FIELD OF THE INVENTION
The invention is directed to Lercanidipine Hydrochloride Novel Crystaline Polymorphic Forms (I) (IE) and Crude Forms (A) and (B) and Processes for their preparation. Pharmaceutical compositions comprising the novel crystalline forms are also contemplated.
BACKGROUND OF THE INVENTION
Lercanidipine (methyl l,l,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl 1, 4dihydro-2,6-dimethyl-4-(3-'nitrophenyl)pyridine-3,5-dicarboxylate) is a highly lipophilic dihydropyridine calcium antagonist witii long duration of action and high vascular selectivity. Its mechanism of antihypertensive activity is attributed to a direct relaxant effect on vascular smooth muscle, which lowers total peripheral resistance. The recommended starting dose of lercanidipine as monotherapy is 10 mg daily by oral route, with a drug titration as necessary to 20 mg daily. Lercanidipine is rapidly absorbed following oral administration with peak plasma levels occurring 2-3 hours following dosing. Elimination is essentially via the hepatic route.
By virtue of its high lipophilicity and high membrane coefiicient, lercanidipine combines a short plasma half life with a long duration of action. In fact, the preferential distribution of the drug into membranes of smooth muscle cells results in membrane-controlled pharmacokinetics characterized by a prolonged pharmacological effect. In

comparison to other calcium antagonists, lercanidipine is charactsiized by gradual ansst and long-lasting duration of action despite decreasing plasma levels. In vitro studies show that isolated rat aorta response to high K+ may be attenuated by lercanidisine, even after the drug has been removed from tiie environment of the aortic tissue for 6 bonrs.
Lercanidipioe is commercially available from Recordati S.pA. (Molan, Italy) and has been described along with methods for making it and resolving it into individual enantiomers in U.S. Patents 4,705,797; 5,767,136; 4,968,832; 5,912,351; and 5,696,139. A process for preparing lercanidipine described in U.S. Patent No. 4,705,797 involves the following scheme:



chromatography columns.
However, the isolation of iercanidipine hydrocbloride in crystalline form is again quite complex. After evaporating the solvent from the reaction mixture and dissolving the residue thus obtained in ethyl acetate, the solution is washed first -with brine, then washed further five times with a 10% solution of sodium carbonate, five times with IN hydrochloric acid, and eventually once again with brine.


authorities) that the product is always the same.
Further research by the present inventors revealed batch-to-batch differences in bioavailability in animals, and diSrerences in crystal size, ha ihe course of researching the causes of the variability problem, the inventors surprisingly discovered novel lercanidipine hydrochloride polymorphs. They also discovered more suitable processes for the preparation and isolation of crystalline lercanidipine hydrochloride products from the reaction mixture. It was surprisingly determined that lercanidipine hydrochloride shows polymorphic features and crystallizes into different crystalline forms depending on the process followed and on the solvents used. Furthermore, the isolation of each of individual crystalline polymorphs has become possible, thus decreasing the possibility of batch to batch variability of lercanidipins, which the present inventors deiermined was due to mixtures of different solid forms being present by the same batch and to such mixtures of


The present invention provides novel crude forms and crystalline farms of lercanidipine hydrochloride and processes for making them.
In one embodiment, the invention provides novel crude lercanidipine hydrochloride Form (A), which has a melting point of about 150-152°C (DSC peak) and comprises about 3-4% (w/w) ethyl acetate.
In another embodiment, the invention provides novel crude lercanidipine hydrochloride Form (B) which has a meltmg point of about 131-135°C (DSC peak) and comprises about 0.3-0.7% (w/w) ethyl acetate.
Methods are provided for the independent syntheses of crude lercanidipine hydrochloride Form (A) and crude lercanidipine hydrochloride Form (B), making possible to obtain each crude form in isolated form.
In a further embodiment, isolated lercanidipine hydrochloride crystalline Form (I) is provided which has the following X-ray diffraction pattern, at wavelengh Ka -wherein distances between peaks (D in X), relative intensity ratios (I/Io) ratios^ and angiles of significant peaks (28) are:


The lesrcanidipine hydrochloride crystalline Form (I) has a melting paint of about I97-2OPC5 when said melting point is determined as DSC peak.



Also provided are methods of syntheses in whihc each of isolated lercanidipine hydrochloride crystalline Form (I) and Form (II) may be obtained, indspsodenfly, from the starting material of lercanidipine hydrochloride crude Form (A) or crude Form (B).



BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of DSC analysis carried out on crystalline Farm (I), aocording to the working conditions described in Example 12. The ordinate indicates heat flow in mW and fee abscissa temperature in °C.



Figure 5 is a graph of solunility at 40°C of Fcrms (I) and (II) ia ethanol at
increasing water concentrations. The tests are described in Example 15. The ordinate indicates % solubility expressed as w/w and tiie abscissa % by weight of water in ethanol-
Figure 6 shows 13C NMR spectra in solid phase of crystalline Form (I). The signals
and attributes of the corresponding carbon atoms can be found in Table 4.



DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses novel crude forms and crystalline forms of lercamidipine hydrochloride and processes for making them. Applicants have detemined that lercanidipine hydrochloride exhibits polymorphism and crystallizes in different forms depending on the process followed and cm the solvents used, especially for crystallization.


Both lercanidipine hydrochlaride crystalline Fonns (I) and (II) exhibit good stability. Fonn. (I) is characterized by a paler yellow color, smaller crystal size, higher solubility in aqueous media (all compared to Fonn (II)), and a melting point (DSC peak)

within the rage of about 197°C to about 201°C, more specifically, about 198.7°C, and the X-ray diffraction pattern set forth, supra,
i?onn (IT) is characterized by a more pronounced yellow color, larger crystal size slightly lower solubility in aqueous media (all compared to Form (I)), and a melting point (DSC peak) wrfhin the range of about 207-211°C, more specifically about 209.3°C,
Both Form (I) and Form (II) are stable. Form II exhibited higher bioavailability in the dog, and was also non equivalent to form I in man, blowing a higher plasma concentration (AUCo-t) and a delayed time of maximum concenlxation (tmax), compared to Form([).
Previously known methods for prodncing crystalline lercanidipine hydrochloride were inconsistent in producing lercanidipine hydrochioride with predictable physical and chemical characteristics. Hence^ the previously known methods had the undesirable property of producing lercanidipine hydrochloride &at varied, eg., in physico-chemical pn^erties, fiom batch to batch, even among batches produced by the same process and under the same conditions. The present inventors have discovered that the source of inconsistency exhibited by the previously known methods of producing lercanidipine hydrochlcnide is the presence of varying and uzperedictable amounts of crystalline lercanidipme hydrochloride Form (EI). In constrast to previously known methods of producing lercanidipine hydrochloride, the invention provides the novel crystalline Forms (I) and (H) that repersent crystalline forms of lercanidipine hydrochloride of a purity and unifeamity that has not been obtained wifh previously adhfeved solid fbnns of lercanidipine hydrochloride.


farm with bath a rapid onset and sustained biological action.
As used herein, the term "crude form" refers to precipitated solid forms compising crystals of a compound that have not been washed and/or recrystallized to remove impunties (including but not limited to solvent) that may be present in the present specification, the crude forms are referred to as Forms (A) and (B) of lercanidipms hydrochloride.
As used herein, the teem "crystallme form" refers to crystals of a ccanpound that have been washed and recrystallized to remove inpurities- In the present znventian, the


essential hypertnsion, secondary hypertension, isolated systolic hyperteosion , coronary heart disease (e.g., chronic stable angina, myocardial infarction), congestive heart failure. A subject in need of treatment for arterial hypertension may be identified using methods



with thionyl cloride or oxalyl chloride in a mixture of an aprotic di[olar solvent azid of an aprotic polar solvent to yield a chloride compound and


ii) removing water from the reaction, mixture of step -i), preferably by azeotropic distillation under vacuum at 200-300 mmHg at a temperature below about 60°C Owsferably at 40-50°C);


is achieved and (2) a water-castent ,measured according to Karl Fischer, between 0.9 and 1,1% is obtained;



alternatively preferred solvent is a C1-C5 alcohol containing a maximum of 5% water, e.g., anhydrous ethanol Crystalline Form (I) may be added in step (e) as seeds to further promote crystal formation.


Process for M«^rfftg Fnrm dD
The second purification process (5 process), winoh yields crystalline Form (H), comprises the steps of:
d") adding acetonireile to crude lercanidipine hydrochlaride (Form (A) or Form (B)) arid heating the mixture under reflux and stining


first Alternative Process for Making Form (II)
The first alternative method compises the steps of:

Second Altemative Method for Making Form II
The second alternative method of obtaining the Form (II) polymorph comprises the
steps of


conditions 6! -f). In fact, the foregoing processes, especially the γ and δ processes can be used to produce the desired polymorph reproducibly and consistently.


