Polymorphic Forms of Lamivudine : Characterization , Estimation of Transition Temperature , and Stability Studies by Thermodynamic and Spectroscopic Studies

The present study is focused on estimation of transition temperature and stability of various forms of lamivudine. The forms were recrystallized from variety of solvents and preliminarily identification on the basis of SEM revealed existence of three forms (Forms I, II, III). DSC scans of Forms I and III show that these are metastable and undergo heat mediated transformation to Form IH and Form IIIH, respectively. Form II is phase pure with single sharp melting endotherm at 178.6 ◦C. The thermal events are visually observed by hot stage microscopy. Enthalpy of solution of the forms is endothermic and magnitude varies in the order Form II > Form IL > Form IIIL suggesting Form IIIL to be least crystalline which is well correlated with XRPD data. The transition temperature of the polymorphic pairs IL/IH and IIIL/IIIH derived from enthalpy of solution and solubility data revealed monotropy whereas enantiotropy exists in IIIH/II. The slurry experiments showed Form II to be thermodynamically most stable. Forms IL and IIIL though stable in water are converted to Form II in ethanol, acetonitrile, and propanol after 1 day. Form IIIL is converted to Form IL in water after 7 days and the observation is of importance as this instability can effect the pharmaceutical preparations whereas Form IL shows a balance between stability and solubility.


Introduction
The increasing recognition of the importance of polymorphism to the pharmaceutical drug industry has generated a great deal of interest and the field has been evolving rapidly [1].In fact, the systematic evaluation and characterization of the polymorphic forms of the drugs is essential and mandatory because important properties such as solubility and drug stability depend on the solid state structure [2,3].The thermodynamic relationships between polymorphic pairs classified as monotropic or enantiotropic, first developed by Berger and Ramberger, is of utmost importance and must be established carefully and accurately because as drug crystallizes from different solvents the kinetically favoured metastable polymorph appears first which can be isolated and characterized and can be formulated under certain conditions [4][5][6].This metastable polymorph may undergo heat induced or solvent induced transformation to produce thermodynamically stable form at its own expense [6,7].
Therefore, a precise knowledge of the stability of crystal forms and their interrelationship is critical for formulation development [8].Thus, the objective of present work is to fully characterize the different polymorphic forms and to study the complex thermodynamic relationship between the polymorphic pairs of lamivudine.
Lamivudine (Figure 1), (2R, cis-4-amino-1-(2-hydroxymethyl-1, 3-oxathiolan-5-yl)-(1H)-pyrimidine-2-one, is a nucleoside reverse transcriptase inhibitor, used for the treatment of acquired immune deficiency syndrome (AIDS) [9].Lamivudine in combination with other retroviral agents is indicated for the treatment of HIV-1 infection [10].The solid state chemistry of this drug is of significant pharmaceutical interest as the drug is reported to exist in three crystalline forms.The two forms (Form I and II) reported in 1996 were again studied by Harris et al. in 1997 [11, 12].Later in 2007, a new patent showing the existence of another polymorphic form III has appeared [13].Unfortunately, all these studies lack in one or other aspect.The thermodynamic aspects of these polymorphs have not been taken into account and the transformation of one form to another has not been studied extensively.There are many lacunae in the reported literature which need to be filled for complete characterization of these polymorphs.Therefore, the purpose of the present work is to revisit these crystalline forms and the prime objective is to determine transition temperature of a polymorphic pairs and to interpret whether they are monotropically or enantiotropically related.The thermodynamic stability relationship of the polymorphic pair has also been determined from their melting temperature and enthalpy of solution data [14].Besides this, the heat mediated and solvent mediated transformations are also emphasized in the present work.

