Claims:WE CLAIM:
1. A salt monohydrate of structure H formed from compound A and compound B ; wherein said compounds A and B are in a ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

H

A B
2. The salt monohydrate as claimed in claim 1, wherein the salt monohydrate is a medicament for mixed infections of bacteria and microbes.
3. A method of preparation of a salt monohydrate of structure H formed from compound A and compound B comprising the acts of

A B

a) mixing compound A and compound B and ethanol to form a mixture;
b) grinding the mixture to obtain the salt monohydrate of compound A and compound B; and
c) obtaining a salt monohydrateof structure H.

H
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).
4 The method of preparation of compound H as claimed in claim 3, wherein the ratio of compound A and compound B is in the ratio 1:1 (w/w) of A:B.
5. The method of preparation of compound H as claimed in claim 3, wherein the ethanol in the mixture is of volume ranging from about 1ml to about 15 ml.
6. The method of preparation of compound H as claimed in claim 3, wherein the grinding on the mixture ranges for a period from about 30 seconds to about 30 minutes.
7. The method of preparation of compound H as claimed in claim 3, wherein the grinding is carried out at a temperature ranging from about 22 °C to about 35 °C.

8. A salt hydrate of formula H

H
9. The compound H as claimed in claim 8, wherein the compound is characterised by PXRD pattern with peaks at 2? values of about, 23.76, 13.23, 26.07, 19.85, 14.69, 16.92, 21.09, 24.50, 28.43, 25.26, 27.99, and 28.87.
10. The compound H as claimed in claim 8, wherein the compound is characterised by FTIR peaks about 3600 cm-1, 3400 cm-1 and 1450 cm-1.
11.A salt solvate of structure S formed from compound A and compound B ; wherein said compounds A and B are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

S

A B
12. A salt solvate of formula (S),

S

13. The salt solvate of structure S as claimed in claim 11, wherein the salt solvate is characterised by PXRD pattern with peaks at 2? values of about, 23.77, 13.25, 26.08, 19.86, 16.95, 19.65, 24.53, 21.11, 28.46, 22.36, 28.00, and 25.31.

14. A method of preparation of a salt solvate structure S formed from compound A and compound B comprising acts of

A B
a) mixing compound A and compound B andmethanol to form a mixture;
b) grinding the mixture to obtain the salt solvate of compound A and compound B; and
c) obtaining a salt solvate of structure S.

S
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

15. A composition of comprising pharmaceutically acceptable amount of salt hydrate of claim 1 along with pharmaceutically acceptable amount of excipients in a pharmaceutically acceptable ratio.

16. The composition as claimed in claim 14, wherein the excipient is selected from group comprising gelling agents, tabletting agents, antioxidants, stabilizing agents, nanoparticles, nanocapsules, micellar and liposomal formulations.
, Description:Field of invention

The invention relates to novel pharmaceutical solid forms and to the studies of physicochemical and biological properties of these solid forms. The invention provides a composition comprising Norfloxacin and Sulfathiazole for the management of mixed infections. In particular, the composition relates to a monohydrate salt of Norfloxacin and Sulfathiazole and to a salt solvate of methanol of Norfloxacin and Sulfathiazole. The invention also provides a method of preparation of the compositions.

