966, 135, 686–710).However, serious concerns have been raised on the utility of these FDC products due to quality problems such as stability which adversely affects product quality and bioavailability of the drugs (H. Bhutani, T. T. Mariappan, S. Singh, Int. J. Tuberc. Lung Dis.2004, 8, 1073–1080). The chemical and physical instability of FDCs is found to occur mainly due to two reasons. One is the direct interaction of Rifampicin with Isoniazid, the mechanism of which involves interaction of imine group of Rifampicin with amino of Isoniazid to yield isonicotinyl hydrazone (HYD) in the solid formulation environment (Scheme1) (H. Bhutani, S. Singh, K.C. Jindal, A. K. Chakraborti, J. Pharma. Biomed.Anal. 2005, 39, 892–899). The other reason is the creation of an acidic hydrolytic environment upon moisture gain by Ethambutol dihydrochloride (S. Singh and B. Mohan, Int. J. Tuberc. Lung Dis., 2003, 7, 298–303). Generally, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also to delay the development of resistance to the individual components of the combination. An example for antimalarial therapy which fit the criteria of synergistic fixed-dose combinations is sulfadoxine-pyrimethamine combination in that neither of the individual components is effective alone (J. K. Trigg, H. Mbwana, O. Chambo, E. Hills, W. Watkins, C. F. Curtis, Acta Tropica, 1997, 63, 185–189). Similarly, combinations of anti TB drugs are prescribed due to drug resistance but the quality of the FDC is the main concern and hence there is an immediate urgency to develop stable FDC formulations.

The schematic diagram of the drugs to show the cross reaction between isoniazid (INH) and Rifampicin (RIF) is shown in Scheme 1.

Scheme 1

Therefore, the major concern of the scientific community is to improve the physical and chemical stability of the individual anti-tubercular drugs so as to eliminate the cross reactions between the drugs viz., rifampicin and isoniazid and thus improving the overall physical and chemical stability of the fixed dose combinations (FDC) comprising the same.

Object of the invention:
Thus it becomes the main object of the invention to improve the physical and chemical stability of TB drugs present in fixed dose combination (FDC).

The other object of the invention is to develop solid state forms (corystals/eutectics) of the pure drugs to control the cross reaction between the FDC drugs such as rifampicin and isoniazid.

Summary of the invention:
In accordance with the above objectives, to address the reactivity of isoniazid and the hygroscopicity of ethambutol dihydrochloride, the instant invention provides cocrystals and eutectics of these anti-tubercular drugs by using crystal engineering approach.

Accordingly, in an aspect, the present invention provides FDC cocrystal composition comprising Pyrazinamide, Ethambutol Dihydrochloride, Rifampicin, INH-CFA Cocrystal with improved stability.

In another aspect, the present invention provides FDC eutectic composition comprising Pyrazinamide, EDH-FA Eutectic, Rifampicin, Isoniazid with improved stability.

In yet another aspect, the present invention provides FDC eutectic and cocrystal composition comprising Pyrazinamide, EDH-FA Eutectic, Rifampicin, INH-CFA Cocrystal with improved stability.

In yet another aspect, the present invention provides a Fixed Dose Combination cocrystal composition which comprises 400 mg pyrazinamide, 150 mg rifampicin, 275 mg ethambutol dihydrochloride and 173 mg INH-CFA cocrystal, in association with one or more pharmaceutical excipients that may be prepared by mixing them together along with pharmaceutical excipients..

In a further another aspect, the present invention provides a FDC eutectic composition which comprises 400 mg pyrazinamide, 150 mg rifampicin, 390 mg EDH-FA eutectic, and 75 mg isoniazid that may be prepared by mixing themtogether along with pharmaceutical excipients.

In a further aspect, the present invention provides a FDC eutectic and cocrystal composition which comprises 400 mg pyrazinamide, 150 mg rifampicin, 390 mg EDH-FA eutectic and 173 mg INH-CFA cocrystal that may be prepared by mixing them together along with pharmaceutical excipients.

In yet another aspect, FDC reference composition, FDC cocrystal composition, FDC eutectic composition, FDC cocrystal and eutectic composition which were prepared in our studies characterised by PXRD, SEM, photographic images, and DSC.

