AU4478399A - Enhancement of the efficacy of drugs by deuteration - Google Patents

Description

1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

2 Name of Applicant/s: Actual Inventor/s: Address for Service: Invention Title: Isotechnika Inc.

Robert R Foster and Richard Lewanczuk and Gilles Caille BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 'ENHANCEMENT OF THE EFFICACY OF DRUGS BY

DEUTERATION

Details of Original Application No. 19441/95 dated 27 MAR 1995 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 22414.00 la ENHANCEMENT OF THE EFFICACY OF DRUGS BY DEUTERATION BACKGROUND OF THE INVENTION The present invention relates to a process for enhancing the efficacy of known pharmaceuticals or drugs, and to the enhanced drugs so produced, by changing the isotopic form of the molecular structure of the known drug. More particularly, the present invention relates to the modification of the molecular structure of known drugs containing one or more hydrogen atoms by deuterating one or more of the hydrogen atoms to deuterium atoms. The resulting drug is significantly altered and has greatly improved activity over the known drug. Most particularly this invention relates to a method of deuterating a dihydropyridine (eg, nifedipine) whereby the deuterated nifedipine has an increased hypotensive effect and an increased duration of action on mammals at lower concentration than does nifedipine.

W" hen pharmaceuticals are synthesized, a carbon back-bone is assembled having various substituents including carbon, hydrogen, oxygen, nitrogen, etc. Pharmaceuticals have been designed and synethesized by a number of modes including, for example, serendipity and molecular modification. These and other methods have generated a vast number of drugs over Sthe course of time. As such modifications have allowed individual companies to keep a competitive edge in the marketplace, a -2 sihnfant part of the industry's time and rsource is went wrChing for novel asezi, within =tain haaolgic claztihfictions, antihypertamsves. Such novel xgents of'=n IM! diftent actvitie from the prototype compounds, thus justifying the monies smat ftx heirdelan It is bwwn that virtUay al drugs now mau1d inchud a number oa hydrogen 'toms, each of which bas a inolecuiar mas ofone. It has now been found that when Oft Or mor of the hydroge aMoms on a drug anrn odified so that their molular mass b,=eomu two, the activity of the drug ii sivgnicantly altered and is even greefly improv44. Thug, for enampl, izotopc modiciann of dihydropyridine, such as Uifediae, has resulted in an unxece change in the hypoeensive (blood presur in mammals cmpared to nifedpine per se, and much effects shoul also be achieved with humans.

:.Nifiedipi is makmd worldwide as aM mportt drug used in the tZ3Md (if aniaadhpgu~LIs ucture i as lk 20 13y modfyig nifeip by replacing one or more hydrog=i of the Inethyl. JTOMS with daitermm or by rqplcing aor mome of the methyl wit CD3, the. theruent properdie Of niftiin can be altmvd and can even be stinfnly iM aio For example, by modifyin the nifdipin by replacig the two Methyl Stups al the 2 and *6 posddons an the fing with two deutwated poupi La., -3replacing 6 hydrogen atoms with six deuterium atoms, the structure of the deuterated nifedipine is as follows:

D

3 CC

CD

3

II

CH

3 0OC

COOCH

3

H

NO

2 I Both of the above molecules are nifedipine and the latter structure is an isotopic form of the former.

According to a first aspect the invention consists in a calcium channel blocking compound having one or more deuterium atoms and wherein the compound has an extended duration of action and/or an enhanced use dependency when compared with a compound that is identical 10 except in that the one or more deuterium atoms are each substituted by hydrogen.

According to a second aspect the invention consists in a method of extending the duration of action of a calcium channel blocking hydrocarbon compound including the step of replacing at least one hydrogen atom of the calcium channel blocking compound with a deuterium atom.

15 According to a third aspect the invention consists in a method of enhancing the use dependency of a calcium channel blocking hydrocarbon compound including the step of a replacing at least one hydrogen atom of a calcium channel blocking compound with a deuterium atom.

According to a fourth aspect the invention consists in a method of modifying the dose-response relationship of a calcium channel blocking hydrocarbon compound 3a comprising the step of replacing at least one hydrogen atom of the calcium channel blocking compound with a deuterium atom.

According to a fifth aspect the invention consists in a method of detecting whether a pharmaceutical compound is identical and/or bioequivalent to a known pharmaceutical compound comprising the steps of: determining the molecular and isotopic structure of said known pharmaceutical compound by gas-chromatography-isotope ratio mass spectrometry.

determining the molecular and isotopic structure of said pharmaceutical compound subject to said detection by gas chromatography-isotope ratio mass spectrometry and comparing the results of said two determinations to detect any isotope variation in the molecular structure of said pharmaceutical compound over that of the known pharmaceutical compound.

So -3b- Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

BRIEF DESCRIPTION OF THE DRAWING The present invention will be further understood with reference to the drawings, wherein: Figure 1 shows the hypotensive effect of the various concentrations of the deuterated nifedipines on the treated rats as compared with nifedipine per se; Figures 2 and 3 show use dependent inhibition of control nifedipine (Nifedipine B) and deuterated nifedipine (Nifedipine D) on T type calcium channels; Figure 4 shows the effect of control nifedipine and deuterated nifedipine on calcium current inhibition as a function of pulse frequency; Figure 5 shows the effect of control nifedipine and deuterated nifedipine on use dependency; S 15 Figures 6(a) and show the effect of control and deuterated nifedipine on mean arterial pressure; Figures 7(a) and show concentration-effect relationships for control (Figure and deuterated (Figure nifedipine fitted using an asymmetric sigmoidal model;

C

-4 Figues 8(a) and show n-fetx~laticathips fo cntrol (Fiur 11(ap) and dztered (Figure Ilifcdipiwn lind Using logistic dose meponse MOdW; Figures 9a and 9b sbow fte dose-response effct for the control and deutauzd t) nicaripiees; shows th dufmio of effec was pester fbr doaterAW ni~dpine compared to control nirdip n; Figure 11 shows the hypoteadsve effect of the various cntatioa of the demmed vapmi an the btd ram~ as comrd with Verapsiil per se Figure 12 shows the duration of efec was grm=e for deuteated veapm mppared to control verapainil; Figur 13 shows a thre dimensional Ifingerprinz' of seven nifedipime IF*=u 14 shows a two dimensional mfingcrpdiin of nine nifedipine PZSTCh±ats; ~Figure 15 shows a two dimnsona (oxygn vs. carbon) 'fngerptint- of molatol crude ublets; a a Figure 16 shows "fingerpintso of tr diffmes hair samples from three diffaent pmplce and Figure 17 shows a two diindzonal carbom-oxyg Ofigeprjtu of thre a. 1iquRorL Such modficatio of nihxdh*n (and other dihyroyICSn such as, for ezamoe, si=r#Aw drit~i~ ine, niaiaol ~sdt, aitumdiomn, ftkkftm, ifradipine and amlodipine) can be achieved by dissolving the nifedipine in a deuteration tube in a mixture of deuterochloroform and deuterium oxide, and then adding a minor amount of trifluoroacetic anhydride and deuteroacetone thereto and mixing therewith. The solution is then frozen within the tube, preferably by immersing the tube in liquid nitrogen and then sealing the tube. The sealed tube is then heated at a temperature within the range of from about 500 to about 0 C, and preferably within the range of about 550 to about 60 0 C and maintained at that temperature for a period of time sufficient to deuterate the methyl group at the 2 and 6 positions on the nifedipine to CD 3 A time of from about 150 to about 180 hours is effective to complete the reaction, with a time of about 160 to 170 hours being preferred.