Background of the invention
Infections are generally caused by various pathogens like bacteria, fungi, viruses, prions, and the like. The pathogens may enter an organism through the respiratory gastrointestinal, or urogenital tracts or through an injury on a surface of the body. The infections usually target the mucosal surfaces of the body, multiplying rapidly and often survive an attack by the human immune system. A course of antibiotics is normally given to the patient when an infection sets in. Antibacterials are often classified based on their mechanism of action, chemical structure, or their activity patterns. Most antibacterials target bacterial functions or their growth processes. However, when an infection is composed of a variety of pathogens, the infection is termed as a mixed infection and the treatment regime needs to be modified. Typically, as when there is the presence of a gastrointestinal infection along with a pelvic infection, a formulation containing a combination of an antibacterial and antimicrobial is administered. Such combinations are given generally to target multiple pathogens. Combinations of antibacterials and antimicrobials have been generally administered as a physical mixture of the drugs to counter mixed infections. However, the vastly differing physicochemical properties of the constituents in the mixed formulations often lead to incoherent pharmacokinetic behaviour causing compromised inhibition. The physical mixture needs to be administered at high doses to get remedial action. Besides, the combination often acts with significant delay leading to deterioration of the condition. Salts like hydrochlorides, citrates, fumarates, and so on of BCS class IV or class III pharmaceuticals have been made in an attempt to increase solubility and bioavailabiIity of the pharmaceutical. Literature provides some examples regarding synthesis and usage of drugs in the form of co-crystals. EP2353594 relates to a compound of tramadol and a co-crystal of celecoxib ? L-proline. The invention is related to structural studies of the cocrystals. WO2012090224 relates to cocrystals of mesalamine. WO2007080362 relates to pharmaceutically acceptable co-crystalline forms of sildenafil with aspirin wherein the cocrystal of sildenafil is seen to enhance the solubility of the drug in the acidic conditions of the gastrointestinal tract and thereby enhance bioavailability. US20120258170 relates to pharmaceutical co-crystals of quercetin wherein the cocrystal of quercetin exhibited enhanced physical properties and bioavailaibity in comparison to the pure form of the of the drug. However, none of the examples mentioned contain an antibacterial -antimicrobial salt or solvate for the treatment of acute mixed infections a condition very common in poor rural populations .The present invention addresses such a need.

Summary of the invention:
Accordingly the present invention provides, a salt monohydrate of structure H formed from compound A and compound B ; wherein said compounds A and B are in a ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

H

A B
A method of preparation of a salt monohydrate of structure H formed from compound A and compound B comprising the acts of

H

A B
a) mixing compound A and compound B and ethanol to form a mixture;
b) grinding the mixture to obtain the salt monohydrate of compound A and compound B; and
c) obtaining a salt monohydrate of structure H.
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

A salt hydrate of formula H

H
A salt solvate of structure S formed from compound A and compound B ; wherein said compounds A and B are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

S

A B

A salt solvate of formula (S),

S
A method of preparation of a salt solvate structure S formed from compound A and compound B comprising acts of

A B
a) mixing compound A and compound B and methanol to form a mixture;
b) grinding the mixture to obtain the salt solvate of compound A and compound B; and
c) obtaining a salt solvate of structure S.

S
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B); and a composition of comprising pharmaceutically acceptable amount of salt hydrate of Norfloxacin and Sulfathiazole along with pharmaceutically acceptable amount of excipients in a pharmaceutically acceptable ratio.

Brief description of the drawings:

The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.
Figure 1: gives the graph for the powder X-Ray diffraction (PXRD) range for the salt hydrate of Norfloxacin and Sulfathiazole structure
Figure 2: the Single crystal X-Ray diffraction (SCXRD) shows the crystalline structure of the salt hydrate of Norfloxacin and Sulfathiazole as a single lattice
Figure 3: exhibits differential scanning calorimeter (DSC) endotherm and Thermogravimetric analysis (TGA) of Norfloxacin-Sulfathiazole salt hydrate showing a sharp melting endotherm at about 174 °C indicating a single homogeneous phase of the drug-drug salt.
Figure 4: shows the Fourier Transform infrared spectroscopy (FTIR) spectra of Norfloxacin-Sulfathiazole salt hydrate, the bands appearing at about 3600 cm-1 and about 3400 cm-1 corresponding to the NH and O-H stretch respectively.
Figure 5: shows the solubility and co-solvent solubility plots. Solubility of the salt hydrate Norfloxacin-Sulfathiazole in pH 7.4 buffer. (b) Solubility of the salt hydrate Norfloxacin-Sulfathiazole in cosolvent (10% ethanol +7.4 buffer).
Figure 6: shows the Diffusion study plots of the Norfloxacin-Sulfathiazole salt hydrate complex.
Figure 7: shows inhibition studies on Gram +ve, Gram –ve bateria and on aspergillus (images).
Figure 8: shows Hirshfeld interaction plots. (a) Hirshfeld 2D finger print plots of the interactions present in NF, ST and its salt hydrate(b) Contributions of the percentage of intermolecular contacts to the Hirshfeld surface area in Norfloxacin, Sulfathiazole and its salt hydrate. Percentages are given on the histogram only for the major atom type/atom type contacts.
Figure 9: shows the single crystal of Norfloxacin-Sulfathiazole-methanol solvate compound.
Figure 10: explains the PXRD plot for the salt hydrate of Norfloxacin-Sulfathiazole compound with Methanol solvent.
Figure 11: shows the DSC endotherm of Norfloxacin-Sulfathiazole compound with methanol solvent.
Figure 12: illustrates the FTIR spectra of the antibacterial-antimicrobial salt with the methanol solvent.

