U.S. patent application number 11/450009 was filed with the patent office on 2006-10-12 for artemisinins with improved stability and bioavailability for therapeutic drug development and application.
Invention is credited to Apurba K. Bhattacharjee, Mark G. Hartell, Rickey P. Hicks, Wilbur K. Milhous, John E. VanHamont.
Application Number | 20060229279 11/450009 |
Document ID | / |
Family ID | 27805252 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060229279 |
Kind Code |
A1 |
Hartell; Mark G. ; et
al. |
October 12, 2006 |
Artemisinins with improved stability and bioavailability for
therapeutic drug development and application
Abstract
A stable form of artemisinin wherein an artelinic acid or
artesunic acid is complexed with cyclodextrin analogs, preferably,
.beta.-cyclodextrin. The complexed cyclodextrin artemisinin
formulation shields the peroxide portion of the artemisinin
backbone from hydrolytic decomposition rendering it stable in
solution. Artelinic acid and cyclodextrin are placed into contact
with one another to yield a 2:1 molecular species. Artesunic acid
and cyclodextrin yield a 1:1 molecular species. The complexed
cyclodextrin artemisinin formulation is effective for the treatment
of malaria and is stable in solution for long periods of time.
Inventors: |
Hartell; Mark G.; (Laurel,
MD) ; Bhattacharjee; Apurba K.; (Silver Spring,
MD) ; Hicks; Rickey P.; (Woodbridge, VA) ;
VanHamont; John E.; (Fort Meade, MD) ; Milhous;
Wilbur K.; (Germantown, MD) |
Correspondence
Address: |
NASH & TITUS, LLC
21402 UNISON RD
MIDDLEBURG
VA
20117
US
|
Family ID: |
27805252 |
Appl. No.: |
11/450009 |
Filed: |
June 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11113546 |
Apr 25, 2005 |
7084132 |
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11450009 |
Jun 9, 2006 |
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10376387 |
Feb 27, 2003 |
6951846 |
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11113546 |
Apr 25, 2005 |
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60362985 |
Mar 7, 2002 |
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Current U.S.
Class: |
514/58 ;
514/452 |
Current CPC
Class: |
Y02A 50/411 20180101;
Y02A 50/30 20180101; A61K 47/6951 20170801; A61K 31/724 20130101;
Y10S 514/895 20130101; B82Y 5/00 20130101; A61K 31/55 20130101 |
Class at
Publication: |
514/058 ;
514/452 |
International
Class: |
A61K 31/724 20060101
A61K031/724; A61K 31/335 20060101 A61K031/335 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The invention described herein may be manufactured, used and
licensed by or for the U.S. Government.
Claims
1-57. (canceled)
58. A method of treating a patient having malaria: comprising
administering to said patient a composition comprising a complexed
cyclodextrin formulation of artemisinin, wherein said cyclodextrin
is complexed with artelinic acid in a 2:1 molar ratio in aqueous
solution.
59. The method of claim 58, wherein said administering is by
intravenous injection, oral dose, sublingual dose, or
suppository.
60. The method of claim 58, wherein said administering to said
patient is by a dose of 4-6 milligrams of artelinic acid per
kilogram of body weight.
61. The method of claim 58, wherein said 40 milligrams of said
artemisinin complexed with cyclodextrin is dissolved per milliliter
of aqueous solution.
62. A method of treating a patient with malaria: comprising
administering to said patient an antimalarial composition
comprising: a complexed cyclodextrin formulation of artemisinin,
wherein said cyclodextrin is complexed with artesunic acid in a 1:1
ratio in an aqueous solution.
63. The method of claim 62, wherein said administering is by
intravenous injection, oral dose, sublingual dose, or
suppository.
64. The method of claim 62, wherein said administering to said
patient is by a dose of 4-6 milligrams of artesunic acid per
kilogram of body weight.
65. The method of claim 62, wherein said 40 milligrams of said
artemisinin complexed with cyclodextrin is dissolved per milliliter
of aqueous solution.
Description
[0001] This application claims priority of provisional application
No. 60/362,985 filed Mar. 7, 2002.
BACKGROUND OF THE INVENTION
[0003] 1. Field Of The Invention
[0004] A novel form of artemisinins that are complexed with
cyclodextrin for solving stability problems associated with
previous forms of artemisinins.
[0005] 2. Brief Description Of Related Art
[0006] Artelinic acid is an effective antimalarial agent when in
contact with the malarial parasite. However, artelinic acid has
poor stability in solution and, thus, has limited bioavailability
in vivo. Artemisinins, as a class, include such analogs as
artelinic acid and artesunic acid among many others. Currently, no
analog of the artemisinin class of compounds exists which can
remain stable in solution. Injectable formulations of artemisinin
analogs, such as artelinic acid and artesunic acid, are not FDA
approved due to their instability in solution. All artemisinins
contain a peroxide bridge susceptible to hydrolytic cleavage.