Deuterated nifedipine was synthesized in the following manner: EXAMPLE 1 80 mg of nifedipine in powder form was placed into a special deuteration tube and dissolved therein in a mixture of 2 ml of deuterochloroform and 0.5 ml of deuterium oxide after which 0.2 ml of trifluoroacetic anhydride and 2 ml of deuteroacetone were added and mixed therewith. The solution was frozen in liquid nitrogen and the tube flame sealed under nitrogen.

The tube was then heated at 57°C for 168 hours after which is was cooled and opened. The contents of the tube were transferred to a round-bottom flask and the solvent was removed in vacuo on a rotovap. All operations were conducted under reduced intensity of light. Using

S

conventional 'H nuclear magnetic resonance (NMR) the deuterium substitution was calculated to S.o.

20 be 95% of the C-2 and C-6 methyl groups shown above. 05* The effect of the deuterated nifedipine on the blood pressure in rats was then determined as follows: 6- SpouanMouy 'yprtV- rats (SHIR) were anesdwied with patwbarbi~a (65 mg/kg, intraq, honeally) and a carotid'artey =Wd jugular vein cmnnil ad. Blood pmasure was .ontinoasly noreud via the carotid armuy cannula.

Nlfeipn 3amples, both deatcred. and non-dewted, dissolved in dimethylsulfozizie (DISC) 9we diluted in saline so that the final injected roaceaxritm of DMSO was lIan than 0.025% by volume. Aliquots of appropria dilutions were dh= injected iniaverou sly in the SHR and blood mmsure effects monitored for at loas two hamr followig injection. Doses used wer 0.00001, 0.00002, 0.000025, and 0.00005 millimcte per rat in the control group and in the tent goup. All rats were within gramns of hody weight of each othar.

Mae reslts are sbown in Figure 1. At the three low= ccautos the hyporwisbre affect of dectmiaed nifedipne was growte than that of regular nifedipine (p-0-08 Ivy Wilcoxon rank-sum tein). Effictive doses of 50% of thera= (ED, 0 1 s) wam on the basis of the results ficim the above damn and results were: Log 'EDw dwsated nifeipn -4.48 (-4M3 to -4.43, 95% canfidence interval); arid Log EDn regular ni~pine: -4.36 (-4.40 to -4.31, 95% coafide interval). As the cnfideo interals do no ovea*, there is a notistil differene in the potency of the *wood two niled pine products, with the driterated nifidipine unexpectedy having the greats 20 POI2O I.

Teffect of doutrmted nilftpne an calcium cbannl blakin activiy was saidiei and the Wtde were c=Wi out. using the whole cell version of the patch clamp metbad, as follows: -7- EXAMPLE III NIE- 115 cells (neuroblastoma cell line) were used and these cells were cultured using conventional tissue culture techniques. For study, cells were used six to eight hours after trypsinization and replating. In this state the predominant calcium channel expressed was the Ttype channel, which was used for the current studies.

Patch clamping was carried out using the following external and internal solutions: External solution (in mM): BaCI 2 20, Tris 105, KCI 5, CsCI 5, HEPES 20, glucose 20 and tetrodotoxin 0.0005. Internal solutions (in mM): CsCI 130, ATP-Na 2 2, HEPES 20, glucose MgCI 2 5, cAMP 0.25, and EGTA 10. Osmolarity of all solutions was adjusted to 310-320 mOsm and the pH adjusted to 7.4 using HCI, NaOH, CsOH or Ba(OH) 2 as required. The Petri dish containing the cells was mounted on the stage of an inverted phase contrast microscope.

Pipettes fabricated from thin-walled borosilicate glass, and containing the internal solution, were *o advanced to the selected cell surface using a micromanipulator. Suction facilitated the formation of a membrane patch with resistance in the range of 20-30 gigaohms. Test pulses in increments t 15 of 10 mV were applied for 200 msec with at least 5 sec allowed for channel recovery between pulses. Basal channel activity was measured based on the peak inward currents. After addition of the nifedipine solutions to the appropriate concentration, 3 minutes were allowed for the drug to reach equilibrium concentrations. After this time current-voltage relationships were re-tested and the results expressed as the percentage of control current obtained (ie, 100% indicates no 20 channel blocking activity).

Preliminary results at concentrations of 1 x 10- 6 and 1 x 10- 5 showed no statistical difference between channel conductance for deuterated and non-deuterated nifedipine (70% vs.

77%, and 35% and 43%, respectively, p-not significant). During 8dumes ivs, however, it was noed that a difference did exist between the c-hanxwei acdvaica~ chepcodency of the two mifadipin conipousids. As usual, nornma nifrgipine showed olccking actviy which 13 depaidelt on the state of activation of the channels (normally 111ocdng is bciiamxed when channeti arm moum -active-). This effec was not see for domersted nifedivine; rather, it seemed to show continual maxima~l eff=c regardess of channel stats. This sugests that the binding of deutmiaed nifedipine to the caliuri2 channel is enhanced rimn thuigh theme may be no diftece in potency.

Clinically, ths minm that dwutcmtd nifedipinet may hav a longer haf-ife of the receptor mid/ar that it mazy have a constant effec across blood pressure ranges (normally the hier the blood pressure, the greater the hypotensive effiect: of cal~um channel blocev). It is expected tha the dauterated nifedipine would act in a similar marninm humans hain high blood pressur.