Preparation of Polymorphs.
The commercial sample of lamivudine was recrystallized from a variety of solvents and their mixtures to generate different crystal forms.Saturated solutions of lamivudine were prepared by heating in different solvents under constant stirring until most of lamivudine dissolved; the solutions were filtered and kept at room temperature until crystallization was complete (slow recrystallization).The further analysis of the recrystallized product by DSC and XRPD techniques showed that the crystallization ultimately resulted in only three polymorphic forms.Table 1 summarizes the different solvent systems leading to the formation of the three distinct crystal forms of lamivudine.

Thermal Methods of Analysis.
The DSC thermograms were obtained on DSC Q20, TA Instruments-Waters LLC, USA.The calorimeter was calibrated for temperature and heat flow accuracy using the melting of pure indium (mp 156.6 • C and ΔH of 25.45 J g −1 ).A mass between 2 and 8 mg was taken into the aluminium pan, covered with lid and sealed.DSC curves were obtained under a nitrogen purge of The TGA scans were obtained on TGA, TA Instruments-Waters LLC, USA.TGA traces were recorded at heating rates of 10 • per minute under a nitrogen purge of 50 mL per minute.Samples with masses between 1 and 10 mg were analyzed using platinum pan.Mass loss (%) was calculated based on the mass of the original sample.

X-Ray Powder Diffraction Analysis.
The powder diffraction patterns were recorded on an X-ray diffractometer (XPERT-PRO, PANalytical, Netherlands) with Cu as tube anode.The diffractograms were recorded under following conditions: voltage 40 kV, 35 mA, angular range 5, and fixed divergence slit.Approximately 200 mg of samples were loaded into the sample holder for analysis.

Scanning Electron Microscopy.
A Jeol JSM-6100 scanning electron microscope was used to obtain photomicrographs of lamivudine and its polymorphs.Samples were mounted on a metal stub with an adhesive tape and coated under vacuum with gold.

Microcalorimetric Study.
Calorimetric studies were performed on Microreaction calorimeter obtained from Thermal Hazards Technology, UK.Phosphate buffer (pH 7.0) was used to determine the enthalpy of solution and the measurements were performed at 25 • C and 37 • C. The size of sample used in this study ranged from 1 to 10 mg and was weighed (Sartorius Model CP225D) into a cylindrical glass tube covered with parafilm on one side.This cylindrical tube was submerged into the ampoule containing the solvent.A plunger with a cap was put from the open end of the tube.The same solvent was put into the reference ampoule.These were put into the sample and reference holes until both rest on the base of the holes.The parafilm was shattered mechanically by means of plunger.flask containing 10 mL of phosphate buffer pH 7.0 (50 mM).

Aqueous Solubility
The mixture was shaken at 37 • C for 24 h.The aliquots were filtered through 0.45 µm membrane filter and analyzed spectrophotometrically at 270 nm.The standard plot of lamivudine was prepared by dissolving a weighed amount of sample in phosphate buffer pH 7.0, suitably diluted and absorbance taken at wavelength 270 nm on a spectrophotometer.E 1% 1 cm was calculated.

Solvent Mediated Transformation Studies.
The stability relationships and transformation between polymorphs were assessed at 25 • C by slurry conversion method.Forms I L and III L and mixtures of these two forms prepared by mild blending of the solids in 1 : 1 ratio were suspended in water, ethanol, acetonitrile, and propanol and were maintained under constant shaking.To assess the conversion kinetics, the samples were withdrawn at 24 h and after 7 days.The suspensions were then filtered and analyzed by DSC and XRPD and compared with the starting material.