Artemisinins have been found to yield an inferior class of
antimalarials due to these severe limitations in chemical
stability. Artemisinins are limited to only being packaged as
solids for oral dosing, as previous patents have claimed. U.S. Pat.
Nos. 6,326,023; 6,307,068; 6,306,896; 5,834,491; 5,677,331;
5,637,594; 5,486,535; 5,278,173; 5,270,037; 5,219,865; 5,021,426;
5,011,951.
[0007] Application of an antimalarial formulation must be specific
to administration in hot, humid tropical regions native to the
malarial parasite. Thus, chemical stability under drastic
environmental conditions is essential. Attempts to produce a more
stable form of artelinic acid have been accompanied by critical
limitations. A soluble sodium salt of artelinic acid has been
successfully formulated, but eventually degrades over time. This is
presumably due to a re-formation of the insoluble acid. Numerous
attempts at preventing this precipitate have been unsuccessful.
[0008] The osmolality of the salt solution is significantly less
than the predicted value indicating possible inter-molecular
complexation that may be responsible for eventual precipitation
over time. An amine-based buffer of artelinic acid has been
successfully formulated, but yields a higher pH solution (>8.0)
that induces significant vein irritation upon injection. Additional
localized redness and swelling surrounding the injection site is a
notable contraindication to a preferred intravenous formulation.
Additionally, amine-based buffers have been observed to take on a
strong yellow hue over time. The mechanism of color formation has
not been deduced, but implies a modification of the artelinate
formulation, which is not conducive to pharmaceutical preparations
where a defined constant state of purity is essential.
[0009] U.S. Pat. Nos. 6,326,023; 6,307,068; 6,306,896; 5,834,491;
5,677,331; 5,637,594; 5,486,535; 5,278,173; 5,270,037; 5,219,865;
5,021,426; 5,011,951 are only directed to be packaged as solids for
oral dosing.
[0010] Therefore, there is a need to provide a form of artemisinins
that solve the stability problems associated with previous
formulations.
[0011] It is an object of the present invention to provide a form
of artemisinins, such as but not limited to artelinic acid and
artesunic acid that solves the stability problems associated with
previous formulations.
[0012] It is another object of the present invention to provide a
stable form of artemisinins that is injectable.
[0013] It is still another object of the present invention to
provide a stable form of artemisinins that does not develop a
yellow hue over time.
[0014] It is still another object of the invention to promote
bioavailability and membrane permeability while decreasing the
likelihood of localized inflammation at the route of entry, thus
increasing its therapeutic activity.
[0015] These and other objects of the invention will become
apparent upon a reading of the entire disclosure.
SUMMARY OF THE INVENTION
[0016] The present invention is directed to cyclodextrin complexed
with artelinic acid or artesunic acid to form complexed
cyclodextrin-artemisinin formulations in a 2:1 ratio of
cyclodextrin per artelinic acid molecule or in a 1:1 ratio of
cyclodextrin per artesunic acid molecule. The formulation is stable
in solution, bioavailable, membrane permeable and does not cause
inflammation upon injection.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 is a plot of the hypsochromic shift observed with
increasing concentrations of cyclodextrin. Artelinic acid
concentration=10 mM.
[0018] FIG. 2a is an absorption spectrum of 10 mM artelinic acid
with and without 1 mM .beta.-cyclodextrin;
[0019] FIG. 2b is an absorption spectrum of 10 mM artelinic acid
with and without 4 mM .beta.-cyclodextrin;
[0020] FIG. 3 is a 600 MHz WATERGATE-TOCSY NMR spectrum of 1.2 mM
artelinic acid with 2.5 mM .beta.-cyclodextrin in PBS (pH 7.4);
[0021] FIG. 4 is a 600 MHz WATERGATE-ROESY NMR spectrum of 1.2 mM
artelinic acid with 2.5 mM .beta.-cyclodextrin in PBS (pH 7.4);
[0022] FIG. 5 is a 600 MHz WATERGATE-ROESY NMR spectrum of 1.2 mM
artelinic acid with 2.5 mM .beta.-cyclodextrin in PBS (pH 7.4);
[0023] FIG. 6 is a 600 MHz WATERGATE-ROESY NMR spectrum of
artesunate with an excess of .beta.-cyclodextrin in PBS (pH
7.4);
[0024] FIG. 7a is the aromatic region of the 600 MHz proton spectra
of 1.2 mM artelinic acid;
[0025] FIG. 7b is the aromatic region of the 600 MHz proton spectra
of 1.2 mM artelinic acid complexed with 2.5 mM .beta.-cyclodextrin
in PBS (pH 7.4);
[0026] FIG. 8a is the alkyl region of the 600 MHz proton NMR
spectra of 1.2 mM artelinic acid;
[0027] FIG. 8b is a 600 mHz proton NMR spectrum of 1.2 mM artelinic
