Solid state characterization of E2101, a novel antispastic drug

E2101, a novel antispastic drug, was found to exist in at least two polymorphs that were confirmed by X-ray powder diffraction (XRD). These two species are designated forms I and II. The physicochemical and thermodynamic properties of these polymorphs were characterized by variable temperature XRD, thermal analysis, hygroscopicity measurements, and dissolution studies. The transition temperature was also estimated from the solubilities determined at various temperatures. The E2101 polymorphs were anhydrous and adsorbed little moisture under high humidity conditions. The melting onsets and heats of fusion for form I were 148.1 +/- 0.2 degrees C and 38.2 +/- 1.0 kJ/mol, respectively, and for form II were 139.8 +/- 0.4 degrees C and 35.2 +/- 0.5 kJ/mol, respectively. The intrinsic dissolution rate of form II in JP 2 medium was 1.5-fold faster than that of form I, corresponding to the rank order of the aqueous solubility and the enthalpy of fusion. Accordingly, form I was thought to be thermodynamically more stable than form II and thus suitable for further development. According to the thermal analysis and variable temperature XRD results, the recrystallization of form I occurred at approximately 145 degrees C after form II melted, however, no crystal transition behavior was observed below the lower melting point. The DSC thermograms at various heating rates and van't Hoff plots from the solubility studies indicated that the polymorphic pair would be monotropic.     

Energetic aspects of diclofenac acid in crystal modifications and in solutions--mechanism of solvation, partitioning and distribution

Temperature dependency of saturated vapor pressure and heat capacity for the diclofenac acid (Form II) were measured and thermodynamic functions of sublimation calculated (DeltaG(sub)(298) = 49.3 kJ x mol(-1); DeltaH(sub)(298) = 115.6 +/- 1.3 kJ x mol(-1); DeltaS(sub)(298) = 222 +/- 4 J x mol(-1) x K(-1)). Crystal polymorphic Forms I (P2(1)/c) and II (C2/c) of diclofenac acid have been prepared and characterized by X-ray diffraction experiments. The difference between crystal lattice energies of the two forms were obtained by solution calorimetry: DeltaDeltaH(sol)(I --> II) = 1.6 +/- 0.4 kJ x mol(-1). Temperature dependencies of the solubility in buffers with pH 2.0 and 7.4, n-octanol and n-hexane were measured. The thermodynamic functions of solubility, solvation, and transfer processes were deduced. Specific and non-specific solvation terms were distinguished using the transfer from the "inert" n-hexane to the other solvents. The transfer of diclofenac acid molecules from the buffers to n-octanol (partitioning and distribution) is an entropy driven process.     

Towards an understanding of the molecular mechanism of solvation of drug molecules: a thermodynamic approach by crystal lattice energy, sublimation, and solubility exemplified by hydroxybenzoic acids

Temperature dependencies of saturated vapor pressure and heat capacities for the 2-, 3-, and 4-hydroxybenzoic acids were measured and thermodynamic functions of sublimation calculated (2-hydroxybenzoic acid: DeltaG(sub) (298) = 38.5 kJ/mol; DeltaH(sub) (298) = 96.6 +/- 0.8 kJ/mol; DeltaS(sub) (298) = 191 +/- 3 J/mol . K; 3-hydroxybenzoic acid: DeltaG(sub) (298) = 50.6 kJ/mol; DeltaH(sub) (298) = 105.2 +/- 0.8 kJ/mol; DeltaS(sub) (298) = 180 +/- 2 J/mol . K; 4-hydroxybenzoic acid: DeltaG(sub) (298) = 55.0 kJ/mol; DeltaH(sub) (298) = 113.3 +/- 0.7 kJ/mol; DeltaS(sub) (298) = 193 +/- 2 J/mol . K). Analysis of crystal lattice packing energies based on geometry optimization of the molecules in the crystal using diffraction data and the program Dmol(3) was carried out. The energetic contributions of van der Waals, Coulombic, and hydrogen bond terms to the total packing energy were analyzed. The fraction of hydrogen bond energy in the packing energy increases as: 3-hydroxybenzoic (29.7%) < 2-hydroxybenzoic (34.7%) < 4-hydroxybenzoic acid (42.0%). Enthalpies of evaporation were estimated from enthalpies of sublimation and fusion. Temperature dependencies of the solubility in n-octanol and n-hexane were measured. The thermodynamic functions of solubility and solvation processes were deduced. Specific and nonspecific solvation terms were distinguished using the transfer from the "inert" n-hexane to the other solvents. The transfer of the molecules from water to n-octanol is enthalpy driven process.     