The invention also contemplates a method of treating and preventing atherosclerotic lesions in arteries of a subject, the method comprisingadministeing a therapeutically

effective amount of isolated lercamdipine irydrocbioride crystalline Farm (I), isolated lercarddipine hydrochloride crystalline Form (II), or comhinations therof to a subject in need of such treatment Pharmaceutical Compositions



substancss, and the like.
Suitable dispersing and suspending agents include, but are not limited to, synthetic and natural gums, soch as vegetable gum, txagacanth, acacia, alginate, dextran, sodiun
carboxymethylcellulose, methylcellulose, polyvinyi-pynolidone and gelatin.


Suitabis edibie oils include, but are not limited to, cottansesd oil, sesame oil, coconut oil and peanut oil
P.-ramplsR of additional additives include, but are not limited to, sorbitol, talc,
stearic acid, dicaldum phosphate and polydesctrose.



For liquid dosage forms, the active substaacss or their physiologically acceptable salts are brought into solution, suspension or emulsion, optionally with the usually employed sobstances such as solubilizers, emusifiers or other auxiliaries. Solvents for the


The polymorphs of the present invention also may be couples with soluble •polymers as targdable drug carriers. Such polymers include, but are not limited to, polyvinyi-pyrrolidone, pyran copolymer, polydroxyprqpylmethacryl-amidephenol.


The phamaceutical composition or unit dosage forms of the present inventioii maybe administered by a variety of routes such as intevenous, intratracheal, subcutaneous, oral, mucosal parenteral, buccal, sublingual, ophthalmic, puhnonary, transmucosal, transdermal, and intramuscular. Unit dosage farms also can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches known to those of ordimary skill in the art Oral



Far combination therapy the compounds may initially be provided as separate dosage forms until an optimum dosage combination and administration regimen is


IN a preferred embodiment of the present invention, the composition is administered daily to the patient Preferably in said embodiment, the pharmaceotical compoosition has


was then added, the whole was stirred for 30 min and after setting the aqueous phase was discarded The organic phase was washed again wife water (340 ml).


filtered and dried in an oven at 70°C, thus obtarning 133 g of crode lercanidipine hydrochloride Form (B), DSC peak 13M35°C; 75% yieli


then temperature was set at 30°C, and stirring was continued at said temperture for another 24 hours. The solid was filtered at 30°C and washed with 50 ml of 2-propanol, then dried

in an over at 70°C undsr vacumn for 24 hours. Weigh of dry prodnct in sach base was (lercanidipme HCl (I)) 90 g (HPLC purity of the product in Form (I) > 99.5%).


far 24 hours. 20.2 kg of product was obtained, corresponding to a yield of 81%; HPLC purity in Form (I) > 99,5%. comparable results are obtained with Form (C) as starting material.



Watts, always at 25°C. Whichever the choice, after 48 hours under stirring the precqritate thus fonned was collected and dried, with yields of 80-85% with respect to stoichiotEietiic


hours at lO°C. Comparable results axe obtained using crude Forms (A) or (B) or lercanidipine hydrochlaride crystalline Form (I) as starting material


EXAMPLE 12 DSC analysis of lercanidipine hydrochioride crystalline Forms (I) and
(II)
DSC analysis measures changes that occur in a given sample with heating, wherein the changes identify transition phases. Enthalpy variations taking place in a transition
phase are calculated on the basis of the area under the curve. The most conunon transition phases are melting and sublimation. The temperature at which transition starts, onset T, is given by the point in which the curve starts to deviate fom the base line (flex point).
DSC of Form (I): 3.8 mg of Form (I) was placed in a golden pan of the apparatus Perkin Elmer DSC7, The heating speed durng the test was 1 Q°C/min
DSC Form (11): 4.6 mg of Form (II) was placed in a golden pan of the apparatus Perkin Ehner DSC7. The heating speed during the test was 1 O°C/min.
The data are shown in Figures 1 and 2 and the characteristic points of the ngures are briefly summarized in the following Table 1,


The results obtained wife crude lercaniciipine hydrochloride Form (A) are shown in Figure 19, where a weight loss of 3.4% can be observed in the temperature range 25-153**C, The volatile compound has been identified by its corresponding IR spectrum and is efcyl

acetate. During degradation (T > 170°C a small amount of ethyl acetate in gas phase could be observed
The results obtained with crude lercanidipine hydrochloride Form (B) are shown in Figure 20, where a weigh loss of 0.5% in tempsrature range 25-153°C can be observed.