acid complexed with 2.5 mM .beta.-cyclodextrin in PBS (pH 7.4);
[0028] FIG. 9a is a 600 MHz proton NMR spectrum of 2.5 mM
.beta.-cyclodextrin in PBS (pH 7.4);
[0029] FIG. 9b is a 600 MHz proton NMR spectrum of 2.5 mM
.beta.-cyclodextrin with 1.2 mM artelinic acid in PBS (pH 7.4);
[0030] FIG. 10a is a 600 MHz proton NMR spectrum (protons number 2
to 6) of 2.5 mM .beta.-cyclodextrin in PBS (pH 7.4);
[0031] FIG. 10b is a 600 MHz proton NMR spectrum (protons number 2
to 6) of 2.5 mM .beta.-cyclodextrin complexed with 1.2 mM artelinic
acid in PBS (pH 7.4);
[0032] FIG. 10c is a 600 MHz proton NMR spectrum (protons number 2
to 6) of artesunate with an excess of .beta.-cyclodextrin in PBS
(pH 7.4);
[0033] FIG. 11 is a 600 MHz proton NMR spectrum of 2.5 mM
.beta.-cyclodextrin and 1.2 mM artelinic acid in PBS buffer at pH
7.4 with 1:9 D2O/H2O;
[0034] FIG. 12 is a 2D NOESY spectrum of 2.5 mM .beta.-cyclodextrin
and 1.2 mM artelinic acid in PBS buffer at pH 7.4 with 1:9
D.sub.2O/H.sub.2O;
[0035] FIG. 13 is a 600 MHz proton NMR spectrum of artelinic acid
BN BPI 1387, WR#255663;
[0036] FIG. 14 is a 600 MHz proton NMR spectrum of 2D TOESY
spectrum of 2.5 mM .beta.-cyclodextrin and 1.2 mM artelinic acid in
PBS buffer at pH 7.4 with 1:9 D.sub.20/H.sub.2O;
[0037] FIG. 15 is a 600 MHz proton NMR spectrum of 2D ROESY
spectrum of 2.5 mM .beta.-cyclodextrin and 1.2 mM artelinic acid in
PBS buffer at pH 7.4 with 1:9 D.sub.2O/H.sub.2O;
[0038] FIG. 16 is a 600 MHz proton NMR spectrum of artesunate with
an excess of .beta.-cyclodextrin in PBS buffer at pH 7.4;
[0039] FIG. 17 is a 600 MHz proton NMR spectrum of 2D ROESY
spectrum of artesunate with an excess of .beta.-cyclodextrin in PBS
buffer at pH 7.4;
[0040] FIG. 18a is the electrostatic potential map of the primary
face of .beta.-cyclodextrin looking into the molecule from the
top;
[0041] FIG. 18b is the electrostatic potential map of the primary
face of .beta.-cyclodextrin as shown in FIG. 18a rotated to the
left;
[0042] FIG. 18c is the electrostatic potential map of the secondary
face of .beta.-cyclodextrin;
[0043] FIG. 18d is a molecular model of FIG. 18d illustrating the
positions of specific atoms;
[0044] FIG. 19a is a side view of the electrostatic potential map
of artelinic acid;
[0045] FIG. 19b is a rear view of the electrostatic potential map
of artelinic acid;
[0046] FIG. 20 is the electrostatic potential map of
.beta.-cyclodextrin complexed with artelinic acid in a 2:1
molecular ratio;
[0047] FIG. 21 is a molecular model of .beta.-cyclodextrin
complexed with artelinic acid in a 2:1 molecular ratio showing
degrees of insertion and interaction between each molecule;
[0048] FIG. 22 is an axial view from the primary face of the
electrostatic potential map of .beta.-cyclodextrin complexed with
artelinic acid in a 2:1 molecular ratio indicating the
electrostatic interaction between the benzoic acid moiety and one
of the cyclodextrins;
[0049] FIG. 23 is a plot of osmolality versus concentration of
artelinate in aqueous solution compared to theoretical
determinations based on the complete disassociation of the
salt;
[0050] FIG. 24 is a plot of osmolality versus concentration of a
lysine-artelinate salt preparation in aqueous solution compared to
theoretical determinations based on the complete disassociation of
the salt;
[0051] FIG. 25 is a plot of osmolality versus concentration of a
lysine-artelinate salt preparation with 3 molar equivalents of
lysine in aqueous solution compared to theoretical determinations
based on complete disassociation of the salt;
[0052] FIG. 26 is the linear regression (R=0.994, p<0.0001) of
experimentally measured osmolality of artelinate complexed with
hydroxypropyl-.beta.-cyclodextrin (1:2 mole ratio) in aqueous
solution. Upper and lower 95% confidence intervals and 95%
prediction limits are also indicated;
[0053] FIG. 27a-c are plots of relative deviation between
experimentally measured osmolality and theoretical determinations
based on complete disassociation for 3 aqueous artelinate
formulations: lysine-artelinate prepared with 1 molar equivalent of
lysine, lysine-artelinate prepared with 3 molar equivalents of
lysine, and hydroxypropyl-.beta.-cyclodextrin-artelinate (2:1)
complex;
DETAILED DESCRIPTION
[0054] The present invention is directed to a novel form of
artemisinins that remain stable over time in solution. The
artemisinins may be, but are not limited to artelinic acid and
artesunic acid. This novel form of artemisinins uses a unique
complexed form of the therapeutic agent with cyclodextrin analogs,
such as but not limited to alpha-, beta-, and gamma-cyclodextrin
analogs and their derivatives.