The stability studies were carried out for all the combinations at accelerated humidity conditions of 40 °C and 75% RH, and the physical stability was assessed through various techniques such as PXRD, photographic images, and SEM analysis. Further these combinations were characterized by DSC, and the chemical stability by HPLC analysis. The chemical structures of the drugs and coformers used in this study are shown in Scheme 2.
Scheme 2

Brief description of the drawings:
Figure 1 depicts comparison of PXRD pattern of FDC reference batch (RIF+INH+PZA+EDH) to show the degradation of FDC drugs kept at 40 °C and 75% RH over 4 h.
Figure 2 depicts comparison of PXRD pattern of FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) kept at 40 °C and 75% RH show stability for 7 days.
Figure 3 depicts comparison of PXRD pattern of FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) kept at 40 °C and 75% RH which is to show the stability for 30 days.
Figure 4 depicts comparison of PXRD pattern of FDC eutectic batch (RIF+INH+PZA+EDH–FA Eutectic) for 30 days kept at 40 °C and 75% RH shows the degradation of the product at 30 days.
Figure 5 depicts comparison of PXRD pattern of FDC eutectic and cocrystal batch (RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic) for 30 days kept at 40 °C and 75% RH shows degradation of the product at 30 days.
Figure 6 depicts DSC thermogram of FDC reference batch (RIF+INH+PZA+EDH) at 0 day.
Figure 7 depicts DSC thermogram of FDC reference batch (RIF+INH+PZA+EDH) after 3 h at accelerated conditions of 40 °C and 75% RH.
Figure 8 depicts DSC thermogram of FDCcocrystal batch(RIF+INH–CFA cocrystal +PZA+EDH) at 0 day.
Figure 9 depicts DSC thermogram of FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) after 7 days at accelerated conditions of 40 °C and 75% RH.
Figure 10 depicts SEM images of FDC reference batch(RIF+INH+PZA+EDH) (a) at 0 day (b) after 3 days at accelerated conditions of 40 °C and 75% RH.
Figure 11 depicts SEM images of FDC cocrystal batch(RIF+INH–CFA cocrystal +PZA+EDH) (a) at 0 day (b) after 3 days at accelerated conditions of 40 °C and 75% RH.
Figure 12 (a) X-ray crystal structure of INH-CFA stabilized by hydroxyl O–H···O hydrogen bonds, acid-pyridine O–H···N hydrogen bond, and C–H···O interactions in a layer structure. (b) The layers were connected through N–H···O hydrogen bonds.
Figure 13 Images of progressive degradation after keeping the FDC samples at accelerated conditions of 40 °C and 75% RH. Left side is petri dish containing the reference batch FDC which was found to degrade within 3 h whereas INH–CFA cocrystal FDC batch was stable at 7 days (Right petri dish).Reference batch (RIF+INH+PZA+EDH, left petri dish) and cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH, right petri dish).
Figure 14 HPLC Chromatograms showing changes in the purity of drugs and formation of by-products on storage of the FDC reference batch RIF+INH+PZA+EDH at accelerated conditions of temperature and humidity (40 °C, 75% RH) for 2 h, 4 h, 6 h, 24 h). Out of four drugs FDC, three are observed in the HPLC chromatogram (RIF, INH, PZA) and fourth (EDH) does not appear in the solvent system used due to lack of a chromophore.
Figure 15 HPLC Chromatograms showing changes in the purity of drugs and formation of by-products on storage of the FDC cocrystal batch RIF+INH–CFA cocrystal+PZA+EDH at accelerated conditions of temperature and humidity (40 °C, 75% RH) for 2 h, 4 h, 6 h, 24 h. Out of four drugs FDC, three are observed in the HPLC chromatogram (RIF, INH, PZA) and fourth (EDH) does not appear in the solvent system used due to lack of a chromophore. Coformer CFA is also present in the HPLC trace.

Abbreviations:
RIF- rifampicin
INH- isoniazid
PZA- pyrazinamide
EDH- ethambutol dihydrochloride
CFA- caffeic acid
FA- fumaric acid
INH-CFA- isoniazid and caffeic acid cocrystal
EDH-FA Eutectic- ethambutol dihydrochloride and fumaric acid eutectic
HPLC- high performance liquid chromatography
FDC cocrystal batch- RIF+INH–CFA cocrystal +PZA+EDH
FDC Eutectic batch- RIF+INH+PZA+EDH–FA Eutectic
FDC eutectic and cocrystal batch- RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic

Detailed description of the invention:
The invention will now be described in detail as certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

The reduced bioavailability of rifampicin in the fixed dose combination (FDC) comprising pyrazinamide, ethambutol dihydrochloride, rifampicin, and isoniazid is due to several reasons such as hygroscopicity of ethambutol dihydrochloride, and drug–drug interactions etc. The isonicotinyl hydrazone (HYD) of 3-formyl rifampicin and isoniazid was the major degraded product from the anti-tuberculosis FDC composition. To address this issue, FDC reference batch (RIF+INH+PZA+EDH), FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH), FDC eutectic batch (RIF+INH+PZA+EDH–FA Eutectic), FDC eutectic and cocrystal batch (RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic) were prepared.

In an embodiment, the present invention discloses preparation of FDC cocrystal composition comprising pyrazinamide, ethambutol dihydrochloride, rifampicin, INH-CFA cocrystal.