Detailed description of the invention:

The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the drawings, description and claims.

It may further be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.

Various embodiments of the present invention provide a salt monohydrate or methanol solvate composition comprising an antibacterial compound and an antimicrobial compound, characterized in that the antibacterial, antimicrobial compound comprises Norfloxacin and Sulfathiazole respectively said compound being used for the treatment of mixed infections. The invention also provides for a method of preparation of the salt monohydrate of Norfloxacin and Sulfathiazole and methanol solvate of Norfloxacin and Sulfathiazole.

The present invention is directed to a composition of a salt monohydrate and methanol solvate of Norfloxacin and Sulfathiazole, for its use in the treatment of mixed infections of bacteria and fungi. The composition is beneficial for the management of mixed infections through synergistic transport of the antibacterial and antimicrobial components. The invention also provides a method for the preparation thereof.

Norfloxacin, (NF) chemically known as 1-ethyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid, an antibacterial agent, belonging to the fluoroquinolones group of drugs, is commonly used in the treatment of both acute and chronic bacterial infections. It inhibits DNA gyrase thereby preventing the replication of bacterial DNA. They have a broad spectrum of activity against gram +ve as well as gram -ve bacteria.

Sulfathiazole (ST), chemically known as, 4-amino-N-(1,3-thiazol-2-yl)benzenesulfonamide, an antimicrobial agent. Sulfathiazole blocks the synthesis of dihydrofolic acid by inhibiting the enzyme dihydropteroate synthesis.

The present invention is related to a salt monohydrate of structure H formed from compound A and compound B ; wherein said compounds A and B are in a ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

H

A B

In another embodiment of the present invention, the salt monohydrate is a medicament for mixed infections of bacteria and microbes.

The present invention is also in relation to a method of preparation of a salt monohydrate of structure H formed from compound A and compound B comprising the acts of

A B

a) mixing compound A and compound B and ethanol to form a mixture;
b) grinding the mixture to obtain the salt monohydrate of compound A and compound B; and
c) obtaining a salt monohydrate of structure H.

H
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

In another embodiment of the present invention, the ratio of compound A and compound B is in the ratio 1:1 (w/w) of A:B.
In still another embodiment of the present invention, ethanol in the mixture is of volume ranging from about 1ml to about 15 ml.
In still another embodiment of the present invention, grinding on the mixture ranges for a period from about 30 seconds to about 30 minutes.
In another embodiment of the present invention, grinding is carried out at a temperature ranging from about 22 °C to about 35 °C.
The present invention is also in relation to a salt hydrate of formula H

H
In another embodiment of the present invention, the compound is characterised by PXRD pattern with peaks at 2? values of about, 23.76, 13.23, 26.07, 19.85, 14.69, 16.92, 21.09, 24.50, 28.43, 25.26, 27.99, and 28.87.
In still another embodiment of the present invention, the compound is characterised by FTIR peaks about 3600 cm-1, 3400 cm-1 and 1450 cm-1.
The present invention is also in relation to a salt solvate of structure S formed from compound A and compound B ; wherein said compounds A and B are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).