[0055] The present invention is directed to cyclodextrin complexed
with artelinic acid in a 2:1 ratio which is a form of artemisinin
that alters the electron cloud surrounding the artemisinin molecule
in such a way as to stabilize this agent to promote bioavailability
and membrane permeability while decreasing the likelihood of
localized inflammation at the route of entry. Thus, this form of
artemisinin increases its therapeutic activity. Artesunic acid was
complexed with cyclodextrin, but in a unique 1:1 ratio in such a
way as to stabilize the agent yield similar increases in its
therapeutic activity.
[0056] The stability of the artemisinins is achieved by changing
the physiocochemical properties such as but not limited to electron
density, electrostatic potential and charge transfer mediated
complexation.
[0057] The complexed cyclodextrin formulation of the artemisinins
described deliberately shields the peroxide bridge of the
artemisinin backbone from hydrolytic decomposition. Additionally,
the aromatic benzoic acid portion of the artelinate molecule is
also complexed with a second cyclodextrin molecule. This unique 2:1
complexation with cyclodextrin is not intuitively obvious because
artelinic acid alone is unstable in aqueous solution. Simply
placing cyclodextrin in solution with artelinic acid would not
achieve these results, as the artelinic acid would not be in
contact with the cyclodextrin to form complexation. Further,
cyclodextrin is know to form complexes with itself and thus may not
be readily available in solution to interact efficiently and
effectively with the artelinic acid. The inventors have placed
artelinic acid and cyclodextrin into contact with one another and
have complexed them in such a manner as to yield a stable 2:1
molecular species. The inventors have also placed artesunic acid
and cyclodextrin into contact with one another and have complexed
them in such a manner as to yield a stable 1:1 molecular
species.
[0058] The present molecules are stable under ambient or
physiologically relevant conditions.
Materials And Methods
[0059] .beta.-cyclodextrin was obtained from Sigma-Aldrich Corp.,
St. Louis, Mo. Artelinic acid was alkalinized with NaOH to yield
the sodium salt. Standardized PBS buffer at a pH of 7.4 was
obtained from Invitrogen Corp., Carlsbad, Calif.
[0060] Absorption Spectroscopy Studies.
[0061] Mixtures of artelinate (10 .mu.M) were prepared with
increasing concentrations of .beta.-cyclodextrin (0.0, 1.0, 4.0,
6.0, and 9.0 mM). Absorption spectra were collected on a Beckman DU
Series 600 Spectrophotometer.
[0062] The spectra collected indicated a clear hypsochromic or blue
shift in the absorption maximum at 230 nm with increasing
concentrations of cyclodextrin. Hypochromic effects were also
notable at 230 nm, as well as the broader transitions observed at
275 and 382 nm (FIG. 1). This combined observation is consistent
with inclusion interactions of the benzoic anion of artelinate with
cyclodextrin.
[0063] Changes in observed isosbestic points at higher cyclodextrin
concentrations indicates a complicated molecular species containing
greater than a simple 1:1 molecular species (FIGS. 2a and 2b).
[0064] .sup.1H NMR Studies.
[0065] Mixtures of .beta.-cyclodextrin (2.5 mM) and artelinic acid
(1.2 mM) were prepared in PBS (pH 7.4) and incubated at 37.degree.
C. for 2-3 hour to promote complexation prior to analysis.
[0066] All .sup.1H NMR data was collected using a Bruker DRX-600
spectrometer operating at a proton frequency of 600.02 MHz at a
temperature of 25.degree. C. Solvent suppression was accomplished
by application of the WATERGATE (WATER suppression by GrAdient
Tailored Excitation) pulse sequence developed by Sklenar and
co-workers. This sequence provides excellent suppression of the
water resonance by a combination of rf pulses and a series of
gradient pulses. The sequence combines a non-selective 90.degree.
pulse with a symmetrical echo formed by two short gradient pulses
in conjunction with a 180 selective (on water) pulse train.
[0067] The two-dimensional WATERGATE-TOCSY experiment employed a
modified MLEV-17 spin-lock sequence for a total mixing time of 80
ms, including the 2.5 ms trim pulses at the beginning and the end
of the spin-lock. The spectrum was collected with a spectral width
of 7183.91 Hz (11.972 ppm) using 2K data points with 32 scans per
256 t.sub.1 increments with a 1.5 s recycle delay. The data was
processed by multiplication with a 90.degree. shifted sine-bell
window function in each dimension, with one zero fill in the
f.sub.1 dimension before transformation to produce matrices
consisting of 512 data points in both dimensions.