Estimation of initial dissolution rate of drug substance by thermal analysis: application for carbamazepine hydrate

We have proposed a theory indicating the correlation between the dissolution rate and the heat of solution of drug substances. The initial dissolution rates of the drug substances containing amorphous were predicted accurately from their heats of solution. In this report, the possibility for the theory to estimate the dissolution rates of hydrate and polymorphs was examined using thermal analysis. The initial dissolution rates of carbamazepine dihydrate and polymorphs (forms I, II, and III) were measured by the rotating disk method. The heats of solution and the heats of fusion of samples were measured by microcalorimetry and Differential Scanning Calorimetry (DSC), respectively. The logarithm of the initial dissolution rate of the sample was correlated linearly with the heat of fusion as well as the heat of solution. The obtained correlation would be applicable for the quality control of the drug substances the contained hydrate and/or polymorphic forms.     

Contribution of solid-state properties to the aqueous solubility of drugs.

This study investigates the influence of the solid-state properties melting point (T(m)), enthalpy of melting (DeltaH(m)) and entropy of melting (DeltaS(m)) of a drug on its intrinsic solubility (S(0)). For this purpose, 26 chemically and structurally diverse drugs covering the oral drug space were selected and the S(0), T(m), DeltaH(m) and DeltaS(m) were determined experimentally. The influence of T(m), DeltaH(m) and DeltaS(m) on S(0) was studied using regression analysis. The overall improvement of the predictions were 0.3 log units, however, five compounds (astemizole, glyburide, fenbufen, gliclazide and griseofulvin) were improved by more than one log unit. T(m) and DeltaH(m) had a larger effect than DeltaS(m) on the solubility predictions. The well-known general solubility equation (GSE) and the Dannenfelser semi-empirical equation for the calculation of DeltaS(m) were evaluated using our data set. While predictions of drug solubility obtained using the GSE were acceptable, the use of the experimental DeltaS(m) values instead of the constant 56.5 J mol(-1)K(-1) improved the accuracy of the prediction. The Dannenfelser equation underestimated the DeltaS(m) for most compounds with on average 15 J mol(-1)K(-1). Our results show that solid-state properties should be considered for improved performance of future models for prediction of drug solubility. In addition our study provides accurate experimental data on intrinsic solubility for 26 compounds, supplying a useful external data set for validation of drug solubility models.     

Determination of lignocaine and amprolium in pharmaceutical formulations using AAS.

The ion-associate complexes of lignocaine hydrochloride (Lig.Cl) with ammonium reineckate (Rk) or sodium cobaltithiocyanate, and that of amprolium hydrochloride (Amp.Cl) with ammonium reineckate, have been prepared. The precipitated ion-associates were subjected to elemental analyses, infrared and nuclear magnetic resonance spectroscopy and determination of the metal content for elucidation of their structures. The solubilities of the solid ion-associate complexes have been studied and their solubility products were determined at different temperatures at the optimum pH for their quantitative precipitation. The thermodynamic parameters DeltaH, DeltaG and DeltaS for the dissolution of the ion-associate complexes were calculated. These ion-associate complexes have been used for the quantitative determination of the above mentioned drugs by precipitating them with an excess of the inorganic metal complex ions and determining the excess metal complex ions using atomic absorption spectrometry. The method was applied for the determination of the above drugs in pure solution and pharmaceutical preparations. 0.135-135.4 and 0.158-157.6 mg of lignocaine and amprolium, respectively, can be determined with mean relative standard deviations (R.S.D.) 0.92-1.20% and recovery values of 99.18+/-0.48 to 100.12+/-0.34% indicating high precision and accuracy.     

