U.S. patent application number 10/240827 was filed with the patent office on 2004-02-05 for bupivacaine enantiomers and levobupivacaine.
Invention is credited to Russo, Elisa M. S., Russo, Valter F. T., Sudo, Roberto T..
Application Number | 20040024021 10/240827 |
Document ID | / |
Family ID | 37515791 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040024021 |
Kind Code |
A1 |
Sudo, Roberto T. ; et
al. |
February 5, 2004 |
BUPIVACAINE ENANTIOMERS AND LEVOBUPIVACAINE
Abstract
The prevent invention describes a new method for the separation
of bupivacaine enantiomers consisting in a continous separation
process performed without heating, by the selective precipitation
of their diastereomeric salts with tartaric acid. This heatless
process avoids the degradation of the reagents granting a
continuous process feature to the procedure. Another embodiment of
the present invention is related to the enantiomeric manipulation
of bupivacaine enantiomers in order to obtain pharmaceutical
compositions presenting several enantiomeric excess of
levobupivacaine to quantify and determinate the role of the
dextrobupivacaine on its anesthetic and cardiotoxic effects. These
enantiomeric manipulated compositions showed to present an
expressive improvement on its anesthetic properties that has shown
to be similar to racemic bupivacaine presenting a cardiotoxic
profile similar to enantiomeric pure levobupivacaine.
Inventors: |
Sudo, Roberto T.; (Rio de
Janeiro, BR) ; Russo, Valter F. T.; (Sao Paulo,
BR) ; Russo, Elisa M. S.; (Sao Paulo, BR) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
37515791 |
Appl. No.: |
10/240827 |
Filed: |
October 7, 2002 |
PCT Filed: |
April 5, 2001 |
PCT NO: |
PCT/BR01/00040 |
Current U.S.
Class: |
514/317 ;
546/225 |
Current CPC
Class: |
A61K 31/445 20130101;
C07D 211/60 20130101; A61P 23/00 20180101 |
Class at
Publication: |
514/317 ;
546/225 |
International
Class: |
A61K 031/445; C07D
211/30 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2000 |
BR |
PI0002246-2 |
Claims
1. Process for obtainment of bupivacaine enantiomers, characterized
by reacting to racemic bipuvicaine with a resolution agent within a
medium of organic solvent a low concentration and at room
temperature, wherein said resolution agent is used in an amount not
lower to 0.6 molar equivalent per racemic bupivacaine molar
equivalent.
2. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized in that the organic solvent employed is
acetone.
3. Process for obtainment of bupivicaine enantiomers according to
claim 1, characterized in that the resolution agent is the acid
(R,R)-tartaric.
4. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized in that the resolution agent is the acid
(S,S)-tartaric.
5. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized inn that the resolution agent used is in
amounts ranging from 0.6 to 1.2 molar equivalent in relation to
racemic bupivacaine.
6. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized by being carried out without heating
employment, at a temperature ranging from 15.degree. C. to
30.degree. C.
7. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized in that the reaction medium of the
resolution phase is considerably diluted, with a concentration
ranging from 0.1M to 0.4M in relation to the base bupivacaine.
8. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized in that the free bases of bupivacaine
enantiomers are directly obtained from tartrate salts, by means of
the treatment of the solutions from such salts with alkali.
9. Process for obtainment of bupivacaine enantiomers according to
claim 1, characterized by the fact that the free bases of its
enantiomers being converted into its chlorohydrate salts.
10. Process for obtainment of bupivacaine enantiomers according to
claim 1, for preparation of the levobupivacaine and/or an
appropriate pharmaceutical salt thereof.
11. Process for obtainment of bupivacaine enantiomers according to
claim 1, for preparation of the dextrobupivacaine and/or an
appropriate pharmaceutical salt thereof.
12. Process for obtainment of pharmaceutical compositions based on
levobupivacaine and/or suitably pharmaceutical salts therof,
characterized by the manipulation of enantiomeric excesses aiming
at obtaining enantiomeric excess inferior to 90% of levobupivacaine
by means of the adequate combination of two pure enantiomers in its
solid forms or in solution, or by mixing the racemic bupivacaine
with the pure levobupivacaine in its solid forms or in solution, or
by manipulation of the enantiomeric excess of the enriched
levobupivacaine, obtained in the process with dextrobupivacaine or
racemic bupivacaine in its solid forms or in solution.
13. Pharmaceutical compositions based on levobupivacaine,
formulated in its free bases forms, and/or in its adequate
pharmaceutical forms, obtained according to claim 12, characterized
by the presence of enantiomeric excess, preferably comprised
between 90% and 20%, and more preferably, comprised between 80% to
30% of levobupivacaine.
14. Pharmaceutical compositions based on levobupivacaine according
to claim 12, characterized by the fact of presenting enantiomeric
excess of 80% of levobupivacaine.
15. Pharmaceutical compositions based on levobupivacaine according
to claim 12, characterized by the fact of presenting enantiomeric
excess of 50% of levobupivacaine.
16. Pharmaceutical compositions based on levobupivacaine according
to claim 12, characterized by the fact of being used in medicine
and veterinary.
17. Pharmaceutical compositions based on levobupivacaine according
to claim 12, characterized by being employed in anesthesia.