0 *While it has been noted tha deuteriing one or more hydrogn aunms in known ph amaccuflal compounds will enbanc or alter the activity of such compounds, it is be~red tha the activity of such compotund may also be altered by substituning a diffewt isotape for one or more of the other atoms in t cmpund. It is known that se~veal VaIfetiCS Of otiu a=3$ms ch as carbon, 1 "ro1ge, 1,ygmn, tin, etc., exist which ~dife in their atomic mass. Thes diffcing species of atom are referrd to as isotope 0 and diiaonly in the number of ne=ton in the nucus.

EAPET

@0 0 Use depezidenicy of calcium charnel inhion-Repetifive activation of calciumi ithameis progressvely reduces the peak inward curruit Such a decrease in onn'ent c~xm nwe makdly the &reuter the frequency of stimulation of the channes.

In t prcu of a 'use dqodent' amgonist the decreaw in curr=1 with repetitive stimulwio, is ahsnced. Use dcpidcncy;;mples that the antagonist bin&s cumulatively in sall imc1a~ts during 3UbqUn channel aidtiiOas. After 2 or 3 channiel activatkcnu in the can of caIcilzm channels,- a steiy saeof inhibition is reached. The undcrling implication of use dpuac is that a giv=i antagonist (drug) binds wor strongly X, the activ channel. A drug which shows greaer ume depmidn channel inhiiu a S presumed to have a greter affnity for the actve calcium channel.

In the following set of examples, calcium channels were depolarZed with rePedlive Aud=e Of Current at the intwvals indicaed, Such studies were carried out using th. wbole CCU Version of the patch-clamp. The reduction of inward cunut as a function 01? the interval between pulses in t abmce of any drug was Wake as the conto1. Control non-deuterated) and deumeated nifedipine were then applied to the celb at a concentmion Of 5 MicruuMolar. The decrease in inward current as a function oi frequency of stimulation (LeC, use dependcem) was td= compared betwee the two abine prepaz==oo.

Fiue 2 shows use dependent ihttnof contrl mxfewpm (N-e~m B) and detterated nifedipine (Nifedipine D) on T type ciumz channels in NiB 115 cells. 7Tii. figur repreamts inward Curret ffiw and its inhibition -by the nifedipine.

As frequ icy of stimulatio ihnae, inward current upon repetitive stimulaf=o 0O*em [Us dq dnc) M fedipin is ame to &ecease inward current, but at incivaseI :dmulzatinn fzhuucy dzatccnd nifedipine is more effective tn control 00: nifedipie Panel These differences are further shown in Figures 3-s.

Figure 3 represcats the inhibitory afea of the two nifedipines on inward calcium cutnt for repetitive I second poulses (depobzitnm), At this pulse frequency dew=3m~d nihdipine Js more effective at blocking calcium curren thn control cwmu 10 (p o~5 by repeated measu ANOVA). rCmn~tutimn-s of niftfipifl were 3iom1ar in both csa.

Figure 4 shoWS die effect of the two nifedipines on calcium curM inhibificin us a functio of pulse frequency. At 1 and 0.3 second intervals deueme1 nifedpie was moe effectkve dhan control nifuipn in blocking the calcium channels.

iFigre s shows te effect of the two nifedipines on use dependency. At all fr pucics deutezazed nifaipine showed gZ==e use dependnt calcium channel inbibiu~m. Such inhibtion was even mor mared as frequency of stimulatin increaed Basd on the above data, detczuted nifdiin is Imto hav PC=te Use dependilnt inhibto of clium channels. This means that doutu~ated nifedipine is more effctvp tn regular nifdiin at blcing the calcium channel as the frequemc of stimuladai krases. Becmuse channel activation is greater in pathologiecal conditions such 0 brvpatgnsion or angina, dvubmrald nifodipine would be expectd to be more .00. effleacioul/patat in these dsorden than regular nifedipine. in non-pathological comidiimx where ebannu actiaio j3 not as pmt, however, the actvity of deutezated nifrdime would appoch that Of regUlar 1ifedipine Such a chazctisc is extremey :::o:appealag as it man tha te Idwaive potanc of the drug deuteated nifediin) would vs-y &dly with the invedt of the condition. Thus, for examplc, the dnit wMul mmngly -kw how much to red=c the blood prmmur in order to achieve a purticu blood preaur gaL. Ciuaietdy, as the severity of a condition ~hyperznz in, an*=n) incm=ae, th dome of aifedipine necessary to trWa such a coU'lition alsoincrzame. Such may not be the came for dmuzated ifediin.

11 EFff= of dcnraned and control nifedipines on blood PTm=i nwrmotsmze Speague-Dawley ra. Control and deut~zed nifedipine were dissoived in mnini=,a vohnme of ethanol and dited =61i the final co~a==o Of ethanol was Ie= dhan 0.04%. One Millilie do90I of the two nifeipne 81 the indiae oo==-:-atons w=r tha injected =nt pe tbaratl-Luesthized Sprague-Dawley r=t wei~ghing letwc 300 arid 350 gzzins. The mwdmum change in mean ateial pressur, aU wel.as the duation of hyponsvc respon, w=r ms~ured directy by Means of an mar~a cathfter. Ony one drug dose was givu to each ra and a minimum of minutes was allowed for blood prsure to re=ur to baseline. Resu.=r shown in the BV=ze diicuse below. At each time period the du==io of effec was pgraer for 0 detmd nifedipine compared to control nifedipine. Noweover, bemause the duranon of response might be dependent on the mapinide of derese in blood pressure, IS.. etM= do=e of the two ft=4132ans wee comnpared. In this compar., control nifed e at a cocntration of 2 x 10W molar and deuterated nifedipine at a @1 0mwm co~ im of 1 x 1013 molar were compared (relative powences -45.38 vs. -40.3 mmrng control va. deuzed, p At thmdozes, despite an euivalent blood press=e effect, tht., duration of &=mo of cwrdl nifedipine was 46.5 min and the duraton of acon of Se~e nifedipine wa 67.2 min Thus, the duration of actio of detOM nffdfi~c is Vre 11 hn tha of control nifedipine indquidWi of blood prew lowerfng dffcr (Le, both potacy and duration of action differ).