The exposure of Form (II) to humidity in DVS causes a negligible mass vaiiatioii f< 0:05%) in the whole RH range tested.
EXAMPLE 15 Solubility of crystalline Forms (I) and (II)
15 .1 Solubility in -water and in ethanol at room temperature
The solubility at 23°C of both crystalline Forms (I) and (II) was evaluated by UV-Visible spectroscopy in bi-distilled water (at the pH value spontaneously reached by the system) and in absolute ethanol. The molar absorptivity had been previously determined in acetonitrile. The same molar absotptivity was considered for the deteamination in water and in ethanol. Solubility in water certainly depends on pH The residual solid obtained by filtration of the suspension was immediately analyzed with Raman spectroscopy. The results are shown in the following Tables 2 and 3.


Form (IT) is less soluble than Form (T) in both solvents. 15.2 Solubility in mixtures of water-ethanol at 25°C and at 40°C, with increasing water


' Total suppression of sidebands(TOSS). TOSS was psrfonned using spin-echose synchronized with the rotation of the sample to cause phase alteration of the spinning sidebands, resulting in cancellation when successive spectra were added together.
Crystalline Fonns (I) and (II) show diFFERENT 13C-NMR spectra in solid phase. The signals (chemical shift ) and attribution of the corresponding carbon atoms (as numbered in the formula of lercanidipine hydrochloride shown below) are represented in the following Tables 4 and 5, respectively.

EXAMPLE 17 IR Stadies
The IR. (infrared). spectra were recorded in KBr powder by Diffuse Refiectance Technique using a Pe±m Elmer Spectrum-one instrument IR spectra, whose wave lengths and corresponding attribution are shown in the following Tables 6 and 7, are clearly different for the new Forms (T) and (II).


EXAMPLE 18: Raman Spectra
A Bruker FT-Raman RFSIOO Spectrophotometer was utilized under the following -typical-conditions: about 10 mg sampie (without any previous treatment), 64 scans 2 cm-1 resolution 100 mW laser power, Ge-detector,
The following Tables 8 and 9 show the most significant peaks of Raman spectra of Form (I) and Form (E), respectively.



EXAMPLE 19 BioavaSability of crystalline Forms (I) and'(II) Example 19a-Dog
A study was carried out on six Beagie dogs to evaluate the bioavailability of crystalline Forms (I) and (E).
The products, in micronized farm, were administered orally by hard gelatin capsules filled 15) with the active agrent, Form (I) and (IT), at a dosage of 3 mg/kg, administered once in the morning of the day of the experiment

Blood samples were taken at given times and plasma concentrations of lercanidipine were determined with a stereoselective analytical method HPLC-MS/MS, according to the —following-working conditions;
Lercanidipine was extracted from dog plasma by means of a liquid-Iiquid extraction with a mixture of n-hexane and ethyl ether. The dry residne of the organic phase was taken up with a mixture of methanol and water and a liquid-phase chromatograhic separation (LC) was carried old; the two enandamets of lercatddipine were separated an a CHIROBIOTIC V column (Vancomycin) (particle size 5 m, column size 150 x 4.6 mm (ASTEC, NJ, USA)) and were detected with a mass spectrometer (MS/MS) by nsiog an electrospray technique..
The analytical method was validated in a concentration range between 0.1 and 20 ng/ml of plasma for both enantiomers. The method has shown to be specific with an accuracy of 15%. The average concentrations of lercanidipine in the table represent the sum of both enantiomers.
The profiles referring to the average cancentrations of lercanidipine for both forms are shown in Figure 10. The following Tables 10 and 11 show single values referring to AUC, Tmax, Cmax and to plasma concentrations.

TABLE 10. Mean values (n=5) of AUCo Cmax and Tmax of learcanidipne hydrochloride (S+R) crystalline Form (I) and crystalline Fonn (II), in dogs, after oral administration at a


Table 11. Average concentration in plasma of lercanidipine hydrochloride (S+R) crystalline Form (I) and crystalline Form (IT), in dogs, after oral administration at a dosage


The frrmulation containinng Form (II) is more bioavailable than the one containing crystalline Form (I) in 5 animals out of 6.


same composition. of Zanedip R 10mg, starting from micronized Form (IT) having the same particle size of Fonn I (Test-T). Blood samples were taken at 15 points from time 0 to 24 h post-dosing and plasma concentrationsof lercanidipine were determine with a stereoselective analytical method HPLC-MS/MS. The pharmacokinetic parameters obtained are given in the following table


(without aoy previous treatment) with application of a slight pressure to obtain a fiat surface, Ambient air atmosphere. O.O2°2θ stepsi2e,2 secstep-l5 2-5O28.
The obtainsd spectra are given, in Figures 11 and 12 and the corresponding main
peaks are dsscribed in Tables 12 and 13. The data are clearly diSerent for new isolated Forms (I) and (II).