Statistical properties of thermodynamic quantities for cyclodextrin complex formation

Literature values of DeltaG degrees (change in Gibbs free energy), DeltaH degrees (change in enthalpy), and TDeltaS degrees (temperature times change in entropy) for 1:1 complex formation by alpha-, beta-, and gamma-cyclodextrins constitute normally distributed populations with the following statistical parameters (all energy quantities in kcal mol(-1); n is the number of data points; mu is the population mean; sigma is the standard deviation): for alpha-cyclodextrin, n = 512, micro(DeltaG) = -2.85, sigma(DeltaG) = 1.23, micro(DeltaH) = -4.77, sigma(DeltaH) = 2.98, micro(TDeltaS) = -1.96, and sigma(TDeltaS) = 2.72; for beta-cyclodextrin, n = 415, micro(DeltaG) = -3.67, sigma(DeltaG) = 1. 37, micro(DeltaH) = -4.24, sigma(DeltaH) = 2.89, micro(DeltaS) = -0. 56, and sigma(TDeltaS) = 2.63; for gamma-cyclodextrin, n = 42, micro(DeltaG) = -3.71, sigma(DeltaG) = 1.19, micro(DeltaH) = -3.10, sigma(DeltaH) = 3.39, micro(TDeltaS) = +0.69, and sigma(TDeltaS) = 3. 29. The temperature is 298.15 K. The mean DeltaG degrees values correspond to binding constants of 123, 490, and 525 M(-1) for alpha-, beta-, and gamma-cyclodextrins, respectively.     

Polymorphism of paracetamol: relative stabilities of the monoclinic and orthorhombic phases inferred from topological pressure-temperature and temperature-volume phase diagrams

The thermodynamic relationships between the two known polymorphs of paracetamol have been investigated, and the subsequent pressure-temperature and temperature-volume phase diagrams were constructed using data from crystallographic and calorimetric measurements as a function of the temperature. Irrespective of temperature, monoclinic Form I and orthorhombic Form II are stable phases at ordinary and high pressures, respectively. The I and II phase regions in the pressure-temperature diagram are bordered by the I-II equilibrium curve, for which a negative slope (dp/dT approximately -0.3 MPa x K(-1)) was determined although it was not observed experimentally. This curve goes through the I-II-liquid triple point whose coordinates (p approximately 234 MPa, T approximately 505 K) correspond to the crossing point of the melting curves, for which dp/dT values of +3.75 MPa x K(-1) (I) and +3.14 MPa x K(-1) (II) were calculated from enthalpy and volume changes upon fusion. More generally, this case exemplifies how the stability hierarchy of polymorphs may be inferred from the difference in their sublimation curves, as topologically positioned with respect to each other, using the phase rule and simple inferences resorting to Gibbs equilibrium thermodynamics.     

Multimodal inclusion complexes between barbiturates and 2-hydroxypropyl-beta-cyclodextrin in aqueous solution: isothermal titration microcalorimetry, (13)C NMR spectrometry, and molecular dynamics simulation

Multiple types (structures) of inclusion complexes between barbiturates and 2-hydroxypropyl-beta-cyclodextrin (HPCD) were evaluated by isothermal titration microcalorimetry and (13)C NMR spectroscopy. The geometries of the inclusion complexes were suggested by molecular dynamics simulation. Barbituric acid (BA), barbital (B), amobarbital (AB), pentobarbital (PB), secobarbital (SB), cyclobarbital (CB), and phenobarbital (PHB) were used as barbiturates with different substituents on the barbituric acid ring and compared for inclusion types in aqueous solution. The association constants (K), stoichiometries, and thermodynamic parameters change in free energy (DeltaG) change in enthalpy (DeltaH), and change in entropy [DeltaS] for each type of complex were determined from the calorimetric data. The inclusion complexation was largely entropy driven because of hydrophobic interactions. The values of K increased in the order BA相似文献   