S

A B
The present invention is also in relation to a salt solvate of formula (S),

S
In another embodiment of the present invention, the salt solvate is characterised by PXRD pattern with peaks at 2? values of about, 23.77, 13.25, 26.08, 19.86, 16.95, 19.65, 24.53, 21.11, 28.46, 22.36, 28.00, and 25.31.
The present invention is also in relation to a method of preparation of a salt solvate structure S formed from compound A and compound B comprising acts of

A B
a) mixing compound A and compound B and methanol to form a mixture;
b) grinding the mixture to obtain the salt solvate of compound A and compound B; and
c) obtaining a salt solvate of structure S.

S
wherein said compounds are in ratio ranging from about 33% w/w (A/B) to about 67% w/w (A/B).
The present invention is also in relation to a composition of comprising pharmaceutically acceptable amount of salt hydrate of present invention along with pharmaceutically acceptable amount of excipients in a pharmaceutically acceptable ratio.

In another embodiment of the present invention the excipient is selected from group comprising gelling agents, tabletting agents, antioxidants, stabilizing agents, nanoparticles, nanocapsules, micellar and liposomal formulations.

In another embodiment, the antibacterial compound may include, but is not limited to, compounds such as ciprofloxacin, difloxacin, levofloxacin, lomefloxacin, nadifloxacin, ofloxacin, and the like.

In still another embodiment, the antimicrobial compound may include, but are not limited to, compounds such as metronidazole, tinidazole, ornidazole, nimorazole, secnidazole, azanidazole and the like.

In another embodiment of the invention, the preparation of the Norfloxacin- Sulfathiazole composition includes grinding the compounds of Norfloxacin and Sulfathiazole which together is termed the mixture, taken in a ratio of 1:1 (W/W) along with an organic solvent whose volume is ranging from about 1ml to about 15ml. Ratios of Norfloxacin: sulfathiazole may range from 33%NF/ST to about 67%NF/ST. The organic solvent used maybe selected from a group comprising ethanol, methanol, isopropanol, acetonitrile and the like. The mixture maybe ground for a period ranging from about 30 seconds to about 30 minutes until a homogenous mixture of the composition is obtained. During grinding, the temperature is maintained at a range from about 22°C to about 35°C. The ground mixtures is confirmed by analytical/spectroscopic methods like PXRD, SCXRD, spectral data, and DSC.
In an embodiment of the present invention a suitable concentration of actives in the composition i.e., Norfloxacin and Sulfathiazole may range from about 33-67% w/w of NF/ST. Other ranges may also be used to arrive at the composition of the invention.

In a preferred embodiment, the antibacterial-antimicrobial salt monohydate composition of Norfloxacin and Sulfathiazole exhibits enhanced solubility, flux and diffusion as compared to the parent compounds, Norfloxacin and Sulfathiazole. An embodiment of the invention is related to the striking synergistic and uniform diffusion, permeation and flux of the components in salt hydrate form. Another embodiment of the present invention is related to the exemplary inhibition studies of E. coli, staph aeureus and aspergillus species by the salt monohydrate compound.