[0068] The two-dimensional WATERGATE-NOESY spectra were collected
with a spectral width of 7183.91 Hz (11.972 ppm) using 2K data
points with 128 scans per 512 t.sub.1 increments with a 1.5 s
recycle delay. The data was processed by multiplication with a
90.degree. shifted sine-bell window function in each dimension,
with one zero fill in the f.sub.1 dimension before transformation
to produce matrices consisting of 512 data points in both
dimensions. Two different experiments were conducted with mixing
times of 50 and 600 ms.
[0069] The two-dimensional WATERGATE-ROESY spectrum was collected
with a spectral width of 7183.91 Hz (11.972 ppm) using 2K data
points with 256 scans per 512 t.sub.1 increments with a 1.5 s
recycle delay with a spin-lock mixing pulse of 400 ms. The data was
processed by multiplication with a 90.degree. shifted sine-bell
window function in each dimension, with one zero fill in the
f.sub.1 dimension before transformation to produce matrices
consisting of 512 data points in both dimensions.
[0070] Two-dimensional NMR methods were used to determine the
degree of capping or complexation of artelinic acid by
.beta.-cyclodextrin. The 2D WATERGATE-TOSCY spectrum of artelinic
acid (FIG. 3) clearly indicates that the individual spin-spin
coupling networks of a mixture of artelinic acid and
.beta.-cyclodextrin can be resolved. In FIG. 3, the spin-spin
coupling network for .beta.-cyclodextrin is shown at A and the
spin-spin coupling network for the alkyl ring of artenilate is
shown at B. The 2D-rotating frame NOE spectrum, WATERGATE-ROESY, of
artelinic acid was collected at a mixing time of 400 ms and is
shown in FIG. 4. The labeled intermolecular ROE interaction between
the aromatic protons of artelinic acid with both the anomeric and
ring protons of .beta.-cyclodextrin proves that this region of
artelinic acid is complexed with one molecule of
.beta.-cyclodextrin. In FIGS. 4, A, B and C indicate the
intermolecular dipolar ROE coupling between the aromatic protons of
artelinate with the glucose ring protons of .beta.-cyclodextrin.
The ROE between the meta protons are more intense than those
observed for the ortho protons indicating that meta protons are
inserted deeper into the cavity. D and F indicate the dipolar
coupling between the ortho protons of artelinate with the two
benzyl protons of artelinate. E indicates the dipolar coupling
between the meta protons of artelinate with the anomeric protons of
.beta.-cyclodextrin. FIG. 5 shows the alkyl region of this same
spectrum. The labeled intermolecular ROE's between the alkyl ring
protons of artelinic acid with both the anomeric and ring protons
of .beta.-cyclodextrin indicate that this region of artelinic acid
is complexed with one molecule of .beta.-cyclodextrin. These
observations are similar to those reported by Nishijo (Nishijo, J.;
Nagai, M.; Yasuda, M.; Ohno, E.; Ushiroda, Y. J. Pharm. Sci. 1995,
84, 1420-1426) and by Redenti (Redenti, E.; Ventura, P.; Fronza,
G.;Selva, A.;Rivara, S.;Plazzi, P. V.; Mor, M. J. Pharm. Sci. 1999,
88, 599-607) in similar NMR .beta.-cyclodextrin complexation
studies. In FIG. 5, A represents a region that contains the dipolar
coupling between the ring protons of .beta.-cyclodextrin and the
alkyl ring proton of artelinate; and B represents the region that
contains the dipolar coupling of the anomeric protons of
.beta.-cyclodextrin with the alkyl protons of artelinate.
[0071] Two 2D WATERGATE-NOESY spectra were collected at mixing
times of 50 and 600 ms (data not shown). The NOESY spectrum
collected at 600 ms gave similar intermolecular and intramolecular
NOE's to those observed in the ROESY spectrum, however the observed
intensities were reduced. The NOESY spectrum collected at 50 ms did
not exhibit the intermolecular NOE's between artenilate and
.beta.-cyclodextrin. This observation is consistent with what one
would expect due to the fact that intermolecular NOE's require a
longer mixing time to develop as compared to intramolecular
NOE's.
[0072] The 2D ROESY and NOESY data clearly indicate that both the
alkyl and aromatic regions of artelinic acid are complexed with one
individual molecule of .beta.-cyclodextrin.
[0073] In FIG. 6, the spectrum of artesunate with an excess of
.beta.-cyclodextrin in PBS is shown. This data clearly indicates
that the artesunate is capped by .beta.-cyclodextrin in a 1:1
ratio. The region that is represented by A contains the
intramolecular dipolar coupling the alkyl ring proton of
artesunate. The region that is represented by B contains the
intermolecular dipolar coupling the alkyl ring proton of artesunate
with the ring protons of .beta.-cyclodextrin. The region that is
represented by C contains the intermolecular dipolar coupling the
alkyl ring proton of artesunate with the anomeric protons of
.beta.-cyclodextrin. The region that is represented by D contains
additional intramolecular dipolar coupling the alkyl ring proton of
artesunate. The region that is represented by E contains the
intramolecular dipolar coupling of the .beta.-cyclodextrin.