Figure 6 shows the afetof control and deuteraed nifedipit on mean arteial pvwure: 7gur 6(a) shows acal values ploned, Figur 6(b) shows =ve fite t damn by logistic doe-an reesimc A total of six rats wem este at eac& 12 doam. As can be ate, at all dome deutemsed nifeipn was more potn than cntrol nifeiie (p .05 by repeated mesures ANOVA). MAP mmen m= al preggmr F'g4r 7 shows coc~nram-ff~±me osbish fbr control (FIgur and dmr~fd (Figure ni dpn fitted using an asymmetrc Sigmoidal mdeL Fitted vahm a plus 95 S confidence intervals ame shown. From thes two grahs, based on a lack of ovcrbp betwe the confidence itavals, it is evident that the Mwntim-effict zektionships for the two Wfedipine differ. To be more precis, cMWv &ungi was cad out using the equation y-a+b(l-(1 +exp((x+dln(?L-l.C)/d)eOn this basis the parmeters a, b and c all differed statistically at p Figure 8 shows cm etzio-effec relationships for courol (Figur 8(a)) and deutsm ed (Figure nifedipine Atted using logistic dose response model. Fitted values PLus 95 confide=c intevals are shown. From thms two grqbs, based on a lack of ,verlap between the canfidence intervals, it is .evident that the mW aX-4 mationuzup fbr the two nifedipines Ma ob nme prw) =mrv filling was catrid Out using the *quatio: on ths basi the pezane~sa and b all differed statistically at p EXASI y Effectm of deugeztd- control nicdpino n blood pressure in norinotiensiye Sprague-Dawley rats. Control and deuterated nicardipines (prepared, as described t cla, n manner analogou to the prepajon of dentmrad nifeihinn) vwe dissolved Mn minimal volumes of ethano and diluted until the final __cetmtion of eftnol Mis less than Doses Of the two nicardq4ne as indicated were then l*td in'OD pe tbarbital-anesdbedzud Sptague-Dawley rans weighing between 300 and 350 grarn. The maximum chance in umen art=ia pressure, as well as the &durtm of 13 hYPOMIdvt: !!RML'OS, Wut MCInauld directy by mean of in intr-artcdW a1zbt.

only on& erug doae was gIven D iCwh WW± nd aMinimum of 60 minutes was allowed for bl ;nm=u to rw=r to binelin POcY-IpuW 9a and 9b show the dow-response effet for the 000oro1 and dmac (tes) nicrdipmrie. Although the =,fide in~vals of the c=ve oveiap in ums sttisical cflmpriscm of ]ED14, ED,N, and ED, 4 showed te following .9 9* ]od~ ccazrol 6. 1 1XlO mninols 95% al: 2.2-16. 8xiOld deutratod 46.2x10 4 minoles 3 7 3 -5 7 .2xiOY4 ED5 0 cc efl 2 .54xl1O3 mmales 95% CI: 1.

5 3 -4.2041 deutatted 2 1X10,3 mmoles l.48-3.O0xi0W

ED"

4 cxmtr 1.06 mmolm 95% CL 0.38-2.91 deutented 0.09 mzral 0.077-0.119 From the above table ut cm be see tha the confide in ls ame exclusive Jor both EDu and ED,4. This indim s the nhr of the dose tapsple reLadom,i diffis. This is codnned by dffcdng slope functons of 416 for ootrol nirdpi~and 46 for dcutam ficardipiz (aus clclated by Lixchfrd-Wicom 2 0 medlod Ibus, the Potencies of the two formlation diffw.

Two-way analysi of vatinc usig dos and fuzmulatio as independent vaiables and reducdon in men arteiul pressurea the dependait variable reveulW the followutg ANOVA Ubl&c 14 factor d.f S=m of mean1 F p value squares square drug 1 323. 323 .3.11 .092 dose 17 5531 3255 30.8 .0001 drug x dose 17 6464 380 3.6 .0001 AlhohJI ttwc is no differvnoe at a p<.05 level for drug differences (ther is a diff~c at the .1I level, however), the lack of differece is likey due to the mny (Loses clustered around the ED5 where the curVe do not differ. There is a marki~d diug x dose interction, howeme, which does implies a differcnce in the ature of the dose-response curves Durution of action-At most tune periods the duratio of Pffec was greatcr for deL-tmated nirdipine comared to control nieadiie (Figure 10). Because the duratioui cf response might be dependnt on the magninida of demmese in blood pressure, howevtz, equipotent dones of the two foirmulations were compared. In tW3 comparzz, conrl n kardipine at a dose of 1 x 10' moles and deuterated nicardipine at a conewnicm of 3 x 104' moles were compaed (relative Potencies 13.8 va -13.8 Tmmng *20 control v3 d=uterted, p NS). At these doses, despite an euivalent blood prm.sur effect, tdm- durtion of acton of control, niesdiine was 5.4 3.8 (SD) mn and the duration )f action of deutcrated nicardipine was 15.0 6.4 (SD) min (p=.049 by Mann-'Wlimney U-test). Thus, the duration of action of deutrated. nicardipine is greater 99 9 than that Df control nicardiie independent of blood prexau lowering -Effeivm Com2pmnhon of dwimtow of action by two-way ANOVA usnq dose and formlaim as independent variables uid duration of action as the dependent variable reveaidb 'hUolowing ANOVA ?able: factor d.f. sum of mean F P value squares square drug 1 5697 5697 24.0 .0001 dose 14 68292 4878 20.6 .0001 drug x dose 14 10678 237 3.2 .0002 These results show significant differences in the duration of action of the two drugs as well as implying a difference in the nature of the dose-duration of action relationship.

Based on the above data, deuterated nicardipine differs from control nicardipine both in the nature of the blood pressure-lowering dose-response effect as well as in duration of action.

EXAMPLE VII Preparation of deuterated nicardipine. Such modifications of nicardipine (and other dihydropyridines) can be achieved by dissolving the nicardipine in a deuteration tube in a mixture of deuterochloroform and deuterium oxide, and then adding a minor amount of 15 trifluoroacetic anhydride and deuteroacetone thereto and mixing therewith. The solution is then frozen within the tube, preferably by immersing the tube in liquid nitrogen and then sealing the tube. The sealed tube is then heated at a temperature within the range of from about 500 to about 65°C, and preferably within the range of about 55° to about 60 0 C, and maintained at that temperature for a period of time sufficient to deuterated the methyl group at 2 and 6 positions on o 20 the nicardipine to CD 3 A time of from about 150 to about 180 hours is effective to complete the reaction, with a time of about 160 to 170 hours being preferred.

Deuterated nicardipine was synthesized in the following manner: 80 mg of nicardipine in powder form was placed into a special deuteration tube and dissolved 16 aborcn in a inixzs of 2 rAl Of dw Or-lnr-Ow. and 0.5 ml of deuml oxide afte which 0.2 ml of uif*iru.Canhydride and 2 ml of d~t.~O wer added and mixed therovith. Mhe 3oion was F in liquid nitrogen and the tube flamed 3aWe under nirogeil. The at was then heated at 57'C for 168 hours aft which it was cooled anD cpaed. The cann.2s of the ut wem reusfred to a round-bottom flask and the soblnt was resnoved m ivCzw an a rotovup. All oprnrkm were conducted under reduoid, inteuisity of ligbL tiling conventional 1 H nudesn mDagnetic rexonan (NMR) the detium mubitstion was clculated to be 95% of t C-2 and C-6 mgthyl grou sbov'n above.