Norfloxacin-Sulfathiazole salt monohydrate of the invention exhibits striking improvements in physicochemical properties compared to the parent drugs. In one embodiment, a significant enhancement in the buffer (pH:7.4) solubility of the dual-drug salt monohydrate of the invention is observed compared to the parent antibacterial or antimicrobial (Figure-5). In some embodiments, the solubility of the salt hydrate ranges from about 1000 mg/ml to about 1500mg/ml. In other embodiments solubility values range from about 800 to about 1300 mg/ml. In some embodiments, the solubility values range from about 900 mg/ml to about 1200 mg/ml. In some embodiments the solubility values of the salt monohydrate show an enhancement of 7 x of the parent Norfloxacin solubility. In other embodiments the solubility values show an enhancement of 9 x of the parent Norfloxacin solubility. The solubility enhancement seen in the studies for the salt monohydrate are about 3 x compared to the parent Sulfathiazole. In other embodiments the solubility enhancement seen in the studies for the monohydrate are about 4 x compared to the parent Sulfathiazole. Increase in solubility often leads to increase in bioavailability and higher drug concentration at the biological site of action. The diffusion (as measured in a Franz diffusion cell) of the salt monohydrate of the invention shows an increase of about 3x the release rate of Norfloxacin and in some embodiments and in others 4 x Norfloxacin. The diffusion rate of the salt is found to be more than double that of the antibacterial-antimicrobial mixture rate and sometimes even 3 times (Figure 6).
It is also found that the minimum inhibitory concentration on E. coli bacteria is reduced to half the concentration for the salt form compared to the physical mixture of antibacterial and antimicrobial. Further, the antibacterial activity (MIC) of the compounds of the invention has been analysed on microorganisms such as E. Coli. These salt monohydrate exhibit higher antibacterial activity even at lower concentration when compared to parent molecule thus indicating good synergy among parent antibacterial and the antimicrobial. In some embodiments the MIC values were half that seen for the parent antibacterial both for gram positive and gram negative bacterial types. In some other embodiments the MIC values (concentration of the drug) seen were about one third that for the parent antibacterial both for gram positive and gram negative bacterial types. In an embodiment the concentration is 1.6 µg/ml to about 0.4 0µg/ml which is half that found when a mixture of the antibacterial and antimicrobial is used (Table-1).
Table 1: Minimum inhibitory concentration (MIC) on E. Coli (Gram –ve) Bacteria ATCC 25922
Broth Control/ BLANK 0.0474
Culture Control- 105 cfu 0.9308
Sample/ Drug Concentration 1 mg/ml
Drug concentration
(µg/ml) 50
25
12.5 6.25 3.125 1.612 0.806 0.403 0.201 0.1007
NF-ST physical mixture
on E. Coli (Gram -ve) bacteria 0.0446 0.062 0.0438 0.0676 0.0709 0.0737 0.1882
(Broad bacteria growth observed) 0.3178 0.6581 0.7398
NF-ST Salt on E. Coli (Gram -ve) bacteria 0.0487 0.047 0.056 0.0546 0.0885 0.0858 0.0812 0.1495
(Broad bacteria growth observed) 0.3756 0.3576

It is also found that the minimum inhibitory concentration on staph. aureus bacteria is reduced to half the concentration for the salt form compared to the physical mixture of antibacterial and antimicrobial. In one embodiment the concentration is 3.1µg/ml to about 0.80µg/ml which is half that found when a mixture of the antibacterial and antimicrobial is used (Table-2).

Table: 2 Minimum inhibitory concentration (MIC) on Staphylococcus aureus (Gram +ve) Bacteria ATCC 29213.
Concentration: 1.8 mg/ml (NF: 1 mg + ST : 0.8 mg)
Drug concentration
(µg/ml) 50 25 12.5 6.25 3.125 1.62 0.806 0.403 0.201 0.1007
NF-ST physical mixture
on staph aureus (Gram +ve) bacteria 0.0896 0.0851 0.0868 0.0851 0.0793 0.1876
Broad bacteria growth observed 0.3356 0.4663 0.4011 0.3477
NF-ST Salt on staph aureus (Gram +ve) bacteria 0.0859 0.0873 0.0972 0.0939 0.0968 0.0992 0.2688
Broad bacteria growth observed 0.2774 0.2778 0.3403

The antimicrobial action seen against aspergillus is seen to be significantly higher for the salt monohydrate of the invention. Such an inhibitory action is noticed by taking comparative photographs as shown in Figure-7. The antimicrobial MIC seen against aspergillus is about 30% less for the salt hydrate of the invention when compared to the mixture. In some cases the reduction is about 40%.