In an embodiment, the present invention discloses preparation of FDC eutectic composition comprising pyrazinamide, EDH-FA eutectic, rifampicin, isoniazid.

In an embodiment, the present invention discloses preparation of FDC eutectic and cocrystal composition comprising pyrazinamide, EDH-FA eutectic, rifampicin, INH-CFA cocrystal.

In an embodiment, the present invention discloses FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) comprising pyrazinamide, ethambutol dihydrochloride, rifampicin, INH-CFA cocrystal with improved stability at accelerated conditions for one month which was monitored by PXRD.

In another embodiment, the present invention discloses FDC eutectic batch (RIF+INH+PZA+EDH–FA Eutectic).

In yet another embodiment, the present invention discloses FDC eutectic and cocrystal batch (RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic).

In yet another embodiment, the purity of different FDC drugs analyzed by HPLC for the reference FDC (PZA+EDH+RIF+INH) and cocrystal FDC (PZA+EDH+RIF+INH-CFA Cocrystal) are listed in Table 6. Table 6 refers to percent drug remaining after storage of FDC reference batch (PZA+EDH+RIF+INH) and FDC cocrystal batch (PZA+EDH+RIF+INH-CFA Cocrystal) at accelerated conditions of temperature and humidity (40 °C, 75% RH) for 2 h, 4 h, 6 h, 24 h.

As is evident from Table 6, fixed dose combination of PZA+EDH+RIF+INH-CFA Cocrystal apparently exhibits more stability/ purity/availability of the active drugs than the reference Fixed dose combination consisting of PZA+EDH+RIF+INH.

Thus the provision of isoniazid as co-crystal along with caffeic acid increases the stability of the fixed dose combination and also reduces the formation of isonicotinyl hydrazone (HYD), which is the result of direct interaction of Rifampicin with Isoniazid, in the solid formulation environment.

Accordingly, the present invention provides a pharmaceutical composition comprises fixed dose combination of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid in association with one or more pharmaceutical excipients.

Thus the fixed dose combination of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid is characterised by; (i) PXRD at 0 days with characteristic peaks of 2 theta at 6.97, 9.28, 11.09, 12.08, 12.56, 15.63, 18.93, and 19.87 degree, (ii) DSC at 0 h with characteristic melting endotherms at 76.45 °C, 132 °C, 144.98 °C, 160.98 °C and 177.73 °C in DSC thermogram.

The fixed dose combination of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid is further characterised by; (i) PXRD after 15 and 30 days with characteristic peaks at 2 theta 6.97, 9.28, 11.09, 12.08, 12.56, 15.63, 18.93, and 19.87 degree, (ii) after 1 week with characteristic melting endotherms at 74 °C, 130.2 °C, 139.40 °C, 158.23 °C and 161.72 °C in DSC thermogram.

In a more preferred embodiment, the invention provides a pharmaceutical composition comprising fixed dose combination of Rifampicin150 mg, ethambutol dihydrochloride 275 mg, Pyrazinamide 400 mg and cocrystal of isoniazid-caffeic acid 173 mg in association with one or more pharmaceutical excipients, for the treatment of tuberculosis.
In yet another embodiment, the fixed dose combination of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid may be prepared by as a single dosage form by combining all the active ingredients in association with pharmaceutical excipients/carriers.

The pharmaceutical excipients/carriers may be selected from the group consisting of diluents, binders, disintigrants, lubricants, polymers, colors, flavours, etc. and the dosage forms may be prepared in accordance with the methods disclosed in the art.

Alternately, the fixed dose combination of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and co-crystal of isoniazid-caffeic acid may be provided as a kit containing individual tablets in a single strip.

In yet another embodiment, all the active ingredients are provided as individual granules and filled into a hard gelatine capsule.

In yet further embodiment, the invention provides method for treating a subject suffering with tuberculosis which method comprises administering the subject a fixed dose combination comprising of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid in association with one or more pharmaceutical excipients.

In yet another aspect, the invention provides a fixed dose combination comprising of Rifampicin, ethambutol dihydrochloride, Pyrazinamide and cocrystal of isoniazid-caffeic acid in association with one or more pharmaceutical excipients, for use in the treatment of tuberculosis.

In another embodiment, the present invention discloses use of supramolecular approach which helps to prevent the degradation and cross reactions of FDCs and hence the invention also relates to stable anti-tuberculosis FDC products.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1
Preparation of FDC batches

Method of preparing INH-CFA cocrystal (1:1)
The bulk material of INH-CFA cocrystal(Form 1 of 3 known polymorphs) was obtained upon extensive grinding of INH (isoniazid) and CFA (caffeic acid) for 1-2 h by adding catalytic amount of CH3CN solvent. The formation of cocrystal was confirmed by FT-IR, FT-Raman, PXRD and DSC. 30 mg of the ground material was dissolved in 8 mL hot THF: n-heptane (1:1, v/v) and left for slow evaporation at room temperature. Colorless plate crystals suitable for X-ray diffraction were obtained after 3-4 days upon solvent evaporation.