Description
[0001] The present invention is basic related with pharmacological
field, more precisely with the anesthesiology field.
[0002] Enantiomeric concept is related to molecules that when
presenting one or more asymmetric carbons, said chiral carbons,
these molecules are not superimposable upon their mirror images.
The different optic isomers are denominated enantiomers. The
enantiomers of the same substance on their pure forms present the
property of rotating the plane-polarized light in a certain number
of degrees. The enantiomer that rotates the plane-polarized light
to the right is called dextroisomer and it is able of doing that in
the same numbers of degrees that the enantiomer that rotates the
plane-polarized light to the left, so called levoisomer.
[0003] Racemic mixtures are mixtures of equal quantities of both
enantiomers. These mixtures are optically inactive (as they do not
present specific rotation) because rotations of opposite sides
cancel each other.
[0004] The separation process of the enantiomers from a racemic
mixture is denominated resolution.
[0005] Chemical reactions when give rise to chiral molecules
originate racemic coumpounds, except in cases related to asymmetric
synthesis. Most of the active pharmaceutical ingredients that
present chiral molecules are nowadays used as racemic mixtures.
[0006] By the evolution of 3D-Biochemistry, mainly in the
stereoisomeric field, advances had come into view that made
possible a better understanding of the interaction between
enantiomer-receptor once those enantiomers are able to produce
different pharmacological effects, although having similar
physical-chemical properties.
[0007] These different pharmacological effects are due to different
selectivity from the enantiomers in relation to specific receptors
and/or enzymes. These enantiomers are metabolized in different
rates and with different affinities from tissues and docking sites
of proteins. This is due to the spatial arrangement of the chiral
carbon, where atoms or atoms groups are linked in different
positions in space, forming 3D-relationships with an ambient not
less asymmetric from the receptors or enzymes made from chiral
amino acids, the L-amino acids (Simonetti M P B, Batista, R A,
Ferreira, F M C--Esterioisomeria: a interface da tecnologia
industrial de medicamentos e da racionaliza.cedilla.o teraputica.
Rev. Bras. Anestesiol., 48(5): 390-399 (1998)).
[0008] When an enantiomer exhibits a high degree of affinity for
the activity site (eutomer) it may interferes in the action of its
antipode, inactivating it (distomer). To this phenomenon Ariens E J
called isomeric ballast (Stereochemistry, a basis for sophisticated
nonsense in pharmacokinetics and clinical pharmacology" Eur. J.
Clin. Paharmacol., 26: 663-668(1994)). This is the case of atropine
that is naturally produced as an S-enatiomer and during its
extraction process racemizes resulting in the relationship S/R
50:50, being the R-enantiomer completely inactive as
anticholinergic. In the clinic it is used as a racemic
compound.
[0009] Twenty five percent of drugs nowadays in medicine use have
one ore more chiral carbons, being 80% marketed in their racemic
form (Calvey T N--Chirality in Anesthesia, Anesthesia, 47: 93-94
(1992)).
[0010] The enantiomeric inactive form (distomer), however is not
always a passive component of the mixture and can act as an
agonist, antagonist, exerts actions on other receptors, produces
unpleasant side effects and even contributes to the global efficacy
of the racemate (Williams K, Lee E--Importance of drug enantiomers
in clinical pharmacology, Drugs, 30: 333-354 (1985)).
[0011] Some examples are presented as follows on which we may find
differences in the activities between the enantiomers of racemic
drugs regularly commercialized: ketamine contains the S-ketamine
that is predominantly anesthetic and hypnotic, and the R-ketamine
is the main responsible for its undesirable side effects (psychic
reactions on awakening); in the case of propoxyphene the
(2S,3R)-(-)-propoxyphene is antitussive while the
(2R,3S)-(+)-propoxyphene is analgesic; the prilocaine has the
R-prilocaine isomer that being faster metabolized than S-prilocaine
induces the increase of the plasmatic concentration of o-toluidine
and methemoglobinemia.
[0012] Bupivacaine is a chiral moleclule, and is used nowadays as
its racemic form. It has two enantiomers: levorotatory or
levobupivacaine and the dextrorotatory or dextrobupivacaine (FIG.
1)
[0013] At the beginning it was believed that bupivacaine
enantiomers had the same local anesthetic potency. However, recent
studies in the sciatic nerve of frog conducted by Lee-Son et al
(Lee-Son M B, Wang G K, Concus A, et al--Stereoselective inhibition
of neural sodium channels by local anesthetics, Anesthesiology, 77:
324-335 (1992)), demonstrated that dextrobupivacaine was more
potent than levobupivacaine, inducing a tonic and phasic block with
potency 2 to 3 times greater.