[0074] FIG. 7a shows the aromatic region of the 600 MHz proton
spectra of 1.2 mM artelinic acid and FIG. 7b is the aromatic region
of the 600 MHz proton spectra of 1.2 mM artelinic acid complexed
with 2.5 mM .beta.-cyclodextrin. Upon complexation the aromatic
resonances of artelinate are both shifted upfield. The chemical
shift values and the relative changes in chemical shift values are
given in Table 1. A similar shift of aromatic protons resonances of
ketoconazole on complexation with .beta.-cyclodextrin was reported
by Redenti and co-workers (Redenti, E.; Ventura, P.; Fronza,
G.;Selva, A.;Rivara, S.;Plazzi, P. V.; Mor, M. J. Pharm. Sci. 1999,
88, 599-607). In addition, the intensity of the resonance for
protons 2 and 2' is reduced indicating complexation. TABLE-US-00001
TABLE 1 .sup.1H Chemical Shift Assignments (.delta.) for the
Aromatic Protons and Methyl Protons of Artelinic Acid Chemical
Shift complexed with Proton Chemical Shift .beta.-cyclodextrin
.DELTA..delta. (ppm) 3 and 3' 8.09 7.82 +0.27 2 and 2' 7.42 7.25
+0.17 methyl #1 0.98 1.02 -0.04 methyl #2 0.95 0.95 0.00
[0075] FIG. 8a shows the alkyl region of the 600 MHz proton spectra
of 1.2 mM artelinic acid and FIG. 8b shows 1.2 mM artelinic acid
complexed with 2.5 mM .beta.-cyclodextrin. As seen from these
spectra the chemical shift position and the appearance of the
methyl protons have changed indicating complexation of this region
of the molecule with .beta.-cyclodextrin. The chemical shift of the
resonances for methyl group #1 are shifted upfield by 0.04 ppm
(Table 1). The resonances for both methyl groups were broadened and
less well resolved.
[0076] FIG. 9a is a 600 MHz proton spectra of 2.5 mM
.beta.-cyclodextrin and FIG. 9b is a 600 MHz proton spectra of 2.5
mM .beta.-cyclodextrin with 1.2 mM artelinic acid. These spectra
clearly indicate that chemical values for protons 2 to 6 on
.beta.-cyclodextrin change on complexation with artelinic acid.
Similar shifts in the proton resonances for .beta.-cyclodextrin
have been reported by Nishijo and co-workers (Nishijo, J.; Nagai,
M.; Yasuda, M.; Ohno, E.; Ushiroda, Y. J. Pharm. Sci. 1995, 84,
1420-1426).
[0077] FIG. 10a-10c show the proton spectra (protons number 2 to 6)
of 2.5 mM .beta.-cyclodextrin, 2.5 mM .beta.-cyclodextrin complexed
with 1.2 mM artelinic acid and 1.2 mM artesunate in an excess of
.beta.-cyclodextrin, respectively. These spectra clearly indicate a
different mode of complexation for the two artemisinin analogs.
[0078] Table 2 summarizes the chemical shift assignments for
cyclodextrin compared with the corresponding complexes with
artelinic acid and artesunic acid as derived from FIGS. 9 and 10.
The change in chemical shifts (.DELTA..delta.) clearly demonstrate
that both cyclodextrins of the artelinic acid complex and the
cyclodextrin of the artesunic acid complex coordinate at the 3-H
end or secondary face (FIG. 18) of the cyclodextrin. Further, the
benzoic acid moiety of artelinic acid coordinates deeply into the
cyclodextrin pocket yielding significant changes in chemical shift
for the 3-H, 5-H, and 6-H protons. In contrast, artesunic acid,
which only binds to one cyclodextrin at the peroxide bridge,
produced chemical shift changes of a lower magnitude indicating a
more shallow binding interaction. Lastly, for the
artesunate-cyclodextrin complex the changes in chemical shift
indicate .DELTA..delta. of 6H<5H<3H which clearly
demonstrates this shallow binding interaction compared to the deep
insertion of the benzoic acid moiety of artelinic acid. This data
clearly supports a unique stereochemical arrangement based upon the
physicochemical properties of each molecular species to yield a
specific stable complex. TABLE-US-00002 TABLE 2 .sup.1H Chemical
Shift Assignments (.delta.) for the Cyclodextrin Protons (2 through
6) 2H 3H 4H 5H 6H .beta.-cyclodextrin 3.63 3.94 3.56 3.83 3.86
artelinic acid 3.61 3.83 3.53 3.72 3.74 .DELTA..delta.' 0.02 0.11
0.03 0.11 0.12 .beta.-cyclodextrin 3.63 3.94 3.56 3.83 3.86
artesuate 3.62 3.88 3.55 3.79 3.84 .DELTA..delta. 0.01 0.06 0.01
0.04 0.02
[0079] FIGS. 11 through 17 provide ancillary and supportive data
that was used in elucidating the structural conformation of the
described cyclodextrin complexes.