Method of preparing EDH–FA Eutectic (1:1)
Ethambutol Dihydrochloride (EDH) and Fumaric acid (FA)were taken in an equimolar ratio and subjected to neat grinding using a mortar-pestle for 15 min. The resultant material was found to be eutectic composition by its unique low melting point than both the starting components.

Preparation of FDC reference batch (RIF+INH+PZA+EDH)
Pure drugs were taken in prescribed amounts given by WHO i.e. Rifampicin 150 mg, Isoniazid 75 mg, Pyrazinamide 400mg, Ethambutol Dihydrochloride 275 mg and all the drugs were mixed uniformly and used for further studies.

Method of preparing FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH)
Pure drugs were taken in prescribed amounts given by WHO i.e. Rifampicin 150 mg, Pyrazinamide 400mg, Ethambutol Dihydrochloride 275mg, and Isoniazid-Caffeic acid cocrystal (173 mg) was taken instead of pure isoniazid. All the drugs were used for further studies after proper mixing.

Method of preparing FDC Eutectic batch (RIF+INH+PZA+EDH–FA Eutectic)
Pure drugs were taken in prescribed amounts given by WHO i.e. Rifampicin 150 mg, Isoniazid 75 mg, Pyrazinamide 400 mg, Ethambutol Dihydrochloride-Fumaric acid Eutectic in 390 mg instead of pure Ethambutol dihydrochloride and all the drugs were mixed uniformly and used for further studies.

Preparation of FDC eutectic and cocrystal batch (RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic)
Pure drugs were taken in prescribed amounts given by WHO i.e. Rifampicin 150 mg, Pyrazinamide 400 mg, and EDH-Fumaric acid Eutectic (390 mg) instead of EDH and Isoniazid-Caffeic acid cocrystal (173 mg) in the place of isoniazid. All the drugs were mixed uniformly and used for further studies.

Example 2
(a) X-ray Powder Diffraction (XRPD)
Powder X-ray diffraction technique is a standard method used for the characterization of solid-state forms. Therefore the technique of PXRD is vital and predominant tool for the study of polycrystalline materials, and is eminently suited for the routine characterization and to know the phase changes of solid material.XRPDs were recorded on SMART Bruker D8 Focus Powder X-ray diffractometer using Cu-Ka radiation (? = 1.5406 Å) at 40 kV and 30 mA. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and figure 6 are the XRPDs of FDC reference batch (RIF+INH+PZA+EDH) at 0 day, FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) after 7 days, FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) after 30 days, FDC eutectic batch (RIF+INH+PZA+EDH–FA Eutectic) after 30 days, FDC eutectic and cocrystal batch (RIF+INH–CFA cocrystal+PZA+EDH–FA Eutectic) after 30 days respectively.

All the batches were studied for the stability at accelerated conditions of 40°C and 75% RH and the resulting material was checked for its phase through PXRD. From the comparison of the PXRD patterns (Figure 1) there is a significant difference in powder lines observed for FDC reference batch at 0 h, after 2 h and 4 h. The disappearing of the peaks at 2 theta 16.7, 22.1, 23.0, 24.8, 25.3, 26.7, 28.1, and 32.0 degree indicates that the degradation of the reference batch, and the material was started liquefying after 2 h by showing thick brownish colour. The missing powder lines in PXRD pattern were majorly from the RIF, INH and EDH. After 4 hrsin ICH conditions there was a drastic change in powder pattern of the reference batch, approximately 50% of the peaks were disappeared because of liquefying of the sample. The liquefying of the sample and the color change may be due to the hygroscopic nature of the EDH, andthe cross reaction between the isoniazid and rifampicin.Under similar experimental conditions the cocrystal batch was studied for stability and the Characteristic peaks in PXRD showed at 2 theta 6.97, 9.28, 11.09, 12.08, 12.56, 15.63, 18.93, and 19.87 degree. The comparison of PXRD pattern (Figure 2 and 3) did not show any new peaks or disappearing of existed lines throughout the stability study indicating the stability of the sample. The detailed peak values of the starting components and FDC compositions were given in Tables 1-4. To see the physical stability we also took photographic images of the powder material (Figure 13) in regular intervals in which the appearance of the material was found to be constant which is in a way complementing PXRD observation.