[0014] Recently Valenzuela et al demonstrated "in vitro" the
greater stereoselectivity of dextrobupivacaine, in terms of potency
and affinity by sodium and potassium channels in the hart in
relation with levobupivacaine. The difference related to the
initially observed toxicity by Aberg (Aberg G. Toxicological and
local anaesthetic effects of optically active isomers of two local
anaesthetic compounds--Acta Pharmacol. Toxicol., 31: 273-286
(1972)) was confirmed by the experiments conducted by Gristwood et
al in volunteers comparing levobupivacaine with racemic
bupivacaine, allowing to conclude that dextrobupivacaine acts by
contributing with the toxic effects of this local anesthetic
(Valenzuela C, Snyders D J, Bennet P B--Stereoselective block of
cardiac sodium channels by bupivacaine in guinea pig ventricular
myocytes. Circulation, 92(10): 3014-24 (1995); Valenzuela C, Delpon
E, Tamkun M M--Stereoselective block of a human cardiac potassium
channel by bupivacaine enantiomers, Biophys. J., 69: 418-427
(1995); Gristwood R, Bardley H, Baker H, et al--Reduced
cardiotoxicity of levobupivacaine compared with racemic bupivacaine
(Marcaine): New clinical evidence. Exper. Opin. Ivest. Drugs, 3:
1209-1212 (1994)).
[0015] With the recent advances of synthetic techniques and in the
separation of enantiomers, the pharmaceutical industry is making
possible the obtainment of pure enantiomers, allowing a significant
increase of therapeutic indices of drugs until this moment used as
racemates.
[0016] The obtainment of bupivacaine enantiomers on their pure
forms, allowed a better understanding about the mechanism of action
of bupivacaine and revealed the contribution of dextrobupivacaine
on the cardiotoxic potential of this anesthetic.
[0017] Clinical trials conducted in Brazil also demonstrated that
the reduction of the cardiotoxicity attained by pure
levobupivacaine was followed by the reduction of its clinical
potency, which can lead to unsatisfactory results as mentioned by
Mathias R S (Mathias R S--Levobupi--Uma nova op.cedilla.o de
anestsico local com menor toxicidade, 44.degree. Congresso
Brasileiro de Anesthesiologia 1997--Belohorizonte--MG). With
effect, pure levobupivacaine had demonstrated unsatisfactory
results considering the quality of neural block in large surgical
procedures, requiring a complementation of the anesthetic
technique, as it was observed with ropivacaine, another anesthetic
recently introduced in the market.
[0018] Otherwise evidenced by Mathias in Brazil, world references
show much more reticence in relation to the anesthetic profile of
levobupivacaine. There is an agreement about the cardiotoxic
profile of this enantiomer that actually presents lower
cardiotixicity when compared to racemic bupivacaine. There are some
uncertain results that seam to show a lower anesthetic potential
related to this enantiomer. Among them there are the results
obtained by Dyhre et al (Dihre H, L.sub.oang M, Wallin R &
Renck H--The duration of action of bupivacaine, levobupivacaine,
ropivacaine and pethidine in peripheral nerve block in the
rat--Acta Anaesthesiol. Scand., 41(10): 1346-1352 (1997)), from
Lyons et al (Lyons G, Columb M, Wilson R C & Lohnson R
V--Epidural pain relief in labor: potencies of levobupivacaine and
racemic bupivacaine--Br. J. Anaesth., 81(6): 899-910 (1998)) and in
the Patent WO 99/04771 reporting that the duration of motor block
with levobupivacaine is relatively inferior when compared with
racemic bupivacaine.
[0019] Although the majority of international studies about the
efficiency of neural blocks obtained with levobupivacaine appear to
be vague, it seams to have a growing tendency on promoting the
utilization of higher concentrations of this new drug (equal or
superior than 0.75%), probably trying to compensate the inferior
activity of levobupivacaine. This tendency may be observed in
several patents and in the studies of some researchers like Cox et
al and Kopacz et al (Cox C R, Faccenda K A, Gilhooly C, Scott N B,
Bannister J & Morrinson L M--Extradural S-(-)-bupivacaine:
Comparision with recemic RS-bupivacaine--Br. J. Anaesth. 80(3):
289-293 (1998); Kopacz D J, Allen H W & Thompson G
E--Double-blind randomized trial of 0.75% levobupivacaine compares
to 0.75% bupivacaine for epidural anesthesia in patients undergoing
major elective abdominal surgery--Anesth. Analg., 86, 2S
(1998)).
[0020] However, increasing in concentrations of the compositions
prepared with levobupivacaine should conduct directly to the
increase of their cardiotixicity, with the consequent disappearing
of the initial clinic advantage of a minor cardiotoxicity inherent
to levobupivacaine.
[0021] Among the related published patents there are the patents
numbers WO 95/10276 and WO 95/10227 identified in Brazil as PI
1100590 and PI 1100586 respectively. These patents disclose about
lower cardiotoxicity of levobupivacaine in relation with
dextrobupivacaine and racemic bupivacaine. Their authors claim the
use of levobupivacaine with an enantiomeric excess preferably
higher than 90%, more preferably higher than 99%. The
concentrations of the pharmaceutical compositions prepared with
this active pharmaceutical ingredient are from 0.25% (m/v) to 0.75%
(m/v).
[0022] Patent number WO 96/32109 identified in Brazil as PI
9604891, also discloses about the cardiotoxicity of racemic
bupivacaine an the lesser cardiotoxicity of its levoisomer. As in
the previous patents the enantiomeric excess are preferably at
least 90%, more preferably at least of 99% in levobupivacaine. This
patent is directed to its use in pregnant women anesthesia, being
the concentrations of those compositions higher than the
concentrations used for racemic bupivacaine. These concentrations
oscillate from a minimum of 0.75% (m/v) and may be increased to
concentrations of 2.0%(m/v) of levobupivacaine.