Results and Discussion
3.1.Recrystallization Product.The preliminary investigation of recrystallized product of lamivudine from various solvents by visual inspection and particle morphology by SEM categorized these into three distinct forms.The forms obtained from water, methanol, ethanol, 1,4-dioxane, ethyl acetate, mixture of toluene and ethanol, and a mixture of DMF and methanol showed elongated needle shaped acicular crystals and are designated as Form I (Figure 2(a)).The forms obtained from dry ethanol, dry methanol, 2-propanol, toluene, and a mixture of 1,4-dioxane, methanol, and water showed prismatic crystals indicating these to be similar and are designated as Form II (Figure 2(b)).Form III was obtained from chloroform, a mixture of heptane and 1butanol, and a mixture of hexane and propanol.The crystals of Form III were found to be very fine needles which were easily distinguishable from Form I (Figure 2(c)).Thus on the basis of morphology existence of only three new polymorphic forms was confirmed after screening from various solvents.existence of low melting metastable forms before they are converted into higher melting stable forms in Form I and III.Thermodynamic relationship between different phases (enantiotropic or monotropic) and transitions between different crystal forms are determined by employing these techniques.The hot stage microscope provides the visual confirmation of events occurring during the transformation of one form to another.

Thermal Analysis of Polymorphic
DSC scan of Form I showed two endotherms peaking at 117.5 • C and 178.2 • C separated by an exotherm at 123.8 • C (Figure 3).Prior to these events there is a very small endotherm in the temperature range 70-100 • C. The thermogravimetric scan confirms the presence of unbound water at a level of 1% by weight (corresponding approximately to 1 mole of water for every 10 moles of lamivudine) in the temperature range 70-100 • C (Figure 4).
A broad endotherm at 117.4 • C is not accompanied by any mass loss in TGA scan, suggesting this endotherm to be a melting event and negate the presence of any solvents.This melting is immediately followed by a small exotherm representing either a solid-solid transition or a liquid-solid transition.The exact nature of transition is confirmed by hot stage microscopy which shows that the crystals of Form I start melting at 118 • C; however, the new crystal form starts to grow at 124 • C from the melt of previous crystals.These crystals finally melt at 178 • C to give a sharp melting endotherm in the HSM images (Figure 5).These thermal events represent a liquid-solid transition to generate Form I H (high melting form) from the melt of Form I L (low melting form).This phenomenon suggests this conversion to be heat mediated transformation.The very low enthalpy of fusion indicates that the low melting endotherm is immediately or simultaneously accompanied by exothermic crystallization into other polymorphic form.The Form I prepared in the present study is comparable to some extent with Form I reported in literature [11,12].In both studies the forms showed endo-/exo-events followed by sharp melting endotherm.However, the solvent system used in our study varies from polar to nonpolar whereas the previous authors have used only polar solvents.Moreover, Harris et al. have shown the presence of bound water at a level of 2% by weight but in our study only unbound nonstoichiometric water is found to be associated.The thermodynamic relationship and the solvent mediated transformations of the forms have not been reported by previous workers and are a part of our work.All these parameters are dealt with in the present work.
Form II showed single sharp melting endotherm at 178.6 • C in DSC with no mass loss in the TGA scan up to 180 • C (Figure 4) indicating it to be a phase pure form which is neither solvate nor hydrate.The HSM images show melting around 179 • C (Figure 6).The melting temperature of Form II suggests this form to be thermodynamically more stable form.The literature survey reveals that a similar form with almost similar melting temperature exists; however, the crystallization solvents are different [12].Moreover, other parameters such as enthalpy of fusion and visual inspection of melting by HSM are not reported.
Regarding Form III, the preliminary investigation by DSC shows endo-/exopeaks as were observed in Form I.A  7).This form (Form III H ) finally melts at 175.8 • C. The melting temperature and heat of fusion of the higher melting Form I H is similar to the heat of fusion of Form II suggesting that Form I L might have converted to Form II after melting; however, the melting temperature and heat of fusion of the higher melting Form III H is different which suggests that Form III L has converted to a new polymorphic form after melting (Table 2).