(b) Single crystal X-ray diffraction
Good quality single crystals of INH-CFA obtained from the THF: n-heptane solvent mixture and were mounted on the goniometer of Bruker SMART CCD diffractometer equipped with Mo-Ka radiation (? = 0.71073 Å) source. Data reduction in case of the INH-CFA cocrystal was performed using Bruker SAINT software. Intensities were corrected for absorption using SADABS, and the structure was solved and refined using SHELX-97. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms on heteroatoms were located from difference electron density maps and all C-H hydrogens were fixed geometrically. The afforded cocrystal was solved and refined in monoclinic space group P21/c with one molecule of INH and CFA in the asymmetric unit. In the crystal structure, sheet structure of INH-CFA formed through hydroxyl O–H···O dimer, acid-pyridine O–H···N hydrogen bond and auxiliary C–H···O interactions and such sheets are stacked through N–H···O interactions. Because of the H-bonding and sheet structure in the INH-CFA cocrystal, the reactive amine group of the isoniazid is buried in the crystal face and hence not readily accessible to react with the imine group of Rifampicin (as shown in Scheme 1). The isoniazid-caffeic acid cocrystal is prevents the cross-reaction between isoniazid and rifampicin. Hydrogen bonding and molecular packing diagram in the crystal structure are displayed in Figure 12.
Example 3
Thermal analysis
Differential Scanning Calorimetry (DSC) was used for the thermal analysis of four FDC batches (FDC reference batch, FDC cocrystal batch, FDC eutectic batch, FDC cocrystal and eutectic batch). DSC was performed on a Mettler Toledo DSC 822e module by placing 4-5 mg of samples inaluminiumpans and the temperature range is 30-300 °C atheating rate of 5 °C min–1. FDC reference batch (PZA+EDH+RIF+INH) at 0 hours exhibits characteristic melting endothermic peaks at 76.39 °C, 140.18 °C, 158.27 °C and 172.94 °C in DSC thermogram. FDC reference batch (PZA+EDH+RIF+INH) after 3 h exhibits characteristic melting endothermic peaks at 64.86 °C, 100.68 °C, 119.18 °C, 160.02 °C, and 177.76 °C in DSC thermogram. FDC cocrystal batch (PZA+EDH+RIF+INH-CFA Cocrystal) at 0 hours exhibits characteristic melting endotherms at 76.45 °C, 132 °C, 144.98 °C, 160.98 °C and 177.73 °C, and the same sample which was kept at accelerated conditions showed characteristic melting endothermic peaks at 74 °C, 130.2 °C, 139.40 °C, 158.23 °C and 161.72 °C after one week in DSC thermogram. An endotherm around 75 °C in the DSC thermogramsof FDC batches is ascribed to the polymorphic transformation of ethambutol dihydrochloride (Form II to Form I). Melting points of individual components and the binary complexes used in our studies are provided in Table 5. Melting endotherms in DSC for FDC batches were found to be lower and broader compared to individual components which may be due to impurity effect, because the impurities will disrupt the consistency and organization of the crystal lattice at the molecular level. The disruption weakens the lattice, so that the lattice can be broken down more easily and hence the weakened structure melts more easily at lower temperature.

Example 4
HPLC analysis
The HPLC system consisted of a solvent delivery unit (LC-20AD), an on-line degasser (DGU-20A3), a PDA detector (SPD-M20A), and LC solution software for data acquisition and processing (All from Shimadzu, Japan). Separations were achieved on a Zodiac C18 (250 X 4.6 mm, 5 µm) column (Zodiac Life Sciences, Hyderabad).

FDC reference batch (RIF+INH+PZA+EDH) and FDC cocrystal batch (RIF+INH–CFA cocrystal +PZA+EDH) were taken in an open Petri dish and exposed to accelerated ICH conditions of 40 °C and 75% RH. Samples were withdrawn after 2, 4, 6 and 24 h time intervals and dissolved in methanol solvent. 20 µl of resultant solution was injected to HPLC for analysis. The mobile phase and the gradient programme are listed below.

Time (min) Solution A (%) Solution B (%)
0 100 0
0?5 100 0
5?18 0 100
18?25 100 0

Solution A = Buffer: ACN (96: 4).
Solution B = Buffer: ACN (55: 45).
Buffer composition: 1.4 g of dibasic sodium phosphate (Na2HPO4) in 1 L water (pH 6.8).
ACN: acetonitrile.

Other HPLC conditions were flow rate 1.2 mL/min, detection wavelength 238 nm. Using this procedure, it was possible only to analyse Rifampicin, Isoniazid, Pyrazinamide and HYD. Ethambutol dihydrochloride could not be detected using these conditions due to the lack of UV chromophore.

The purity of different FDC drugs analyzed by HPC for the reference FDC (PZA+EDH+RIF+INH) and cocrystal FDC (PZA+EDH+RIF+INH-CFA Cocrystal) are listed in Table 6.