[0023] Patent number WO 98/38996 also discloses about the inferior
cardiotoxicity of levobupivacaine in relation to its dextroisomer
and racemic bupivacaine. The levoisomer is also used in an
enantiomeric excess of at least 90%, more preferably at least 99%.
The concentrations of the pharmaceutical compositions are between
0.25% (m/v) and 1.5% (m/v).
[0024] Patent number WO 98/38997 discloses about the synergistic
effect observed between opioids and .alpha..sub.2-agonists in
relation to levobupivacaine. Once again levobupivacaine is used in
an enantiomeric excess of at least 90%, more preferably at least
99%. It discloses that the synergistic effect observed allows the
decreasing in concentration of levobupivacaine in the
pharmaceutical compositions.
[0025] Patent number WO 98/38999 discloses the use of
levobupivacaine in an enantiomeric excess of at least 90%, more
preferably at least 99%. The concentrations of the pharmaceutical
compositions are from 0.25% (m/v) to 2.0% (m/v). Its application is
for pediatric usage.
[0026] Patents numbers WO 99/04771 and WO 99/0472 also claim the
use of levobupivacaine in an enantiomeric excess of at least 99%.
The concentrations of the possible pharmaceutical compositions are
from 0.75%(m/v) and 2.0%(m/v) of levobupivacaine.
[0027] From all the patents listed above it is possible to extract
important features. Among them it is possible to verify that the
levobupivacaine used in the studies performed until now presents
high enantiomeric excess, and there is no reference about the
associated effects on using lower enantiomeric excesses. It is
observed the great strictness on studying levobupivacaine
preferably free from its dextroenantiomer, in an enantiomeric
excess of at lest 99%, and there is not any mention to the possible
effects related to the presence of small definite quantities of
dextrobupivacaine. There is no study about the use of
levobupivacaine in enantiomeric excess lesser than 99%.
[0028] The utilization of both enantiomers to reach a desired
pharmacological profile or an ideal therapeutic effect of a drug is
not a novel procedure in the field, and is employed in some
circumstances. Indacrinone was one of the first drugs on which the
manipulation of both enantiomers demonstrated the improvement on
its activity. Its levoenantiomer demonstrated to be a natriuretic
agent more potent than its dextroenantiomer. The relatively high
uricosuric/natriuretic rate of its dextroenantiomer, offered the
opportunity to improve the pharmacological profile of this drug.
The enantiomeric manipulation of indacrinone was conducted
expecting to observe if the increasing in the
dextroenantiomer/levoenantiomer rate could prevent or revert the
hyperuricemic effect of its racemate, without inducing natriuresis.
This study demonstrates that the ideal proportions between its
dextroenantiomer and levoenantiomer was from 60% to 77% of an
enantiomeric excess of its dextroenantiomer (Tobert J A, Cirillo V
J, Hitzenberger G, James I, Pryor J, Cook T, Brentinx S, Holmes I B
& Lutterbeck P M--Clin. Pharmacol. Ther. 29: 344-350
(1981)).
[0029] The second important feature derived from these patents, is
related to the tendency on using higher concentrations in
pharmaceutical compositions. These concentrations are in some
instances higher than the twice the maximum concentration suggested
to be used for racemic bupivacaine. On the other side there is no
reference in the previous literature that states that
levobupivacaine is so lesser toxic as suggested by those patents in
order to justify the use of so high concentrations, mainly in
pediatrics, pregnancy and in cardiac compromised patients.
[0030] Racemic bupivacaine is nowadays marketed in 0.25%, 0.50% and
0.75% concentrations. Higher concentrations are not formulated
because its high toxicity. Even 0.75% concentration is not used on
all procedures due the elevated risk associated to an accidental
intravascular administration.
[0031] From the studies performed on the toxicity of
levobupivacaine, several researchers estimate that it is around 30%
to 40% lower in relation to racemic bupivacaine (Aberg
G--Toxicological and local anaesthetic effects of optically active
isomers of two local anaesthetic compounds--Acta Pharmacol.
Toxicol., 31: 273-286 (1972); Luduena F P, Bogado E F & Tullar
B F--Optical isomers of mepivacaine and bupivacaine--Arc. Int.
Pharmacodyn., 200: 359-369 (1972); Vanhoutte F, Vereecke J, Verbeck
N, Carmellet E--Brit. J. Pharmacol., 103: 1275-1281 (1991)). These
therapeutic indices do not justify the use of so high
concentrations as proposed in the above patents.
[0032] As we emphasized before, these higher concentrations of
levobupivacaine in the pharmaceutical compositions are going to
eliminate its better quality and the reason for its development,
the inferior cardiotoxicity.
[0033] With the objective of obtainment of bupivacaine enantiomers,
there are several available references in the literature. Among
them it is possible to mention the procedure described in the
"Journal of Medicinal Chemistry, 14(9): 891-892(1971)" that
describes a separation process for bupivacaine enantiomers by using
selective precipitation of its diastereomeric salts with natural
tartaric acid, from high concentrated solutions using high
temperatures on dissolution of the reagents. The experimental
execution of this procedure shows to be an extremely delicate
process, depending on features like cooling rates, stirring
conditions, batch size among others that directly interfere on the
stability of the solution and in the purity of the precipitated
product. Besides most of the times the separation of the
diasteriomeric salts do not occur and both tartrates precipitate
together.