Estimating relative stability of polymorphs by generation of configurational free energy phase diagram

Relationship between two polymorphs is described to be either enantiotropic or monotropic with transition temperature/transition point (T(t) ) below the melting point (T(m) ) of the lower melting form in former case and above the T(m) of the higher melting form in latter case. In the present work, a new methodology for assessing thermodynamic T(t) of two polymorphs has been developed. Configurational free energy (G(c) ) of amorphous with respect to each polymorph was calculated to determine the T(t) . This method was used to determine the T(t) and polymorphic relationship of two model drugs, namely, carbamazepine and nateglinide. This method was also compared with the previous methodologies. Deduced T(t) of carbamazepine using this methodology was compared with previously reported values and was found to be in good agreement. A monotropic relationship was established between nateglinide polymorphs based on T(t) obtained by present methodology and previous reported methodologies.     

Binding thermodynamics at the human A(3) adenosine receptor

The thermodynamic parameters DeltaG , DeltaH and DeltaS of the binding equilibrium of six adenosine receptor agonists and five antagonists at adenosine A(3) receptors were determined by means of affinity measurements at six different temperatures (4, 10, 15, 20, 25 and 30) and van't Hoff plots were constructed. Affinity constants were measured on Chinese hamster ovary (CHO) cells transfected with the human A(3) receptors by inhibition assays of the binding of the selective A(3) antagonist [3H]MRE 3008F20. van't Hoff plots were linear for agonists and antagonists in the temperature range 4-30 degree. Their thermodynamic parameters fall in the ranges 21 < or = DeltaH < or = 67kJmol(-1) and 208 < or = DeltaS < or =410 J(Kmol)(-1) for agonists and -52 < or = DeltaH < or = -9 kJmol(-1) and 16 < or = DeltaS < or =81 J(K/mol)(-1) for antagonists, showing that agonist binding is always totally entropy-driven while antagonist binding is enthalpy- and entropy-driven. The results are discussed with the aim of obtaining new details on the nature of the forces driving the A(3) binding at a molecular level.     

Solvation and hydration characteristics of ibuprofen and acetylsalicylic acid

Ibuprofen and acetylsalicylic acid were studied by thermoanalytical methods: sublimation calorimetry, solution calorimetry, and with respect to solubility. Upon measuring the temperature dependences of the saturated vapor pressure, enthalpies of sublimation, DeltaH0sub, as well as the entropies of sublimation, DeltaS0sub, and their respective relative fractions in the total process were calculated. The Gibbs energy of solvation in aliphatic alcohols as well as the enthalpic and entropic fractions thereof were also studied and compared with the respective properties of model substances and other nonsteroidal antiinflammatory drugs (benzoic acid, diflunisal, flurbiprofen, ketoprofen, and naproxen). In all cases, enthalpy was found to be the driving force of the solvation process. Correlations were derived between Gibbs energy of solvation in octanol, DeltaGOct(solv), and the transfer Gibbs energy from water to octanol, DeltaG0tr. Influence of mutual octanol and water solubilities on the driving force of partitioning is discussed. An enthalpy-entropy-compensation effect in octanol was observed, and consequences of deviation from the general trend are also discussed.     

Characterization of polymorphs and solvates of 3-amino-1-(m-trifluoromethylphenyl)-6-methyl-1H-pyridazin-4-one.

The characterization of two polymorphs of the title compound (F2692; 1) by differential scanning calorimetry (DSC), microanalysis, proton nuclear magnetic resonance spectroscopy, thermogravimetry, thermomicroscopy, infrared spectroscopy, and X-ray diffractometry is described. Both polymorphs are crystalline, with form II being more stable at temperatures less than 160 degrees C. The thermal behavior was studied at different rates of heating, and the enthalpies of transition were calculated from DSC data. The transformation of aqueous suspensions of form I to the water-stable form II is described, and the heats of solution and intrinsic aqueous dissolution rates of both polymorphs were determined. 1 also formed solvates with dimethyl sulfoxide and 1-methyl-2-pyrrolidinone. The solvates were studied by thermogravimetry, DSC, and infrared spectroscopy.     