Increase in solubility and diffusion may lead to increase in bioavailability and higher drug concentration at the biological site of action. A singular diffusion of the antibacterial-antimicrobial monohydrate salt which shows useparated components during diffusion is a very important aspect of the invention. Such singular, smooth and un-separated diffusion is often a very important observation and very often advantageous leading to synergistic transport through the gut wall. Such singular, smooth and un-separated diffusion is often advantageous leading to synergistic transport through bacterial cells or fungi. Such exemplary transport behavior of the inventive salt monohydrate is in contrast to separated diffusion patterns of a mixture of the parent antibacterial and antimicrobial compounds and the inventive salt monohydrate may be used to increase in inhibition on bacterial and fungal species typically found in mixed infections. In other embodiments the salt monohydrate may also be used to cause increase in inhibition behaviour on aerobic and anaerobic species that are often found in mixed infections.

In some embodiments, the salt hydrate composition maybe administered as an excipient selected from gelling agents, tableting agents, antioxidants, stabilizing agents, nanoparticles, nanocapsules, micellar and liposomal formulations.

In other embodiments, the salt solvate composition maybe administered as an excipient selected from gelling agents, tabletting agents, antioxidants, stabilizing agents, nanoparticles, nanocapsules, micellar and liposomal formulations.

The Norfloxacin-Sulfathiozole monohydrate salt of the present invention obtained during the experimentation process is identified using the PXRD pattern (Figure-1, 2 and Table 3).

Table -3: PXRD pattern of Norfloxacin-Sulfathiozole monohydrate salt.

Angle
(2 Theta) Relative intensity
9.8806 48.72
10.5975 26.38
10.9382 36.56
11.4417 31.01
12.9719 14.22
13.2338 65.27
14.6976 50.15
16.6813 22.65
16.9267 47.46
17.2398 12.17
18.5145 15.52
18.6847 13.8
19.6356 26.45
19.8509 58.05
21.0966 26.32
21.617 12.11
22.1731 13.37
22.3479 23.39
23.7658 100
24.5076 25.97
25.2654 16.41
25.4592 12.21
26.0706 61.45
27.9974 16.39
28.4347 23.48
28.8795 15.24
36.0054 13.13

The DSC studies (Figure- 3) also indicate the formation of salt hydrate as a single sharp peak at 174° C showing a single melting point. The FT-IR spectrum of the salt hydrate (Figure-4) with bands appearing at about 3600 cm-1 and about 3400 cm-1 corresponding to the NH and O-H stretch respectively. The structure of the salt monohydrate (H) is thus identified.

(H)
In another embodiment of the present invnetion Norfloxacin-Sulfathiozole methanol solvate is obtained. The structure of the methanol solvate (S) is identified by XRD (Figures 9 and 10), FTIR (Figure -12) and DSC studies (Figure 11).

(S)
Table 6: PXRD of Norfloxacin-Sulfathiazole solvate

Angle
(2 Theta) Relative intensity
23.7762 100
13.2588 67.29
26.082 62.52
19.8647 57.02
9.8954 51.87
14.7152 48.54
16.9576 46.12
10.9478 38.38
11.4538 33.15
10.6172 28.04
19.6586 27.44
24.5305 26.34
21.1115 24.9
28.4615 22.98
22.3617 22.57
16.7108 21.17
28.0003 15.96
18.5313 15.64
25.3138 15.23

Figure.8 depicts the Hirshfeld interaction plots. (a) Hirshfeld 2D finger print plots of the interactions present in NF, ST and its salt hydrate (b) Contributions of the percentage of intermolecular contacts to the Hirshfeld surface area in Norfloxacin, Sulfathiazole and its salt hydrate. Percentages are given on the histogram only for the major atom type/atom type contacts. The fingerprint plot indicates increase in NH and OH interactions and salt formation which causes exemplary physicochemical properties and inhibition.