Giveni the profound effects of substitutofi of dautimium for hydrogen in the methyl lpmups attachied to posfloni 2 Wd 6 of the dihydrapyridine ring, and given the fact tat all dihydropyzidine cim channel blockers have at lest one methyl. group m. theme ;asts m fwt, all dihydopyndbMe have 2 mothyls =Mcet amladipn which ~only has a 65 methyl, bimdtkmtiz of the 2 methyl with CH 2

OCH

2

CM

2

NH

2 inuestingy drastically imcreaso the dluma of action of this dihydropyridin), deuteration of thewe groups in atty other dihtydropyddml would be ected to have t atne effect as in niuedip

VM

Effct of Deutezzted and Contro veampaml3~ on Blood presur in 20 Niormotensve; SprAgue-Dawley Rats.

rtrlg fza i-a~ml hydrochloride was added to a soluion of 25 det ternted sulfic acid in dwtMXIt WaOW and deuterated. methanol. The solutian wis xdnd for 140 hours at90*C. The pH was adjusted to 12.0 and the mixure extrcted with etyl acte. The ombne ethyl acetate =zUct were washed, 17 with wzu. dded over magnWIuzu l fhl and evaporated to yied a via~us oiL. This oil was dialved in ether and ewal hydrochloride was added to Wprm the hydroch&OL& salt. The sat was collected by filtration and cryeallized from ethyl acemtei to btin deuated vwspili as a white old.

Control and deutax vempamils (weren the aromatic position of vmpszail (a pbenylakylammie) deuzeraWe) were dissolved in minimal vol=me of chanol ami~ diluted until the final nfif of ftanol was leas than 0.04%. Doses Of the twc' Verpails as indicted then injected into pentab ri l-an d SpragutDawley rats weighing between 300 and 350 grams. The =x=dm change mn mean jartzia1 pressure, as well as the duration of hypotensive reqpons, wer masued directY by means of =n intra-eraial catheer. Only medrug dose was given to each mra and a ininimum of 60 mutes was allowed for blood prsure to ream to baseline Results ar-, shown below.

At each dose, the effect (Potey) of deuterated vm-Aparnil was les ha that of cootrol verapamil. (we figur 11). In terms of dose response, using XP~fOxim~dzey -90v mm14 as mhe mwximiu respom theE aor control verpaini was 2.89 x 104 mM and 2.16 x IV' mM for deutented vexap=i. This difereace, anmg a ~with dffcsm in the end=r dose reqnae curia were diffat, by I jchield-Wilcom anayvis. Indeed, poamey =dto deumined by this technique was .133 deutmd vs conifol(95%wcnfidencentrvals.07 to.25, p<.O0l). Thus,fthedeuzerated verapamil Was leCs I'zent tha control verumil.

in terms of datsion of actinn, howeve, by T itch iold-Wicozon analysis, the dow-zAted verap=Umiad a longer dttioo of action (we 4=ur 12). In addition, the &xieduutio cuves were also signiftantly differet, iin a potecy rato of 4.95 18 The dgn ted vs control (95% confidence interval 2.3 to 10.7, To compare awhe wuy, at equipotet do= (0.3 x IV~ mM for control, 0.3 x 101 mM for deazerabn1; BP reduction -52.8 and -57.0 mmRg rspectively), the duratilon of action for dlsawd vs cocno vcmpmil was 12.5 vs 6 minutes Thm rmults suggest that the potency and duration of action were altered by daitcr=awi As with the dihydropyridines, dwratoc of actmo was prolonged by deuteratto., but pomicy, unmlim dihyroydie, was dweused. Depeding on the site of deuteration, howe@e=, patMic could potentially be incMasd.

Thus, in view of the above, duration of action is prolonged by dammario. (for all drugs tesed); poueny is affected by deuteatinn: it is increased for dihydiopydidine and decreased for vezupamil (a phenylakylamin); and (3) use-dqet=ency is altered for dihydropyridime (it has not beow ted for va-pamil).

Prolonging the Duraton of Action of Drugs Using Isotopes. 1nwxhducman Pharmaceutical manuacm=r ohme spend mveWa minions developing drugs that have long durations of activity. Thw ques for drugs with longer duration of action. atisix for several reason including, for example: prolonation of the bemeficial *0 0 therupitk: effect protection of the drug's inarkas-shart try new patnt issued. for the 0o:new dug-U &sgeoIIUtin J.UhMRan, woigaincmlac W.UAAUmak14I.L A~MA. ing keas tIoL I a once-a-day formulation instead of thre.-, or four-tiines-a--day formulation).

:00.0* 0 20 Each of the three drugs tested nifeipn, nicardipine, and vm%=mil is =ently being used in th =wnamt of hypezianso (bigh. bloodl preure).

As carlIicmacular dimese is a leading cua of death in North Amrica, and hypertna~ ns a signfl= n ik fato, it is imo=n that patients requirig thm fid is hmv drug conceomations In circulaio Which protalct tiem from theme 19 Off-~ of high blood pressure 24 hours a day. Although som manwfawrers offer two, thz=, 3r four dmes a day dosing, wveal ane manuctuing drugs which hzve longer durationi of aaion (longer half lives) by altxn the donae formulation. These dosage forauLatm al clAtio include, fix czample, inclusi of mcits that &low the drug's releae, andng of fomulation, newer formulations osmotic pumps), and coated pramul s- many of these modifcations of the dosage fomulatin are extremely expcn~ve to develop and manubetme.

Using the deuemted drugs disclosed heren, we have shown that we can modify dtriup of diffumt cblam 1-4 dihydropyddinea and phanylalkyhaminus) and believe thit our rnodificadon applies to all drugs, a3 the principle involved is the smne, i=sp*;tVe of the drug. Without being bound by theory, by d==ozting drugs, we have likely pro uced two efects: increased th lipophili natre of the molecule and xendered the C-11 bond more thermodynaxndca~y stable by subgftuting the -H for -D (deutezium atom). The former point may allow the drug to disdibute into more Ndg r fimie depots (sich as adipose) and may linge= in the body for a long=r peiod of time. The latte 1iiat, however, is the point that should be regardd coerefully. By subsdud*g the hydrogen by a donedum atom, the resulting carbon deuterum (D) bond bcuia stronger. Haice, by increasng the mass of hydrogeni one atomic mass iuait and creatn a Di, the C-fl bonds ae probably becoming stronger. T"his is done wiing D as a non-rjdoactive stable isotope of the H. Interestingly, as the C-D bond u~ thermodynamically more stable than the C-H bond, it is less easily cleaved by metbolHe (or destructive) processes. Hence, the elimnation half life of the drug is proloqjed and the drug's thmapetic effects are inesed. Although we have also 20 obsinva that there may be changes in the drug,'s poueny, the poagatiOM of the drug's effec i-s lam~ scng.