Physico-chemical characterisation of the modifications I and II of (R,S) propranolol hydrochloride: solubility and dissolution studies

The crystallisation conditions and the physicochemical properties of the modifications I and II of (R,S) propranolol hydrochloride were investigated. Detailed methods of preparation of the two forms were described. Data from FTIR spectroscopy, X-ray powder diffraction, thermal analysis, solubility and dissolution studies were used for the identification and the characterisation of the two forms. The forms I and II were easily differentiated by their IR spectra, X-ray patterns and thermal behaviour. The two polymorphs were found to be enantiotropically related to each other. Their stability was followed at room temperature over a period of 1 year and under different conditions of temperature, grinding and compression to verify the tendency to solid solid transition and to study the existence range of the two forms. The equilibrium solubilities of the two polymorphs in n-octanol were determined as well as their dissolution profiles as pellets in aqueous medium. These studies showed that form I, the less thermodynamically stable, was more soluble (by more than 34%) and dissolved faster than form II in agreement with the thermodynamic rules (A. Burger, R. Ramberger, Mikrochim. Acta II (1979) 259-271).     

Study of retinoic acid polymorphism

Some authors recently hypothesized the existence of a new retinoic acid (RA) phase in addition to the two already known polymorphs. We investigated RA polymorphism and our results exclude the presence of new modifications and refine the properties of the known forms. By comparison of simulated and acquired X-Ray Powder Diffraction (XRPD) it was possible to identify only the known monoclinic (I) and the triclinic (II) modifications; the same were also characterized by DSC, IR, and Raman spectroscopy. A solubility study associated to DSC allowed establishing an enantiotropic relationship between the two forms, with form II being less stable (DeltaGII/I=0.71 kJ/mol at 37 degrees C) below the transition temperature (136.6 degrees C; DeltaH=3.2 kJ/mol). The intrinsic dissolution rate (IDR) (I=61 microg/cm2xmin-1; II=125 microg/cm2xmin-1) confirmed this energetic relationship. The kinetics of solid transition I-->II was examined and its activation energy estimated (356 kJ/mol). The attempts to produce new phases allowed the development of methods to obtain the two polymorphs with high chemical and polymorphic purity. A validated DSC method is presented that enables detection of the presence of form I at a level of 1% (w/w) when in mixture with form II.     

Receptor binding thermodynamics at the neuronal nicotinic receptor

Simple determination of K(A) or K(D) values makes it possible to calculate the standard free energy DeltaG degrees = -RTlnK(A) = RT lnK(D)(T= 298.15 K) of the binding equilibrium but not that of its two components as defined by the Gibbs equation DeltaG degrees = DeltaH degrees - TDeltaS degrees where DeltaH degrees and DeltaS degrees are the equilibrium standard enthalpy and entropy, respectively. Recently, it has been shown that the relative DeltaH degrees and DeltaS degrees magnitudes can often give a simple "in vitro" way for discriminating "the effect", that is the manner in which the drug interferes with the signal transduction pathways. This particular effect, called "thermodynamic discrimination", results from the fact that binding of antagonists may be enthalpy-driven and that of agonists entropy-driven, or vice-versa. In the past, the thermodynamic discrimination was reported for the beta-adrenergic G-protein-coupled receptor (GPCR) and confirmed later for adenosine A(1), A(2A) and A(3) receptors. Moreover, it has been found that the binding of all ligand-gated ion-channel receptors (LGICR) investigated was thermodynamically discriminated. In particular, affinity constants for typical neuronal nicotinic receptor ligands were obtained by both saturation and inhibition experiments with the radioligand [(3)H]-cytisine, a ganglionic nicotinic agonist. Thermodynamic parameters indicated that agonistic binding was both enthalpy- and entropy-driven, while antagonistic binding was totally entropy-driven. These results have shown that neuronal nicotinic receptor agonists and antagonists were thermodynamically discriminated. On these grounds, the thermodynamic behaviour makes it possible to discriminate drug pharmacological profiles in vivo through binding experiments in vitro.     