[0080] Molecular Electrostatic Potential Mapping and
Docking/Affinity Determinations.
[0081] Molecular Electrostatic Potential (MEP) maps on cyclodextrin
and artelinic acid were developed by calculating electrostatic
potentials on the van der Waals surface of the molecules using the
semi-empirical PM3 molecular orbital theory as implemented in the
SPARTAN software (SPARTAN version 4.0, Wavefunction, Inc., 18401
Von Karman Ave., #370, Irvine, Calif. 92715 U.S.A. 1995
Wavefunction, Inc.). PM3 is a semi-empirical quantum chemical
theory model based on Thiel's integral formalism underlying MNDO/d,
and is used in conjunction with parameters for both transition and
non-transition metals (reference: (a) W. Thiel and A. Voityuk,
Theor. Chim. Acta., 81, 391, (1992); (b) W. Thiel and A. Voityuk,
Int. J. Quantum Chem., 44, 807 (1992).
[0082] Molecular electrostatic potential (MEP) maps and their
electrostatic potential energy isopotential profiles were generated
and sampled over the entire accessible surface of a molecule
(corresponding roughly to a van der Waals contact surface). The MEP
maps provide a measure of charge distribution from the point of
view of an approaching reagent. This is calculated using a test
positive charge as the probe. Thus, these types of profiles can
provide an estimate of electronic distribution surrounding the
molecule so as to enable qualitative assessment of any possible
interaction with an approaching molecule. However, conformation
search calculations using the "systematic search" technique via the
single-point PM3 method of SPARTAN were used to generate different
conformers for each of the molecules. The minimum energy conformer
with highest abundance (a Boltzman population density greater than
70.0%) was chosen for full geometry optimization using the PM3
algorithm. The MEP profiles were generated on the optimized
geometry of the molecules. The computations were carried out on a
Silicon Graphics Octane workstation.
[0083] To further understand the binding affinities between
cyclodextrin and artelinic acid, the complete optimized structures
of both the compounds have been considered and docking calculations
using the Docking/affinity module in Insight II (Accelrys Inc.,
9685 Scranton Road, San Diego, Calif. 92121-3752) were conducted.
See Oprea, T. I. and Marshall, G. R. (1998) Receptor-based
prediction of binding affinities. Perspectives in Drug Discovery
and Design 9/10/11:35-61; and Insight II User Guide, San Diego:
Accelrys Inc. (2002), which are herein incorporated by
reference.
[0084] Docking/affinity module in Insight II allows calculating the
nonbonded energy between two molecules using explicit van der Waals
energy, explicit electrostatic (Coulombic) energy, or both van der
Waals and electrostatic energies. The number of atoms included in
the calculation can be limited by specifying a monomer- or
residue-based cutoff. Other methods known in the art may be used,
for example, the computation can be done using a pre-computed
energy grid.
[0085] These molecular modeling determinations based on unique and
specific physicochemical properties of the artemisinins studied
complexed with .beta.-cyclodextrin produced conceptual models which
clearly rationalized the direct physical measurements of the NMR
experiments. FIGS. 18a-d illustrate the unique electrostatic
potential map of .beta.-cyclodextrin showing the primary binding
faces (FIGS. 18a and 18b) and secondary binding faces (FIGS. 18c
and FIG. 18d). Most notable is the unique net positive region 1 of
the electron cloud at the primary face.
[0086] FIGS. 19a and 19b illustrate the unique electrostatic
potential map of artelinic acid. Most notable is the dense negative
region 2 of the carboxylic acid tail as well as a more subtle
negative region 3 of the peroxide bridge.
[0087] FIG. 20 clearly demonstrates the 2:1 complexation of
.beta.-cyclodextrin with artelinic acid. Two .beta.-cyclodextrin
molecules are shown at 4 and one artelinic acid molecule is shown
at 5. The depth of insertion of the carboxylic acid tail compared
to the peroxide bridge portion of the molecule is more clearly
illustrated in the corresponding ball-and-stick model of the
complex in FIG. 2 lwherein two .beta.-cyclodextrin molecules are
shown at 4 and one artelinic acid molecule is shown at 5.
[0088] Lastly, FIG. 22 directly illustrates the unique
physicochemical interaction of the electrostatc potential map of
cyclodextrin with that of the artelinic acid tail. This axial view
into the primary face of the second cyclodextrin molecule clearly
illustrates this unique and selective electrostatic interaction.
The negative region of the electrostatic potential map is shown at
6 and the positive region of electrostatic potential map is shown
at 7.