[0034] Patent GB 1180712 (1970) from Luduena and Tullar, describes
two different process on the separation of bupivacaine enantiomers,
one of them using isopropanol as solvent and the other using
acetone. Both procedures are conducted at elevated temperatures and
high concentrations, being unfeasible to conduct the process in a
continuous way due the decomposition of the reagents on standing
for long times at high temperatures and also due to the variable
purity of the tartrate salt isolated.
[0035] Patent WO 96/12699 from Mariene Langson alternatively
suggests the use of D-(-)-tartaric acid, precipitating directly the
salt containing the levoenantiomer by a procedure involving high
temperatures. This procedure also could not be conducted in a
continuous operation due its reagents decomposition and besides,
the resolution agent also known as unnatural tartaric acid, is ten
to twenty times more expensive than natural tartaric acid,
elevating considerably its production cost.
[0036] The present invention describes a new process to separate
the enantiomers from racemic bupivacaine. One of the objectives of
the present invention is the process of separating the enantiomers
from racemic bupivacaine. This process consists in the formation of
diastereomeric salts employing a tartaric acid as resolution agent.
The tartaric acid preferably used is the L-(+)-tartaric acid,
however the process can be conducted with similar results by using
D-(-)-tartaric acid, noticing the change in the salts used to seed
the solutions and the inversion in the order of the precipitated
tartrates. The solvent preferably used in this resolution is
acetone, but the procedure can be performed in aqueous ethanol,
methanol and isopropanol.
[0037] In this procedure the obtainment of the diastereomers is
performed in a diluted solution of the organic solvent, which gives
the enough stability to the reaction mixture yielding the tartrates
in a constant enantiomeric purity. Their separation is performed in
small portions, but in a continuous process from the mother
liquors. As this process do not use heating there is neither
thermal degradation nor racemization of the resolution agent and no
thermal degradation of the substrate either (racemic bupivacaine),
being not necessary the substitution of the mother liquors.
[0038] Due those features described above the separation process
acquires a character of continuous process and can be easily
automated in order to simplify its monitoring, lowering the
production costs.
[0039] The resolution procedure consists in dissolving the
substrate on its free base form and the L-(+)-tartaric acid at room
temperature in a suitable solvent. This solution is firstly seeded
with dextrobupivacaine tartrate and is kept under stirring for a
few hours. Dextrobupivacaine tartrate precipitates and it is
separate from the reaction mixture by filtration. The filtrate is
then seeded with levobupivacaine tartrate and kept under stirring
for a few hours, while precipitation of levobupivacaine tartrate
occurs. This salt is separated and the reaction mixture is
reconstituted to its original proportions of tartaric acid and
bupivacaine, and the solvent is completed to its initial amount.
Seeding with dextrobupivacaine tartrate restarts the process and
the above procedure is repeated.
[0040] According to the resolution process described, the molar
relationship between bupivacaine free base and the resolution agent
may be from 1:0.6 to 1:1.2. The concentration of the reaction
mixture may be from 0.1M to 0.4M in relation to racemic bupivacaine
free base. Temperatures may be from 15.degree. C. to 30.degree. C.
and the time necessary to the precipitation of each tatrate salt
may be from 4 to 10 hours.
[0041] The respective free bases (dextrobupivacaine and
levobupivacaine free bases) are obtained by the dissolution of the
respective tartrates salts in water, resulting in solutions with
concentrations from 0.05M to 0.4M, and the subsequent treatment
with alkaline solutions (sodium hydroxide, ammonium hydroxide and
other bases) in order to adjust the final pH of the solution in a
range from 7 to 13. During this treatment precipitates the free
base that is separated by filtration or centrifugation. Enriched
dextrobupivacaine and levobupivacaine so obtained present
enantiomeric excess of about 70% to 80%. The obtainment of pure
enantiomers (ee>99%) may be achieved by simple recristalization
from isopropanol or another suitable solvent.
[0042] The respective hydrochloride salts may be obtained by
dissolution of these bases in suitable organic solvents followed by
the addition of hydrochloric acid in a concentrated aqueous
solution or in gas.
[0043] Another embodiment of the present invention is based on the
prior verification that the dextroenantiomer of bupivacaine seems
to represent an important role in the potency and duration of the
anesthetic effect that may be the complement of the expected
activity of its levoenantiomer. Its absence in levobupivacaine
compositions affects directly the duration and deepness of the
anesthetic effects, being necessary higher dosages of this
enantiomer to reach the desired levels of anesthesia and anesthetic
duration effect.
[0044] We verified that there is no study performed to determinate
the possible contribution of low definite quantities of the
dextroenantiomer on the anesthetic effect of levobupivacaine
compositions.
[0045] In addition, another objective of the present invention is
to provide the enantiomeric manipulation of levobupivacaine, by
lowering the enantiomeric excess of its levoisomer, quantifying the
contribution of the dextroenantiomer on the anesthetic and
cardiotixic effets, in order to improve the anesthetic profile of
levobupivacaine.