In essenCe, therfe, by simply subuuuriing a H for a I) in the three molemula iuvestgatd thus far, we hav creaed a new "modified aeame- drug by way Of incr~awing the duration of th activit. Additionally, a distnc advurnag of this matho w~ that this method obviates ft ne for ad&in sevuca pot=Utay toxi excIpcts which are prsetly included in tbe modified release for ms.

Pinerpntig'of c4opounds Since the subxtihtuet of OhaMUCntia comPOunds are usually element of cubon, hydrogen, niuogc, oxygen, etc., and these CIRMen3 MAY exist in Mott than one isotopic form, the Preise ==uz of these isotpi foms Is deremined by how and geographicLmy where each chemical 13 made, partcularlY ince each Wlon where the drug is Manubcured usually use local chei a supplies fcr its umail for Synthesis. HEk, a given. pharmaceuticai may be develOPed as a molecule with a very distinct paern of isotopes prownt terin.

CompRanacti Of the ksown moIlet manufacturd by owe uwanuftmrr with the molel of a ftlbr pharmceutical fosmd in the marketplamcan establish whather the smcod du WIR wus by dz SIn. MM N or men 4* by anoter. Thi s possible since each molecue IMS a Very dic Par=m (CflgePrnts) Of iSOtopeS. Gas crMatogrAphy- 2~0 ibotpe rajW mas Vwtrnomeoy (GC-IMS) cam be used to asc=zin t matedals tha ae pres: in a pamcuia p~m Altho a generic drug product must pass cerai Vecific ta prior to being ampivd for the nlarkueace, it has been, detemmed tha such geec drug Product ctsmed by a patient may not have the same, quality as that of a drug 21 manticnrrad by the innovatos of tha drug. Diffec s in quality may be relaWe to impudie: or to dffnm in the chemietsi make-up of the drag MOICCUlc, which diflemics are =o dftecte us*n s~ndard tinsfn prucdum~ but ame dexeted by the =s of OIR MS (damncibed below) for demining the mleular S=uetze of the drug molcle.I From the foregoig, it is readily ap"=en that phzz aicaj prepratimn wh"c (oMtein idectical am=t of a drug may not be binequival=it This imeqivjjcny may be etrdey due to the ixotopi mImr FOf f all Of the elemmat which mr included in a particulix pharmaeutical. Such isouopc dffecs betwe a generic drug and one of a knOW:i n~cue made under the nigts of the inovtor of that drug couli regult in ina&'=tent over- or under-doig if owe formulation is substituted fo anothff. Thi *My =Wga, fCr cxazupm, that XzVAiUdy w mAuI a~ngjIn of hyperenson =ayno conttolid upon exchanging cm fo~rmlaltcm for anoter, leaing a phyucian puzzed as to why the drug is no longer performing as epcd.Conversely, pefients who ptviously Cpqienced no advers drug effect may begin to develop side, effects fom 9 the new fermujatiozi. In addition, the dumasm of clinical effect may differ between two &iff==et A =muafimns In accodanc with an emboditment of this invention, a new drug ~Appeing on the mirw and sold as an equivalent of a knmow drug which ha bee n i the mnar= and made by one =mnanuwrer can be readily analyzed for biosquivalency with the kwwn dg.

BAMI OF MHE MMtHOD OF IRMS ISOtope rato mass Vectonmwy (IRMS) is a hi"l precis method of 2alysis W~Iich is able to Measr =uall umples (low nanogm amomtsb). For exaMple n~2:"t~m are detrined on a mono-caibon molecule;- C0I gas. Thlufor,orni 22 carbo hax to be comic to C02 ps by cotubusdon over Cuprus oxide (CuO, on-line combmLico oven or alenal analym) and myogcnly r chrognatographu~ny ptwihtd. The C% 2 gas can thn be iimadiaze]y dix~ed to dhe spwtromejer by man,, of a coadamous &mo Ii (als calwe CF-IRM).

The statistical combinatio of te istopes of carbon ('IC and uC) and Oxygec T7 0 160) to gernt the CO 2 molmcues Sime rise to the form of vanous isxtopom whole molecular wcights =u 44, 45, and 46, respect-ey. Thbus, for MWSU~dg Carbon imotope ralmo, thme mo bums are g.2mzd and recuzde in the UWS, ==~SPondng to the Man"e of t variOus IsotpUae Of C02.

In order to obtain a bigh precision and a high accuracy,_ referenc gase of abuoi~ut~y known isotopic COMPosizio =r ud and a dual inlet systezn allows an ailwuave adnissiof both $mkaud rccc 9U Int the ULmiat source via anna gas-swirch jog valve. The m sr of the vanu ion barn allows for te ~culculatior of the IC enrichment of the inMple. The value of t alublton is given a PC notation. The tC aburinm is expreaued as according to the folowirg: 2 C. $MmlI (UVI LC)B]_1) X 1000 This 31 value mmaures the vafrizons in pw=t per thoUsad of the Carbon isotope r~ato from the standard. For Cuarbn, PDB was sdacted as the inrfnazionl reac PDB is'.Pe-, Dee Belemaitrla (a flossil hrm the pee Dee ztological formaf=o in South CaroliN). The 13CflC ra0 from te III clim boca of this fossi is 0.011237.

23 C C

C

C.

CCC.

C

CC

Compwai to PDR, mow of the nanal oompounds dispay a negat &ha, value. in the abjove iwtinn, 12C ref=r to tte wwtpomers.

noe inxammit of the variou ion buam for LIN is givena 8"(6 notanocxa. The 1N abunidance is aprased as according to the folowing equatict: ("NI 14N =MpleXO{N/I" smnduzd)( L*Ni "N)sndrdJ x 1000 The 6'JN(96) value menures the variatios in parts per dxmuinxi of the nitrogn iwotpe ratio front the standard. For rzihgMu air was kscned as the interatonal referenc.