Heat Mediated Transformation and Thermodynamic
Relationship.Table 2 shows that Forms I H or II and III H obtained after phase-phase transition (liquid-solid) of Forms I L and III L , respectively, show higher melting temperature and higher enthalpy of fusion suggesting existence of monotropic relationship between them.The polymorphic pair III H /II show enantiotropic transition as the high melting form has lower heat of fusion and the low melting form has higher heat of fusion.The liquid-solid transition as shown by HSM also confirms that the polymorphic pairs I L /II and III L /III H are monotropically related.To confirm the occurrence of heat mediated transformation of metastable Form I L and III L to stable forms as well as to determine whether the transition is reversible or irreversible a separate experiment was performed.Forms I and III were heated on a DSC furnace just above the end of crystallization exotherm, cooled to room temperature (28 • C), and heated again to the melting point.It was observed that the first endotherm at 117.4 • C in Form I and at 116.3 • C in Form III disappeared in the DSC graph of the repeated heating curve (Figure 8).The first endotherm corresponding to metastable form does not appear in the DSC scan even after 5 days.These heatingcooling experiments suggest kinetically irreversible transition confirming Forms I L /I H and III L /III H to be monotropes.

X-Ray Powder Diffraction (XRPD).
One of the reliable ways to distinguish the various forms of lamivudine is with   10).This supports the observation made by DSC that Form I L has converted to Form II and Form III L has converted to a new polymorphic form after heat mediated transformation.

Solution Calorimetry.
The enthalpy of solution has been utilized to differentiate the metastable Forms I L and III L from the stable high melting forms I H and III H .Moreover, this data is also required to determine the transition temperature for conversion of metastable forms to stable forms and to determine the stability relationship in a particular polymorphic pair.The molar enthalpy of solution (Δ sol H) determined in phosphate buffer pH 7.0 at 25 • C and 37 • C is given in Table 3.All the forms showed endothermic behavior and the absolute value of molar enthalpy of solution followed the order: Form II > Form I L > Form III L .The highest endothermic Δ sol H associated with Form II and this is very well correlated with XRPD studies.Form I L is less endothermic, less crystalline, and obviously less stable.These results are somewhat contrary to the results reported by Jozwiakowski et al.The authors have determined the Δ sol H of Forms I and II in various solvents ranging from water to alcohols and have shown Form II to be less endothermic  than Form I L whereas in the present study Form II is more endothermic.This variation may be due to the fact that Form I L is a hydrate while in the current work water is not incorporated into the crystal lattice of Form I L .The lowest Δ sol H for Form III L indicates this form to be least crystalline.The molar enthalpy of solution is increased with increase in the temperature (Table 3).The enthalpy of solution was also determined for the high melting Forms I H and III H .The molar enthalpy of solution was found to be more endothermic than their corresponding low melting forms at all the temperatures which is a consequence of lattice energy being more in the higher melting stable crystal form.Moreover, the magnitude of Δ sol H of Forms I H and II is comparable suggesting the similar nature of their crystal lattices.The results further suggest that Form III L is a new polymorphic form.

ΔH(T)
where Δ sol H L is the enthalpy of solution of lower melting form and Δ sol H H is the enthalpy of solution of higher melting form one has where S L is the solubility of low melting polymorph and S H is the solubility of high melting polymorph.
The entropy of transition between the two forms is calculated by the following equation: At the transition temperature, T t , Hence, The negative value of ΔG and ΔS for transition of I L to II indicates that higher melting forms have lower Gibb's free energy as well as lower entropy.However, the dominant factor in the free energy differences for all the polymorphic pairs at both temperatures is the difference in the enthalpy between these two phases.The transition temperature for polymorphic pairs I L /II and III L /III H was found to be higher than the melting temperature of both the forms suggesting them to be monotropically related whereas the transition temperature of polymorphic pair I H /II was between 0 K and melting point of lower melting form indicating this pair to be enantiotropically related.The previous section has shown that Form III L is most soluble which can be further used for formulation development.However, the most suitable form is the one which exhibits an appropriate balance between solubility and stability.Therefore, it is important to establish the stability of these forms.Thus the solvent mediated transformation experiments of forms (I L , II, and III L ) of lamivudine were performed in water, ethanol, acetonitrile, and propanol (Table 5).Form I L is stable in water but undergoes transformation into Form II when suspended in ethanol, acetonitrile, and propanol.On the other hand, mixture of Forms I L and II slowly converts to Form II after 7 days in these solvents.Moreover, water has no effect on Form III L up to 24 h but it undergoes a transformation to Form I L after 7 days.However, it transforms to stable Form II in ethanol, acetonitrile, and propanol.The analysis of forms recovered after suspending solvents revealed that Form I L is preferred over Form II in water whereas in ethanol, acetonitrile, and propanol Form II is the preferred form.