PXRD 2 theta values are listed in Tables 1–4 along with relative peak intensity data of starting materials, Rifampicin, Isoniazid, Pyrazinamide, Ethambutol Dihydrochloride, EDH-FA Eutectic, INH-Caffeic acid cocrystal, FDC reference batch, FDC cocrystal batch.

Table 1 PXRD 2 theta (deg) and relative peak intensity of Rifampicin, Isoniazid, Pyrazinamide.
Rifampicin Commercial Isoniazid Commercial Pyrazinamide commercial
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
7.292 12.113 58.2% 9.760 9.055 3.8% 7.880 11.210 42.1%
8.541 10.345 82.6% 11.931 7.412 77.8% 13.805 6.409 14.2%
9.992 8.845 4.6% 14.263 6.205 76.5% 15.470 5.723 59.0%
11.745 7.529 43.0% 15.529 5.702 38.9% 17.783 4.984 100.0%
13.589 6.511 57.4% 16.764 5.284 100.0% 20.502 4.329 4.5%
14.236 6.216 100.0% 19.631 4.518 93.0% 23.724 3.747 9.9%
16.161 5.480 34.3% 24.042 3.699 15.5% 24.315 3.658 2.0%
18.307 4.842 58.4% 25.073 3.549 30.3% 26.400 3.373 6.6%
19.189 4.622 36.5% 25.955 3.430 11.6% 27.425 3.250 18.8%
20.450 4.339 36.2% 27.181 3.278 21.7% 29.119 3.064 3.8%
21.072 4.213 92.3% 28.783 3.099 21.0% 30.952 2.887 2.2%
22.168 4.007 16.7% 30.472 2.931 2.0% 31.686 2.822 2.2%
23.923 3.717 5.6% 31.998 2.795 5.7% 35.768 2.508 13.0%
24.581 3.619 6.4% 34.224 2.618 11.7% 36.876 2.436 1.6%
25.763 3.455 23.9% 36.532 2.458 10.6% 38.206 2.354 2.5%
27.393 3.253 18.6% 37.389 2.403 10.2% 39.770 2.265 5.3%
29.088 3.067 9.9% 38.661 2.327 21.2% 41.907 2.154 3.1%
31.591 2.830 4.8% 39.821 2.262 2.3%
32.725 2.734 2.8% 41.464 2.176 2.1%
33.634 2.662 2.5% 43.920 2.060 2.1%
36.327 2.471 4.3% 45.702 1.984 4.8%
37.423 2.401 2.7%
38.394 2.343 3.6%
40.324 2.235 3.1%