Th eRSur~et Of the Varios ion beams for 1"0 is iven a8"(P nottin.Th 150 abundanc is CprmMd as 8110(%e) acording to fte ftoowing equation: 68s0(%t)-- uample)-(u0/'O standard)/e(Y 0/O)standadJ x 1000 T-he 8"0(96) value measures the variations in pars per thousand of the oygai isotope 15ratio dust is, uygen derived from CC), from the standard. For oxygen, CO1 was wlcted as the international rz.cr 100d NShMUENTAL, SLrrxGS The isotope ratio mass 3peczUmnrn was a Finniga MAT 252 (Brmen, Gecrmaiiy), equpped with a dual inlt mode and an Amiennal uadyz for introducfion of no-ps umples. Sampla were weighed such that each contained aprimatAly 10-20 i's and tawsl OF d to tin capsuls The closed tin capsule was hoiused in tte automatc introduction system (Oental anayzer mode! NAMSQ NC, Frauns, UK).

SawIes were comibusted at 1OW0C with oxygen and oxcidation catalysts (Cr0 CuO).

The pszdfed C0 2 was analyzed by dre continuous &lw IRMS. lTe C% 2 sadard was 24 0= C02 (dC(PD) w-36.49 96:* dO(PDB) 25.56 %i6; dO(SMOW)-=+ 15.06 obund from 0MCh Trading Co nt~kn, Dellas, TX, USA. Other s~ndards rn wase atropine M0.56%, El, 8.01%; M, 4.84%; 0, 16.59%) and pheanthrene (C, 93.5565'; EE, 5.615 N, 0. 180%; S, 0.456%). Thex pwified nitroen was analyzed by the cninzuous flow IMM. Th. N stadd was 5 N Oztech =M~oen (dN(AIR)=-1.89 obttnmd fron Omtch Trading Corporation, DallaS, TX, USA.

In=umenml setting were high V01129e (OY, 10 MY mthode, 5.74 A; emission, 1-473 mA; trap emissio, 0.937 mA; electron gnergy, 75.0 trap, 50.1 V.

APPLICATION OF ISOTOPE RATIo mAss sFEcTRtohmEY FOR FINGfPflTl4G A number of phama~szcls, including nifedipine,- ciprofloxacin, katoprlan, :zopickxe and acebutolol hav bown ivestigted usng an isotope xa*o mass Veict=2ftar (IRMS) for the Ietmination of source of manuft==. -This method for Producin a 'fingerpidnt0 is based, for example, on raujo of LC/uC which are determijed from C02 ,as oxge and lzrgen jW"rad can als beusdo 'igerp&drting". The organic =an in the phraetclis converted to C02 by combusti ove caprous oxide and punfied cryopmnicy or chromatoaphically. 7Ue resuling C 02 is tranfered directly to the specvomew by continuou flow via a transfer tine to the IRMS.

Inorder to inain ia igh demof pmeisiofor the~ -es tof the 0: ~isotopes 0f C%2, a refece= gas of absolutely kiown ix*Opc compositio is admitted to the IRMS trugh a dual Wet~ syM which enables the altuate input of both a uample p; (from the pharmnaceutical) and a referenc gas The analysis Sanera&= a "C/'2C rafio (6 96) which is ezxpres--eFd as follow: 25 TIu 8 96 (da pa ni vahn um Urn vazido in paru pa fthuaand of the mrbon i*MW ra from the undad. It JS thin Vale which is usedi m -the flnw iag* of ph =i bcau'flaw mauciai (awve nPudlits) as well a fiWue produ tablfts) wte ulydm. The rmlts for *An pbamcaU which have b=e prwegad are as hmTyom :eve*, 0 0 .0 0 '60.9 00 '.00 .6098 *55 on 26 LjLM-s. pisLim roR RAw M.4LL.s AND Fv~m PloDucTs F~fIPsv~n

S

G so 08 1. MOLECIZee NAME/BATCH WCIEAN STD.

RW

DEviATIoNq M.ArDJL

OR

Noaucr Acebwniol A.BC-191-?v2 -30.67 0.10

RM

AicebuIO1oI "359 t02= 0.08

RM

Acewumb~I 157 -30.65 0.06

KM

Zoi~.re M-23079 -30.42 0.05 KM a 9321500 RPP. Zoi~oe M-23 149 -33.93 0.04 RM 9332900 RFR ZopicLor~z M-23077 -31.43 0.08 P.M 9322900 Zopi1oe 6211402= -33.20 0.05 RM Ntfcdic OEFIS SA -26.67 0.10

RM

Sanofi le 39163032 -32.07 0.04

KM

Fifedipiaie 1838'99 -29.41 0.11

P.M

Siegfried M4ifedipime 7113-Sumi3m -39.69 0.03

P.M

Nifedipine, 038-1016B- -30.35 0.14 KM ~Seloc AG Nifecipine NF-1.9010 -35.92 1 0.07 RM Quimichi Aimar NiftLip-Lne C.F.M. -19.61 0.0r7

RM

Wvilanese Nifcc ,ne. 795019 -36.67 0.24 KM Nifoiipine pT24O563H -30.03 0.15

RM

,Baver Nifedize PT257982A -30.50 0.10

RM

Baver AG Nifei inc FT2.57981 3 -30.59 0.09 RM I Bayer AG I Nifediline pT249S77D -31.17 0.15 P.M Bayer AG I I 27 Nifediptae P=27980D -30.66 0.09 Pik Nifedipiras 898156A -30.09 0.13

R-M

Nifedipine 8996L2A -30.51 0.09 I ~Bayer AG__ Niffedipin 899552S -30.45 0.13 RiM ~Bayer AG N*Lfedipine 898553K -30.32 0.10 kIM Bryea AG Nifedipicte 898555C -30.21 0.14 PLM Bayer AG Nifedione 2704 -29.67 .8R De~on Luzel Nifedipine NI-S-89 101 -34.33 .6R China_ Nifedipine 50-OHS SA -28.75 .8R m m ~~~Sanoff Nifed.ipine 038. 1016B -30.05 0.20 RM Seloc AG Switerlanid_______ Dfecipune 150 -28.40 0.05 RiM Cipla Bombay NW667ectirn 208143 -35.67 0.11R Sumi a ou NIft in e K31972400 .28.14 0.14

I

Iing-. Phanrma Nuifeiie 5486 -33.46 0.15 RM MV01I102 Zambon Grop CipIroi 43/93 -34.62 0.09 RM Union Ciproli .udcn 92/18 rak, -35.84 0.07 R.M 1s~nbu1 Ciprwilbiczin 930501, -30.02 0.17 1 Rid ShangWa 28 cpmfxn~ic P3094 Ci~h -34M2 0.06 RM Kecoprmht BP E1344- -27.66 0.29 RM Nifd~ipive 92001 -32.3 F (Anooj 1O1 -32-1 Nif~ied202 -34.0 Ow Pp (AztmL -33.9 Nifipine 2FC -33.9 Fp (Cardolaft -34.6 Nifedipine RABP -32.3 F? (c4nm SC) 32.1_ Niedpin CF109D .32.5 IP Nlfedpine B533 -2s.65w FP Adalat LIZ, 1 _1 mk*only 'dim~ ierennnazio wn made. Menton of a .ztwdwvi deviaton. there. was no: appmptiaie.