[0046] Another embodiment of the present invention is the process
of obtainment of the pharmaceutical compositions based on
levobupivacaine prepared with their free base forms or their
pharmaceutical acceptable salts.
[0047] The enantiomeric manipulation in order to obtain
levobupivacaine with enantiomeric excess lower than 99%
(ee<99%), may be achieved by several procedures known in the
art. For example, but not limited to these procedures, it can be
achieved from the pure enantiomers on their solid states or in
solutions, or can be achieved by the mixture of racemic bupivacaine
with levobupivacaine on their solid states or in solutions. In the
described procedure, enriched levobupivacaine obtained directly
from its tartrate salt may have its enantiomeric excess adjusted by
the addition of pure levobupivacaine or dextrobupivacaine, and even
racemic bupivacaine, in order to achieve the desired enantiomeric
excess. The monitoring of the enantiomeric excess may be done by
HPLC with chiral column to guarantee the achievement of the desired
enantiomeric excess.
[0048] By these enantiomeric manipulation procedures described
above, and analysis of the final enantiomeric excess by HPLC, there
may be prepared several compositions between levobupivacaine and
dextrobupivacaine in order to obtain the most variable enantiomeric
excess on levobupivacaine.
[0049] We could notice that the addition of the dextroenantiomer in
definite quantities in order to lower the enantiomeric excess on
levobupivacaine confer to the compositions using this combined
active ingredient, properties equivalent to those existing in
levobupivacaine in respect to its lower cardiotixicity and
properties equivalent to racemic bupivacaine in respect to the
efficiency on neural blocks.
[0050] The studies we are going to present demonstrate that there
are ideal relationships between the bupivacaine enantiomers, being
these relationships different from the existing 1:1 found in
racemic bupivacaine, and different from the existing in the
levobupivacaine used in the studies published until now, that were
performed using this enantiomer on its almost enantiomeric pure
form (ee>99%).
[0051] According to the present invention the compositions from
levobupivacaine and dextrobupivacaine may have an enantiomeric
excess in levobupivacaine from preferably 90% to 20%, more
preferably from 80% to 30% and more preferably yet from 70% to
40%.
[0052] The resulting active ingredients, comprehended in this
enantiomeric excess range, may be used in several pharmaceutical
compositions on their free base forms or their pharmaceutical
acceptable salts. The pharmaceutical compositions may be prepared
in analogy with the existent in the market for racemic bupivacaine
as well those active pharmaceutical ingredients may be employed in
novel pharmaceutical compositions.
[0053] According to the surprising and significant improvement in
the pharmacological profile of these pharmaceutical compositions,
prepared with the active ingredients manipulated on their
enantiomeric proportions, these formulated pharmaceutical
compositions may be employed in the same final concentrations
nowadays used for racemic bupivacaine, although conferring a lower
associated cardiotoxicity than bupivacaine and equivalent to the
cardiotoxicity observed on levobupivacaine, besides conferring an
equivalent anesthetic effect to the observed with racemic
bupivacaine.
[0054] Another objective of the present invention is the use of the
pharmaceutical compositions based on levobupivacaine formulated
with their free bases or their pharmaceutical acceptable salts.
[0055] The experiments described forward are illustrative but are
not limited on demonstrating the obtainment of bupivacaine
enantiomers as well they exemplify the proportions between the
enantiomers in order to obtain compositions with different
enantiomeric excess in levobupivacaine, illustrating the described
technique. In addition, it is presented two studies performed using
the pharmaceutical compositions prepared with active ingredients
enantiomeric manipulated, studies that demonstrate the improvement
on the pharmacological profile of those compositions, retaining its
lower cardiotoxicity property inherent to levobupivacaine.
[0056] The following examples are illustrative of the obtainment
process of bupivacaine enantiomers:
EXAMPLE 1
Separation of Racemic Bupivacaine Enantiomers in a Continuous
Process
[0057] A reactor fitted with mechanical stirring was charged with
10 liters of acetone, 288.4 g (1.0 mol) of bupivacaine base and
151.1 g (1.0 mol) of L-(+)-tartaric acid. The mixture was kept
under stirring at ambient temperature until complete dissolution of
the solids. To the resulting solution was added 1 g of
dextrobupivacaine tartrate and the system was kept under stirring
for approximately 7 hours. During this period the precipitation of
dextrobupivacaine tartrate was complete. The solids were filtered
and the filtrate was returned to the reactor. It was added of 1 g
of levobupivacaine tartrate and the solution was kept under
stirring for approximately 7 hours for the precipitation of
levobupivacaine tartrate. The solids were separated and the liquid
returned to the reactor. It was charged with 57.7 g of racemic
bupivacaine and 30.0 g of L-(+)-tartaric acid. Acetone was added to
complete the initial volume and the procedure was restarted by the
total dissolution of the solids, addition of dextrobupivacaine
tartrate seeds and keeping the solution under stirring for
approximately 7 hours to complete the precipitation of
dextrobupivacaine tartrate, and so on.
[0058] Dextrobupivacaine tartrate--Each batch yields m.congruent.45
g MP=178-186.degree. C.
[0059] Levobupivacaine tartrate--Each batch yields m.congruent.45 g
MP=110-120.degree. C.