Table 2 PXRD 2 theta (deg) and relative peak intensity of Rifampicin, EDH-FA Eutectic, INH-Caffeic acid cocrystal.
Rifampicin Commercial EDH-FA Eutectic INH-Caffeic acid cocrystal
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
7.292 12.113 58.2% 7.575 11.661 60.4% 8.406 10.510 5.4%
8.541 10.345 82.6% 14.003 6.319 30.5% 10.104 8.748 12.3%
9.992 8.845 4.6% 15.221 5.816 100.0% 12.168 7.268 14.9%
11.745 7.529 43.0% 17.721 5.001 2.2% 12.506 7.072 29.4%
13.589 6.511 57.4% 18.516 4.788 2.3% 14.776 5.991 2.6%
14.236 6.216 100.0% 20.430 4.344 26.0% 15.814 5.599 3.6%
16.161 5.480 34.3% 21.841 4.066 33.8% 17.028 5.203 18.0%
18.307 4.842 58.4% 22.391 3.967 44.3% 17.540 5.052 21.8%
19.189 4.622 36.5% 22.780 3.901 76.8% 18.475 4.799 96.6%
20.450 4.339 36.2% 24.401 3.645 6.2% 19.360 4.581 8.6%
21.072 4.213 92.3% 25.203 3.531 6.2% 20.013 4.433 6.8%
22.168 4.007 16.7% 25.754 3.456 13.1% 20.440 4.341 6.1%
23.923 3.717 5.6% 26.657 3.341 9.2% 22.511 3.947 0.5%
24.581 3.619 6.4% 28.719 3.106 58.6% 24.408 3.644 64.6%
25.763 3.455 23.9% 29.324 3.043 24.2% 25.679 3.466 28.2%
27.393 3.253 18.6% 31.433 2.844 5.5% 27.549 3.235 100.0%
29.088 3.067 9.9% 32.109 2.785 18.3% 29.286 3.047 8.4%
31.591 2.830 4.8% 32.446 2.757 6.5% 29.800 2.996 5.2%
32.725 2.734 2.8% 33.541 2.670 4.1% 35.583 2.521 9.0%
33.634 2.662 2.5% 34.828 2.574 9.5% 38.151 2.357 3.7%
36.327 2.471 4.3% 35.820 2.505 3.2% 40.951 2.202 6.0%
37.423 2.401 2.7% 37.276 2.410 4.2% 41.534 2.172 4.2%
38.394 2.343 3.6% 37.887 2.373 5.3%
40.324 2.235 3.1% 38.473 2.338 2.8%
41.194 2.190 11.8%
42.070 2.146 5.0%
Table 3 PXRD 2 theta (deg) and relative peak intensity of FDC reference batch at 0 h, 2 h and 4 h.
FDC reference batch 0 h FDC reference batch
after 2 h FDC reference batch after 4 h
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
7.658 11.535 42.6% 7.669 11.518 23.6% 7.880 11.211 37.2%
8.669 10.192 6.0% 8.660 10.203 9.7% 13.831 6.398 15.0%
11.898 7.432 6.1% 11.832 7.474 5.7% 15.360 5.764 71.4%
13.634 6.490 18.2% 13.597 6.507 20.3% 17.629 5.027 100.0%
13.938 6.349 21.3% 14.321 6.180 16.1% 18.559 4.777 3.2%
14.321 6.180 7.2% 15.333 5.774 92.8% 19.377 4.577 3.1%
15.219 5.817 100.0% 16.246 5.452 4.4% 20.456 4.338 12.5%
16.264 5.446 3.1% 17.589 5.038 100.0% 21.284 4.171 5.4%
16.714 5.300 2.1% 18.397 4.819 8.9% 23.682 3.754 15.4%
17.642 5.023 79.9% 19.260 4.605 5.9% 24.213 3.673 8.9%
18.436 4.809 4.0% 20.409 4.348 15.3% 26.164 3.403 30.9%
20.434 4.343 19.8% 21.142 4.199 14.2% 27.379 3.255 75.2%
21.157 4.196 7.5% 21.945 4.047 19.0% 29.111 3.065 11.0%
21.885 4.058 12.4% 22.897 3.881 6.5% 30.795 2.901 5.6%
22.120 4.015 2.2% 23.576 3.771 9.0% 31.786 2.813 6.3%
23.031 3.859 25.5% 24.217 3.672 8.1% 35.739 2.510 23.5%
23.574 3.771 6.1% 26.221 3.396 27.6% 36.874 2.436 4.9%
24.841 3.581 3.4% 27.218 3.274 54.2% 38.157 2.357 5.9%
25.370 3.508 27.0% 29.072 3.069 6.7% 39.811 2.262 15.3%
26.273 3.389 9.3% 30.423 2.936 26.4% 41.818 2.158 7.6%
26.754 3.329 8.5% 30.966 2.886 9.8% 42.681 2.117 4.4%
27.393 3.253 16.2% 31.476 2.840 5.2%
29.131 3.063 5.7% 32.418 2.760 7.9%
31.323 2.853 3.7% 34.732 2.581 2.0%
32.036 2.792 4.3% 35.710 2.512 16.4%
32.591 2.745 7.3% 36.853 2.437 2.7%
33.380 2.682 2.1% 38.051 2.363 3.5%
33.922 2.641 2.2% 39.692 2.269 10.4%
35.789 2.507 11.0% 40.938 2.203 2.8%
39.911 2.257 5.8% 41.663 2.166 6.9%
41.292 2.185 8.8% 42.652 2.118 2.8%
42.065 2.146 2.9% 45.778 1.980 12.9%

Table 4 PXRD 2 theta (deg) and relative peak intensity of FDC cocrystal batch at 0 h, 2 h and 4 h.
FDC Cocrystal batch 0 days FDC Cocrystal batch 15 days
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
6.974 12.665 2.0% 6.974 12.665 2.0%
7.614 11.602 48.8% 7.614 11.602 48.8%
8.432 10.478 0.8% 10.005 8.833 3.2%
10.005 8.833 3.2% 11.092 7.971 8.0%
11.092 7.971 8.0% 12.560 7.042 6.9%
12.560 7.042 6.9% 13.716 6.451 15.4%
13.716 6.451 15.4% 13.992 6.324 23.7%
13.992 6.324 23.7% 15.284 5.792 88.3%
15.284 5.792 88.3% 15.631 5.665 32.5%
15.631 5.665 32.5% 17.061 5.193 6.5%
17.061 5.193 6.5% 17.641 5.024 100.0%
17.641 5.024 100.0% 18.526 4.786 12.8%
18.526 4.786 12.8% 18.930 4.684 2.1%
18.930 4.684 2.1% 19.877 4.463 8.2%
19.877 4.463 8.2% 20.467 4.336 18.7%
20.467 4.336 18.7% 21.853 4.064 16.1%
21.853 4.064 16.1% 22.254 3.992 7.4%
22.254 3.992 7.4% 23.023 3.860 23.5%
23.023 3.860 23.5% 23.547 3.775 7.5%
23.547 3.775 7.5% 24.416 3.643 9.9%
24.416 3.643 9.9% 24.805 3.587 7.6%
24.805 3.587 7.6% 25.778 3.453 9.0%
25.778 3.453 9.0% 26.358 3.379 9.3%
26.358 3.379 9.3% 27.323 3.261 26.8%
27.323 3.261 26.8% 29.102 3.066 4.9%
29.102 3.066 4.9% 31.340 2.852 4.1%
31.340 2.852 4.1% 32.486 2.754 8.8%
32.486 2.754 8.8% 33.878 2.644 2.6%
33.878 2.644 2.6% 35.734 2.511 10.5%
35.734 2.511 10.5% 39.799 2.263 5.4%
39.799 2.263 5.4% 41.287 2.185 9.6%
41.287 2.185 9.6% 42.069 2.146 3.7%
42.069 2.146 3.7% 42.581 2.121 2.21%
42.581 2.121 2.21%