Percutaneous absorption of cyclizine and its alkyl analogues.

Cyclizine (I) alkyl analogues (II-IV) were synthesized and their skin permeation parameters evaluated in vitro. It was hoped that these compounds would possess physicochemical properties more favourable for percutaneous delivery than (I). The identification and levels of purity for the compounds were confirmed by mass spectrometry (MS), nuclear magnetic resonance (NMR) spectrometry, and infrared spectrometry (IR) while melting points were determined by an electrothermal digital Bupsilonchi melting point apparatus. Aqueous solubilities (25 degrees C) and partition coefficients were determined and in vitro permeation studies were performed in buffer (37 degrees C) at pH 7.4 over a period of 24 h, using Franz diffusion cells fitted with human epidermal membranes. Generally, the analogues were more lipophilic, but nevertheless possessed higher aqueous solubilities as compared to (I). (II) and (IV) exhibited two- to three-fold increase in aqueous solubility and their melting temperatures dropped by more than 55 degrees C. Compound (III) had similar aqueous solubility to (I), but its melting point dropped by about 35 degrees C. Measured steady-state fluxes indicated that (II) is a far better penetrant (J=6.95 microg/cm(2)/h) of human epidermis than (I). Although fluxes of (III) and (IV) drop off markedly from that of (II), they remained above the flux of (I), which is (0.132 microg/cm(2)/h). In conclusion, (II) was the best skin permeant and also exhibited the highest aqueous solubility and lowest level of crystallinity as compared to (I) and other analogues. (III) and (IV) were more lipophilic. The overall permeation data of this series indicated that the more water-soluble and the lowest melting point compound was the best skin permeant.     

Solid-state characterization of fluconazole

Two polymorphs and three solvates of fluconazole were isolated and characterized by x-ray powder diffractometry, IR spectroscopy, differential scanning calorimetry (DSC), thermogravimetry, and their dissolution rates. The different forms were prepared by crystallization of the original powder in different solvents at different cooling rates. X-ray diffraction patterns of the five solid modifications exhibited substantial differences in both the intensity and position of the peaks. FTIR spectra of the five different solid-state modifications also exhibited differences in the peaks' positions and intensities. DSC thermogram of anhydrate form I showed a single melting point at 139.2 degrees C. Anhydrate form II showed two endothermic peaks at 136.5 and 139.2 degrees C and one exothermic peak in between. The DSC thermogram of acetone 1/4 solvate exhibited two endothermic peaks at 75.5 and 139.2 degrees C. Benzene 1/7 solvate exhibited two endothermic peaks at 131.5 and 138.8 degrees C. Hydrate E exhibited two endothermic peaks at 102.7 and 139.2 degrees C. The DSC thermogram of anhydrate form II showed that this form is sensitive to the application of a mechanical force. The solubility study showed that anhydrate form II and acetone 1/4 solvate have higher solubilities than anhydrate form I while benzene 1/7 solvate and monohydrate have lower solubilities than anhydrate form I. The intrinsic dissolution study confirmed these results.     

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

Pharmaceutical studies on the polymorphism of hydrochlorothiazide

Four polymorphic forms (I, II, III and IV) of hydrochlorothiazide have been characterized on the basis of x-ray diffractometry and differential thermal analysis. Form I was obtained by crystallization from N,N-dimethylformamide and Form II was crystallized from hot methanol. Form III was precipitated from sodium hydroxide aqueous solution by treatment with hydrochloric acid and Form IV was crystallized from 50% methanol. The metastable form I was a most stable form among four polymorphs, which was stable more than ten months at room temperature. The thermodynamic parameters such as heat of solution, enthalpy, entropy, free energy difference and transition temperature were determined by the measurement of intrinsic dissolution rate. The transition temperature and the heat of transition between the metastable Form I and Form II were determined to be 299.15°K and 5.03 Kcal/mole, respectively and free energy difference (δF) was 302.13 cal/mole. Diuretic action of these four polymorphic forms was also evaluated by monitoring the difference in urinary excretion of sodium, potassium and magnesium in rats.