EXAMPLE 21 Melting point determination of various mixtures of lercandipine hydrochloride-crystalline Forms (I) and (II)
The melting points of compositions consisting pf known ratios of lercanidipine hydrochloride crystalline Fonns (I) and (II) were determined manually. Conditions cansisted of using a set point of 177°C and introducing the cspillary into the instrument (Melting Point Apparatus model 535, Buchi Labortechmk AG, Flawil, Switzerland) at approximately 5°C below die melting point Results are shown in Table 14.


melting point of 186-188°C (non DSC) for a lercamidipine product they characterize as "crystals". Hence, the melting point of a preparation of lercanidipine hydrochloride is not sufficient-by-itself to- distinguish the particular-form or forms -present-therein, and many mixtures of different compositions have the same melting point range,
EXAMPLE 22. Micronization of lercanidipine hydrochloride.
Mironization is carried out by a jet-mill process using a MICRONETTE KBOO
from the firm NUOVA GUSEO (Villanova sull'Axda -PC- Italy). Parameters are as
follows: Injection pressure, 5 Kg/cmq; micronization pressure, 9 Kg/cmq; and cyclone
pressure, 2.5 Kg/cmq. Capacity of microziization is 16 Kg/L Particle size is determined by
laser light scattering using a GALAI CIS 1 laser instrument (GALAI, Haife Israel).
Micronization is performed to obtain an average particle size of D (50%) 2-8 µm and D
(90%) < 15 pm.
The present invention is not to be limited in scope by the specific anbodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applicadons, publications, procedures, and the like are cited throughout this application, the disclosures of whihc are incorporated herein by reference in their entireties.

WE CLAIM:
1. A method of producing lercanidipine hydrochloride crystalline Form (I), which
comprises:
d) adding isopropanol containing a maximum of 5% water (v/v) to a crude lercanidipine hydrochloride Form and heating under reflux and with stirring to produce a clear solution;
e) cooling the solution of step d) optionally adding crystalline seeds of the form (I) and stirring until the concentration of lercanidipine hydrochloride dissolved in the crystallization solvent is < 2% at a temperature of from 30 to 40°C; and
f) recovering the solid obtained from step e), and drying said solid to produce the lercanidipine hydrochloride crystalline Form (I).

2. The method as claimed in claim 1, wherein the temperature of step (e) is comprised between 30 and 40°C,
3. The method as claimed in claim 1, wherein step f) comprises filtering the solid obtained from step e), washing the solid with isopropanol and re-filtering the solid before drying.

4. The method as claimed in claim 1, wherein the crude Form is lercanidipine hydrochloride crude Form (A), lercanidipine hydrochloride crude Form (B) or lercanidipine crude Form (C)
5. The method as claimed in claim 1 wherein said step d) further comprises filtering the heated solution.

6. The method as claimed in claim 1 wherein said step e) further comprises stirring for a period of time of 12-48 hours.
7. The method as claimed in claim 1 wherein said drying in step f) takes place in an oven.
8. A method for producing lercanidipine hydrochloride crystalline form (I) in ethanol comprising the following steps:
d') preparing a mixture of lercanidipine hydrochloride and ethanol in a weight/ volume ratio within the range of about 1:4 to 1:6, refluxing under stirring and cooling and adding crystalline seeds of Form (I);
e') further cooling the seeded mixture of step d') up to a temperature between 10 and 15°C and stirring until the concentration of lercanidipine hydrochloride dissolved in the crystallization solvent is < 2%; and
f) recovering the solid of step e') to form lercanidipine hydrochloride Form
(D-
9. The method as claimed in claim 8, wherein said ratio is 1:4
10. The method as claimed in claim 8 wherein in step (e') the mixture is cooled to
20°C.
11. The method as claimed in claim 8 wherein said step d') further comprises filtering the heated solution.
12. The method as claimed in claim 8 wherein the drying in said step f) takes place in
anovenat70°C..

13. The method as claimed in claim 8 wherein authentic seeds of lercanidipine Form (I) are added at the end of cooling in steps e') and d').