Table 5 Melting points of individual drugs and their binary systems.
Compound Melting point ( ºC)
Pyrazinamide 190
Isoniazid 171
Ethambutol Dihydrochloride 202
Rifampicin 183
Caffeic acid 224
INH-CFA Cocrystal 168
Fumaric acid 287
EDH-FA Eutectic 200

Table 6 Percent drug remaining after storage of FDC reference batch (PZA+EDH+RIF+INH) and FDC cocrystal batch (PZA+EDH+RIF+INH-CFA Cocrystal) at accelerated conditions of temperature and humidity(40 °C, 75% RH) for 2 h, 4 h, 6 h, 24 h.
Compound Time intervals % of INH remaining % of PZA remaining % of RIF remaining % of HYD
formed a
FDC-Ref 0 h 100 100 100 0
FDC-Cocrystal 0 h 100 100 100 0
FDC-Ref 2 h 97.03 96.29 96.94 6.03
FDC-Cocrystal 2 h 98.64 99.96 97.81 3.55
FDC-Ref 4 h 89.72 94.09 86.92 23.36
FDC-Cocrystal 4 h 95.66 99.87 94.47 9.87
FDC-Ref 6 h 79.90 92.02 79.24 40.86
FDC-Cocrystal 6 h 93.71 97.76 92.93 13.36
FDC-Ref 24 h 76.38 89.33 74.40 49.22
FDC-Cocrystal 24 h 86.39 93.53 89.58 24.03

aThe structure of HYD by-product is given in Scheme 1.
,CLAIMS:We claim:

1. A fixed dose combination of Rifampicin, Ethambutol dihydrochloride, Pyrazinamide and cocrystal of Isoniazid-Caffeic acid characterised by; (i) PXRD at 0 day with characteristic peaks of 2 theta at 6.97, 9.28, 11.09, 12.08, 12.56, 15.63, 18.93, and 19.87 degree, (ii) DSC at 0 h with characteristic melting endotherms at 76.45 °C, 132 °C, 144.98 °C, 160.98 °C and 177.73 °C in DSC thermogram.
2. The fixed dose combination of Rifampicin, Ethambutol dihydrochloride, Pyrazinamide and cocrystal of Isoniazid-Caffeic acid according to claim 1 further characterised by; (i) PXRD after 15 and 30 days with characteristic peaks at 2 theta 6.97, 9.28, 11.09, 12.08, 12.56, 15.63, 18.93, and 19.87 degree, (ii) after 1 week with characteristic melting endotherms at 74 °C, 130.2 °C, 139.40 °C, 158.23 °C and 161.72 °C in DSC thermogram.
3. A pharmaceutical composition comprising a fixed dose combination of Rifampicin, Ethambutol dihydrochloride, Pyrazinamide and cocrystal of Isoniazid-Caffeic acid according to claims 1 & 2 in association with one or more pharmaceutical excipients, for use in the treatment of tuberculosis in a mammal.
4. The pharmaceutical composition according to claim 3, wherein the composition comprises Rifampicin 150 mg, Ethambutol dihydrochloride 275 mg, Pyrazinamide 400 mg, and cocrystal of Isoniazid-Caffeic acid 173 mg in association with one or more pharmaceutical excipients.
5. The pharmaceutical composition according to claim 3, wherein the pharmaceutical excipients/carriers may be selected from the group consisting of diluents, binders, disintigrants, lubricants, polymers, colors and flavours.
6. A method for treating a mammal suffering with tuberculosis which method comprises administering the mammal a fixed dose combination comprising of Rifampicin, Ethambutol dihydrochloride, Pyrazinamide and cocrystal of Isoniazid-Caffeic acid in association with one or more pharmaceutical excipients.