Materials and methods:
Norfloxacin and Sulfathiazole are obtained from Sigma Aldrich Chemicals, Bangalore, India and used directly for experiments without further purification. All other reagents are purchased from commercial sources and were used directly. Melting points were measured on a Büchimelting point apparatus (Sigma Aldrich,Bangalore, India). Water filtered through a doubledistilled water purification system (Siemens, Ultra Clear, Germany) is used in all solubility/permeability experiments.

Instruments used for characterizations :Powder X-ray diffraction (PXRD) data is recorded using a Philips X’pert Pro X-ray powder diffractometer equipped with a X’cellerator detector at room temperature with the scan range 2?=5 to 40º and step size 0.017º. X'Pert High Score Plus is used to compare the experimental PXRD pattern of the salt with the calculated lines from the crystal structure. Fourier transform IR (FT-IR) spectra are recorded as KBr pellets with a Perkin–Elmer (UK) spectrophotometer (4000–400 cm-1). Differential scanning calorimetry (DSC) is performed on a Mettler Toledo DSC 822e instrument with heating rate 10 K min-1 over the temperature range 303– 573 K under an N2 atmosphere.
Single-crystal X-ray diffraction :
Single-crystal X-ray data for all co-crystals were collected on a Rigaku Mercury 375/M CCD (XtaLAB mini) diffractometer using graphite-monochromated Mo K_ radiation. H atoms were treated by a mixture of constrained and free refinement. H atoms bound to C atoms were placed geometrically and treated as riding, while H atoms bound to N and of the carboxylic acid groups are located in difference Fourier maps and refined with isotropic displacement parameters.
Experimental procedure for solubility and permeability studies
Solubility Study: The absorption coefficient of each NF, ST, and its salt is measured using the slope of absorbance vs concentration of the five known concentrated solutions in pH 7.4 phosphate buffer and in cosolvent (10% Ethanol+7.47.4 phosphate buffer) and measurements are done at 271-272 nm on a PerkinElmer UV-vis spectrometer. The solubility of each solid is measured at 24 h using the shake flask method.
Permeability/Diffusion/Flux Study: The diffusion studies of NF, ST and its salt is carried out using the modified Franz diffusion cell apparatus through a cellulose nitrate membrane (0.45 µm, 11306, Sartorius, Germany). The dialysis membrane is pretreated with 10% of NaHCO3 at 70 °C for 20 min to remove traces of sulfides, followed by 10 mM of EDTA at 70 °C for 20 min to remove the traces of heavy metal and 20 min of treatment with deionized water at 70 °C to remove glycerine followed by soaking the membrane in buffer for 15-20 minutes. The treated dialysis membrane is mounted in vertical static diffusion cells with an effective surface area of 4.5 cm2. The donor compartment contained 50 mg of the drug and its respective cocrystals, and these were suspended in 2 mL of pH 7.4 phosphate buffer. The receptor compartment is filled with 20 mL of phosphate buffer (pH 7.4), maintained at room temperature, and bubbles were removed. Receptor solution is magnetically stirred at 45 ± 5 rpm to ensure medium homogeneity throughout the duration of the experiment. An aliquot of 2 mL of the sample is withdrawn from the receptor compartment at predetermined time intervals and replaced with fresh medium. Diffusion study of NF, ST and its salt is carried out in triplicate. Samples were analyzed by UV-vis spectrophotometer at a ?max of 271-272 nm after suitable dilution.
Diffusion Measurements: The concentration of each of the component and the salt is measured at 1 h intervals for 12 h during the diffusion experiment in pH 7.4 phosphate buffer medium and recorded at 271-272 nm using a UV-vis spectrometer.
Experimental procedure for in-vitro biological studies