Conclusion
The DSC and SEM results suggest that Forms I and III in the present study are sensitive to thermal stress and the heat mediated transformation of these forms leads to generation of stable Form II and a new polymorphic form, respectively.The interpretation of the DSC data is facilitated by visual observations in thermal microscopy.Monotropic relationship has been identified in the polymorphic pairs I L /II and III L /III H while Form III H is shown to be enantiotropic modification of Form II.On the basis of comparison of differences between the enthalpies of solution and XRPD data, Form III L is found to be least crystalline and most soluble.However, the metastable Form I L , which shows better solubility than Form II, though transforms to Form II in solvents but is stable in aqueous suspension and can be categorized as suitable candidate for solid dosage.
Measurement.MSW-275 (Macro scientific works, New Delhi) shaker was used for measuring aqueous solubility of different forms of lamivudine.Solubility studies were performed by adding 10 mg of sample in

Figure 2 :
Figure 2: Scanning electron microscopy of (a) Form I, (b) Form II, and (c) Form III of lamivudine.

Figure 3 :
Figure 3: DSC plots of (a) Form I L , (b) Form II, and (c) Form III L of lamivudine.

Figure 4 :
Figure 4: TGA plots of (a) Form I L , (b) Form II, and (c) Form III L of lamivudine.

Figure 10 :
Figure 10: XRPD patterns of (a) Form I H and (b) Form III H of lamivudine.

Table 1 :
Different solvent systems to prepare different crystal forms of lamivudine.

Table 2 :
Thermal characteristics of Form I, Form II, Form II H , Form III L , and Form III H of lamivudine by differential scanning calorimetry.
• C suggests unbound solvent in nonstoichiometric ratio whereas the two endotherms peaking at 114.3 • C and 173.8 • C separated by an exothermic peak at 118.6 • C are suggestive of phase-phase transition.The HSM images confirmed it to be a liquid-solid transition to generate Form III H (high melting form) from the melt of Form III L (low melting form) (Figure

Table 3 :
Enthalpy of solution and solubility of Form I, Form II L , Form II H , Form III L , and Form III H of lamivudine.
upon heating.However, The XRPD pattern of Form III H showed appearance of new peaks at 8.3 • , 12.8 • , 19.0 • , and 29.6 • indicating formation of a new phase (Figure
3.5.Solubility Studies.Solubility of all the low melting and high melting forms was determined in phosphate buffer (pH 7.0) at 25 • C and 37 • C and is given in Table3.The solubility study suggests that Form II is least soluble while Form III L is most soluble.The solubility of Forms I H and II are almost identical indicating that they have similar free energies.These similarities agree well with thermoanalytical and XRPD data.The solubility of high melting forms (I H and III H ) is lower than their corresponding low melting forms; however, the solubility of Form III H is found to be still higher than Forms I H and II.3.6.Transition Temperature Determination.The transition temperature of polymorphic pairs I L /II, III L /III H , and III H /II has been estimated by utilizing solubility and enthalpy of solution data.The enthalpy of transition (ΔH(T)), free energy of transition (ΔG(T)), and entropy of transition (ΔS(T)) accompanying the transition of lower melting form to higher melting form are also determined at 25 • C and 37 • C using the following equations (Gu and Grant) and are given Table4one has.

Table 5 :
Stability of polymorphs by slurry conversion method.