EXAMPLE 2
Obtainment of Enriched Levobupivacaine
[0060] 45 g of levobupivacaine tartrate were dissolved in 222 mL of
water under stirring. Concentrated ammonium hydroxide was added
under stirring the pH adjusted at 9-10. The solids were filtered
and washed with 222 mL of water. The solids were dried in an oven
with temperature around 45.degree. C. until constant weight. Yield
27.6 g of enriched levobupivacaine free base (70%<ee<80%),
MP=125-132.degree. C.
EXAMPLE 3
Obtainment of Enriched Dextrobupivacaine
[0061] By the same procedure described in the example 2, but using
45 g of dextrobupivacaine tartrate in the place of levobupivacaine
tartrate it was obtained 27 g of enriched dextrobupivacaine free
base (70%<ee<80%), MP=125-132.degree. C.
EXAMPLE 4
Preparation of Pure Levobupivacaine
[0062] 27.6 g of enriched levobupivacaine free base obtained in the
example 2 were recristalized from 138 mL of hot isopropanol,
yielding 22.3 g of pure levobupivacaine free base with ee>99%
and MP=135-137.degree. C. [.alpha.].sup.D.sub.25=-80.degree. (c=2,
MeOH).
EXAMPLE 5
Preparation of Pure Dextrobupivacaine
[0063] 27 g of enriched dextrobupivacaine free base obtained by the
procedure described in example 3, were recristalized from 135 mL of
hot isopropanol, yielding 23.3 g of pure dextrobupivacaine
(ee>99.5%) MP=135-137.degree. C.,
[.alpha.].sup.D.sub.25=+80.degree. (c=2, MeOH).
EXAMPLE 6
Preparation of Levobupivacaine Hydrochloride
[0064] 23.3 g of pure levobupivacaine free base were dissolved in
125 mL of hot isopropanol. To this solution was added 8.8 mL of
concentrated hydrochloric acid. The solution was cooled to room
temperature and kept at a temperature between 4.degree. C. to
6.degree. C. for two hours. The mixture was vacuum filtered and the
solids were stirred with 20 mL of acetone. The mixture was filtered
and the solid was allowed to dry in an oven yielding 24.2 g of
levobupivacaine hydrochloride ee>99.5%, MP=246-250.degree. C.,
[.alpha.].sup.D.sub.25=-12 (c=2, H.sub.2O).
EXAMPLE 7
Preparation of Dextrobupivacaine Hydrochloride
[0065] Dextrobupivacaine hydrochloride may be obtained by the same
procedure described in example 6, by substituting pure
levobupivacaine free base by pure dextrobupivacaine free base
obtained in the example 5. The obtained product presents
ee>99.5%, MP=247-250.degree. C.
[.alpha.].sup.d.sub.25=+12.degree. (c=2, H.sub.2O).
[0066] Studies Conducted with Active Ingredients Manipulated on
Their Enantiomeric Relationships.
[0067] Study 1
[0068] To perform this study it was prepared the compositions with
active ingredients containing the enantiomeric proportions
described in table 1 presented below:
1 TABLE 1 % mass % mass % enantiomeric levohupivacaine
dextxobupivaciane excess Composition A 90 10 80 Composition B 75 25
50 Composition C 100 0 >99.5 Composition D 50 50 0
[0069] This study was designed to investigate the influence from
the presence of pre-determined quantities of dextrobupivacaine in
formulations of levobupivacaine to determine the influence of the
dextroenantiomer on the sciatic nerve of the rat "in vivo". The
final concentration (% on total mass) of the formulations was
0.5%.
[0070] Method:
[0071] 44 male Wistar rats were divided into 4 groups and injected
in the periarticular space of the right hind limb, according to
Truant's modified technique (Simonetti M P B, Valinetti E A,
Ferreira F M C--Braz. Journal Anesthesiol. Int. Issue, 9:65-72
(1998)). The following variables were studied: onset, motor block
duration and sensory block intensity. To evaluate the sensory block
intensity the animals were submitted to a pressoric stimuli (g/sec)
in an Analgesy Meter by the Randall-Selitto test. A limit of 300
g/sec was adopted to avoid tissue injury. The onset of the effect
from the injection of 0.2 mL of each 0.5% solution was evaluated by
hyperextension of the hind limb. Motor block was defined as the
time elapsed between the onset and the disappearance of such
signal. The paw withdrawal reflex (PWR) was assessed in order to
study the sensory block. This parameter was evaluated in the
following periods: t0 (baseline), t30, t60, t90, t120, t150, t180,
t210, t240.