29 The above informron canhrms that IRM can be Used to mfngerprint* phar==tilcals fom various so==e of manufacure. Such infonnain can be used to sou=c -A-rziaxcals, whether as raw materials (active ingrentms) or as finis1W Figuze 13 shows a dmre dimaisional 'fingerprint" of inven nfedipne preandoms. All of thcm preattoO ar ngnicanty diff==an from c od=e. 71his type of th=e dirmensional Ofingesprin'i what would be typicaly done to provide the most 3enzfmv *fngmpnt" of a dr" or miy other mibmrace.

0. Figure 14 shows a two dimensina Ofingerprints of ni=ne jieipn preparaik ns. The error lines are 95 owfdenc intevuls; thus, anl of the popcnaimn ,.oane sigiff ndy different from each other. Although three dimensional fnrprintin givs re precise *fingwixit% in praetice two d 3ensioaal "figrpimigU is o ouually affciet to differentiat fmaerals.

Figure 15 shows a two dimensional (oxygen vs. carbon) Ofingerpnintu of 0 solarol crude WaWt. Modt of the tablets can be diffztetiae from each other by the two dime nsiconl technique though carbon-oxygen two dimensional ngrzntin W is less prds than carbon-n3mm gui WfgtZ -fg.

Fig=r 16 shows 'fingrpsits' of three different hair amles from tr different people. In the case of t flrU patient (pi. a secod sample was obtained one *weec later and compare with the E=st This cowparison gave a stattisically id ial valne to the first ample, thus showing tha this technique is reprodneible and can be. wed tD identify ha& samples.

30 Pigu 17 sbows a two 6200u09 m wl Orbe-Olgn afiu*gUPiflt Of tm Iiqwux Thugha t"o Of th IiqM amO f t M.W "wM'uplnun mn jimtastb agclfto w y of origin.

Fwdtbg V~iadMs md modfkflice of ft foragiang will be ipjrt ID diom ukiMa, in tbe art and such vaiuiz and modlhdowas re mtmded to be e=0npmt by tim claims tin are vpmided bheto

Claims (17)

1. A calcium channel blocking compound having one or more deuterium atoms and wherein the compound has an extended duration of action and/or an enhanced use dependency when compared with a compound that is identical except in that the one or more deuterium atoms are each substituted by hydrogen.

2. A compound according to claim 1 wherein the compound has an extended duration of action. a* 3. A compound according to claim 1 or claim 2 wherein the compound has an 10 enhanced use dependency.

4. A calcium channel blocking hydrocarbon compound according to any one of the S:preceding claims wherein said compound is a dihydropyridine compound. A compound according to any one of the preceding claims which is a dihydropyridine compound having a methyl group in the 2 and in the 6 position and having a hydrogen atom of one or both of the methyl groups in the 2- and 6- ~positions replaced with a deuterium atom.

6. A compound according to claim 5 wherein all hydrogen atoms of each methyl group in the 2- and 6- positions are replaced with deuterium atoms.

7. A compound according to any one of the preceding claims suitable for use as a medicament.

8. A compound according to any one of the preceding claims when used in the treatment of hypertension. -32-

9. A pharmaceutical composition suitable for use as a medicament and comprising one or more compounds according to any one of claims 1-7 as the active ingredient and a pharmaceutically acceptable support. Use of one or more compounds according to any of claims 1-7 or of the composition of claim 9 for the manufacture of a medicament having an antihypertensive activity with an extended duration of action.

11. A method of extending the duration of action of a calcium channel blocking hydrocarbon compound including the step of replacing at least one hyrogen atoms S °of the calcium channel blocking compound with a deuterium atom. S. 10 12. A method according to claim 11 wherein the calcium channel blocking ,hydrocarbon compound of extended duration of action is a dihydropyridine compound according to any one of claims 5 or 6. S13. A compound according to any one of claims 1 to 7 suitable for use as a medicament having an enhanced use dependency.

14. A compound according to any of claims 1 to 7 when used in the treatment of •,hypertension and having an enhanced use dependency. Use of one or more compounds according to any of claims 1 to 7 or of the composition of claim 9 for the manufacture of a medicament having an antihypertensive activity with an enhanced use dependency.

16. A method of enhancing the use dependency of a calcium channel blocking hydrocarbon compound including the step of replacing at least one hydrogen atom of a calcium channel blocking compound with a deuterium atom. -33

17. A method according to claim 16 wherein the calcium channel blocking hydrocarbon compound is a dihydropyridine compound.

18. A method according to claim 17 wherein the dihydropyridine compound is a dihydropyridine according to any one of claims 5 or 6.

19. A method of modifying the dose-response relationship of a calcium channel blocking hydrocarbon compound comprising the step of replacing at least one hydrogen atom of the calcium channel blocking compound with a deuterium atom. A method according to claim 19 wherein the calcium channel blocking hydrocarbon compound is a dihydropyridine. 10 21. A method of detecting whether a pharmaceutical compound is identical and/or bioequivalent to a known pharmaceutical compound comprising the steps of: determining the molecular and isotopic structure of said known pharmaceutical compound by gas-chromatography-isotope ratio mass spectrometry. determining the molecular and isotopic structure of said pharmaceutical compound subject to said detection by gas chromatography-isotope ratio mass spectrometry and comparing the results of said two determinations to detect any isotope variation in the molecular structure of said pharmaceutical compound over that of the known pharmaceutical compound.

22. A calcium channel blocking compound according to claim 1 and substantially as herein described with reference to any one of the examples, but excluding any comparative examples. II i -34-

23. A method of extending the duration of action of a calcium channel blocking hydrocarbon compound substantially as herein described with reference to any one of the examples.

24. A method of enhancing the use dependency of a calcium channel blocking hydrocarbon compound substantially as herein described with reference to any one of the examples, but excluding any comparative examples. A method of modifying the dose-response relationship of a calcium channel blocking hydrocarbon compound substantially as herein described with reference o* o to any one of the examples, but excluding any comparative examples. 10 DATED this 27th Day of August 1999 ISOTECHNIKA INC. Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS S

22414-OO.DOC