Minimum inhibition concentration (MIC): Bacterial MICs were determined using different drug concentrations by microdilution broth method with Mueller-Hinton broth and disc diffusion technique [ref]. Serial twofold dilutions were made by using 50 µlmicrodiluters. An equal volume of a suspension of organisms from a 24 h broth culture is added to each vial to a bacterial density of 105 CFU/ml. After 20 to 24 h of incubation at 37 °C, the MICs were determined as the lowest concentrations of antibiotic that completely inhibited growth using micro plate reader. Each experiment is repeated two times to find the final concentration for the MIC.
Antibacterial and Antimicrobial susceptibility test by Disc diffusion Technique
Disc impregnated with 25 µl of the samples for bacterial and 20 µl for microbial studies (salt and physical mixture) are placed on an agar plate that has been inoculated uniformly over the entire plate with a culture of the bacteria (E. Coli and Staph. Aureus and Aspergillus). The plates is labelled with the name of the culture, sample and stock at the middle of the first plate for anti bacterial test (E. Coli and Staph. aureus) and in the case of Aspergillus control is kept in the corner of disk. The treated disks were incubated for 18 to 24 hours at 37 oC and after incubation the diameter of the zone of inhibition of growth is measured for bacterial studies and in the case of fungi disks were incubated for 3-4 days at room temperature and after incubation the diameter of the zone of inhibition of growth is measured. During this period, the antimicrobial agent diffuses through the agar organism. Effectiveness of susceptibility is proportional to the diameter of the inhibition zone around the disc.
The following examples illustrate the methods of preparation of salt-monohydrate as well as salt solvate of Norfloxacin-Sulfathiazole compound:
and may prevent the growth of the
Example 1: 100mg of Norfloxacin and 80mg of Sulfathiazole are taken in mortar and pestle. About 2-6 ml of ethanol is added to it and the mixture is ground for 30s to 30 min to obtain a homogenous mixture of the salt hydrate.
Example 2: 120mg of Norfloxacin and 80mg of Sulfathiazole are taken in a mortar and pestle. About 2-6ml of ethanol is added to the mixture and the mixture is ground for 30s to 30 minutes to obtain a homogenous mixture of the salt hydrate
.Example 3: 80 mg of Norfloxacin and 100mg of Sulfathiazole are taken in a mortar and pestle. About 2-6ml of ethanol is added to the mixture and the mixture is ground for30s to 30 minutes to obtain a homogenous mixture of the salt hydrate.
Example 4: 80 mg of Norfloxacin and 120mg of Sulfathiazole are taken in a mortar and pestle. About 2-6ml of ethanol is added to the mixture and the mixture is ground for 30s to 30 minutes to obtain a homogenous mixture of the salt hydrate.
Example 5 : 100 mg Norfloxacin and 80 mg Sulfathiazole are taken in a mortar and pestle and the mixture ground using about 1-4 ml of methanol for about 30 seconds - 5 minutes to obtain Norfloxacin-Sulfathiazole methanol solvate.
Example-6 : 120 mg Norfloxacin and 80 mg Sulfathiazole are taken in a mortar and pestle and the mixture ground using about 1-5 ml of methanol for about 30 seconds - 10 minutes to obtain Norfloxacin-Sulfathiazole methanol solvate.
Example 7 : 100 mg Norfloxacin and 100 mg Sulfathiazole are taken in a mortar and pestle and the mixture ground using about 1-8 ml of methanol for about 30 seconds - 15 minutes to obtain Norfloxacin-Sulfathiazole methanol solvate.
Example 8 : 80 mg Norfloxacin and 100 mg Sulfathiazole are taken in a mortar and pestle and the mixture ground using about 1-10 ml of methanol for about 30 seconds – 20 minutes to obtain Norfloxacin-Sulfathiazole methanol solvate.
Example 9 : 80 mg Norfloxacin and 120 mg Sulfathiazole are taken in a mortar and pestle and the mixture ground using about 1-12 ml of methanol for about 30 seconds - 30 minutes to obtain Norfloxacin-Sulfathiazole methanol solvate.
The aforesaid description is enabled to capture the nature of the invention. It is to be noted however that the aforesaid description and appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention may admit other equally effective embodiments.
It is an object of the appended claims to cover all such variations and modifications as can come within the true spirit and scope of the invention.