[0072] Results:
[0073] The results obtained in this experiment was grouped in
tables 2 and 3 presented below:
2TABLE 2 Onset time and duration of motor block with compositions
A, B, C and D. Anesthetic Onset time Motor block duration
Composition A 2,4 .+-. 0,2 (n = 10) 161,5 .+-. 2,8 (n = 10)
Compesition B 4,1 .+-. 0,2 (n = 10) 133,0 .+-. 4,7 (n = 10)
Composition C 4,2 .+-. 0,3 (n = 12) 104,6 .+-. 4,3 (n = 12)
Composition D 9,1 .+-. 0,5 (n = 12) 116,2 .+-. 3,5 (n = 12)
[0074]
3TABLE 3 Sensory block evaluated by the paw withdrawal reflex
(g/sec) with compositions A, B, C and D. Local Anesthetic t0 t30
t60 T90 t120 t150 t180 t210 t240 Composition A 60 .+-. 2 112 .+-. 5
138 .+-. 4 140 .+-. 3 115 .+-. 4 89 .+-. 3 80 .+-. 3 72 .+-. 2 57
.+-. 2 Composition B 70 .+-. 3 129 .+-. 5 148 .+-. 3 161 .+-. 2 153
.+-. 3 144 .+-. 4 130 .+-. 6 117 .+-. 5 94 .+-. 2 Composition C 70
.+-. 3 132 .+-. 4 132 .+-. 5 130 .+-. 5 117 .+-. 5 110 .+-. 5 97
.+-. 4 90 .+-. 4 76 .+-. 6 Composition D 59 .+-. 3 112 .+-. 9 109
.+-. 7 117 .+-. 8 112 .+-. 6 108 .+-. 6 103 .+-. 4 96 .+-. 7 70
.+-. 7
[0075] Discussion:
[0076] The effects of the bupivacaine enantiomers demonstrate to be
stereospecific. The obtained results showed that the mixtures play
an important role on the efficacy of local anesthesia, in terms of
onset and duration of neural block (FIGS. 2 and 3). The FIG. 2
presents the onset time results, where "+" symbol represents the
statistic significant indices P<0.01 in relation to Composition
D, "*" symbol represents statistic significance indices p<0.01
in relation to composition A and the symbol "#" represents
statistic significance indices P<0.001 in relation to
composition C. FIG. 3 presents the results of duration of neural
block, where "+" symbol represents significant difference in
relation to composition D with p<0.001, the "*" symbol
represents significative difference in relation to composition A
with p<0.05 and the "#" symbol represents significative
difference in relation to composition C, with p<0.01. Both
parameters were improved in Composition A (ee=80%). These results
show that dextrobupivacaine must contribute to efficacy of sodium
channels nerve block. On the other hand, the action on fibers
A-delta and C seems to require a relative higher concentration of
dextrobupivacaine, once the greater analgesic activity was observed
with the composition B (FIG. 4). The FIG. 4 presents the results of
sensorial block, where "+" symbol represents statistic significance
indices p<0.05 in relation to composition D and the "*" symbol
represents the statistic significance indices p<0.05 in relation
to composition C. The relevance of this investigation points out
that there are effective differences on activity presented by
pharmaceutical compositions prepared with lower enantiomeric excess
of levobupivacaine studied until this moment.
[0077] Study 2
[0078] The following study had the objective to determinate the
effects of different compositions prepared on the cardiovascular
system, medium arterial pressure (MAP) and heart rate.
[0079] Method:
[0080] 35 rats were anesthetized with sodium pentobarbital (30
mg/kg i.p.). Jugular vein and carotid artery were cannulated for
injection of the local anesthetics and monitoring the medium
arterial pressure; ECG was recorded. Each rat was injected with 2.0
mg/kg in bolus.
[0081] It was used solutions with a final concentration of 0.5% (in
mass) of the active ingredients listed in Table 4.
[0082] The monitoring of the enantiomeric excess of the solutions
was carried out by HPLC analysis.
[0083] The results of the assays over cardiovascular system, mean
arterial pressure and heart rate were submitted by statistic
treatment with one-way ANOVA. p<0.05 was considered
statistically significant.
4TABLE 4 Active ingredients employed in the anesthetic solutions
prepared. Local Anesthetic Composition Solution A Racemic
bupivacaine Solution B Levobupivacaine (ee>99.5%) Solution C
Dextrobupivacaine (ee>99.5%) Solution D 10% in mass of
dextrobupivacaine + 90% in mass of levobupivacaine (ee 80%)
solution E 25% in mass of dextrobupivacaine + 75% in mass of
levobupivacaine
[0084] Results:
[0085] All local anesthetic solutions decreased the mean arterial
pressure and the heart rate 30 seconds after their administration.
After 1 minute, rats that took solutions B, D, and E, had those
parameters back the basal level. Rats that took solution A, had
those parameters back for the basal level only after 4 minutes.
Solution C, prepared with pure dextrobupivacaine induced
cardiovascular collapse and death in five from 7 rats tested.
[0086] The FIGS. 5 to 8 represents the results of the time course
of local anesthetic effects over mean arterial pressure for the
single experiments and FIG. 9 for all experiments. FIGS. 5 to 8
represent the time course of local anesthetic effect (L.A.E) over
mean arterial pressure (M.A.P.), where the "*" symbol is p<0.05
and "#" is p<0.01 and "+" p<0.001 (data expressed by
mean.+-.standard medium error n=9/group). FIG. 9 represents the
L.A.E over M.A.P. where data are expressed by mean.+-.standard
medium error (n=9/group).
[0087] Discussion:
[0088] Solutions D and E did not induce cardiodepressing effects.
The proportions used of the dextroenantiomer in these formulations
showed to be in reality less harmful than racemic bupivacaine
(solution A). The effects of these compositions presenting lower
enantiomeric excess on levobupivacaine seem to be similar to the
effects of pure levobupivacaine over cardiovascular system, besides
inducing a superior neural block.
* * * * *