[0089] Simple docking calculations do not yield these results as
they assume an in vacuo environment. Inclusion complexes with
cyclodextrins are mediated by the release of high-energy water
molecules from the inner core of the cyclodextrin molecule.
Therefore, direct structural measurements of the complex by
techniques such as high resolution multi-dimensional NMR
rationalized by physicochemical property determinations such as but
not limited to molecular electrostratic potential mapping is
specifically required to accurately characterize these
complexes.
[0090] Osmometry Determinations.
[0091] Solutions of hydroxypropyl-.beta.-cyclodextrin and artelinic
acid of varied compositions as indicated were measured at room
temperature using a Fiske ONE-TEN Osmometer (Fiske Associates,
Norwood Mass., USA). The solvent for all experiments was ultra-pure
distilled deionized water (18 M.OMEGA.) filtered through a 0.45
.mu.m filter. Small sample volumes (15 .mu.L) were measured in
units of mOsmol/kg water with an instrument repeatability of .+-.2
mOsmol/kg water in the data range studied (0 to 400 mOsmol/kg
water). The instrument was calibrated routinely with NIST standards
of NaCl and a daily NIST reference of NaCl was verified at the
start of each set of experiments.
[0092] Osmolality is a direct measure of the degree of molecular
dissociation of a species in water. FIG. 23 illustrates the
deviation of measured osmolality in aqueous artelinate solutions
versus theoretical calculations which assume complete dissociation.
This deviation from ideality also appears to have a significant
margin of error as observed by the marked degree of data scatter in
the measurments.
[0093] FIGS. 24 and 25 illustrate a similar relationship between
measured osmolality and ideal dissociation with a lysine salt
formulation and a lysine salt formulation with 3 molar equivalents
excess lysine. All three artelinate formulations appear to deviate
strongly from ideality. Secondly, the measure of osmolality versus
concentration of artelinate appears to be biphasic as demonstrated
most clearly in FIG. 25, but also observed in FIGS. 23 and 24.
[0094] FIG. 26 illustrates the strong linear correlation of the
experimentally measured osmolality of artesunate complexed with
hydroxypropyl-.beta.-cyclodextrin in aqueous solutions.
Hydroxypropyl-.beta.-cyclodextrin was chosen for all osmolality
determinations, as its aqueous solubility is greater than
.beta.-cyclodextrin and its well-established pharmacological
compatibility for future i.v. drug formulations.
[0095] Measured deviation in osmolality of the artelinic
acid-cyclodextrin ( 1:2) formulation after 28 days at room
temperature was <7% in the concentration range of 15-25 mg/mL
artelinate. This 7% deviation was consis tently observed as an
increase in osmolality due to an enhancement of solvation over
time, rather than a decrease in solubility. The more concentrated
solutions of cyclodextrin complexes would need to incubate for
longer periods of time to ensure maximum complexation.
[0096] FIGS. 27a-c illustrate the deviations from ideality of three
artelinate formulations, 1 molar equivalent of lysine shown at FIG.
27a, lysine-artelinate prepared with 3 molar equivalens of lysine
shown at FIG. 27b and cyclodextrin-artelinate (2: 1) complex shown
at FIG. 27c. The artelinate-cyclodextrin formulation clearly
deviates from ideality in a more predictable manner. The decrease
in relative deviation with increasing concentration is mostly
likely due to enhanced complexation due to a Le Chatelier's shift
in solution equilibrium. This is notably contrasted with the other
two formulations which yield solutions that deviate in an
increasing manner (10-15%) from 12 to 30 mg/mL.
[0097] Injectable Formulation:
[0098] The stable form of artemisinin, the cyclodextrin complexed
with artenilate in a 2:1 ratio, may be dissolved in saline,
phosphate buffered saline (PBS), deionized water or any other
suitable aqueous carrier for injection. The pH is preferably about
7.4. Generally, 40 milligrams of artelinate complexed with
cyclodextrin per milliliter of solution is suitable. A dose of
about 4-6 mg of artelinic acid (in complex) per kilogram of weight
for a human is an appropriate dose. An injection of 10 ml of
complex in solution or less is appropriate for treatment.
[0099] The formulation of the cyclodextrin complexed with
artelinate in solution can be prepared and pumped through a filter
into an injection vile, freeze dried for storage and later
rehydrated with sterile water or saline or PBS for injection.
[0100] The cyclodextrin complexed with artelinate in solution can
also be administered orally, sublingually, or in the form of a
suppository.
Toxicity:
[0101] Cyclodextrins and artemisinins are both non-toxic to humans.
However, large doses of cyclodextrins are not implicated in cases
where kidneys are not fully functional.
In Vitro Data
[0102] In Vitro Inhibition Of Plasmodium Falciparum.
[0103] See U.S. Pat. No. 6,284,772, which is herein incorporated by
reference. The in vitro assays were conducted by using a
modification of the semiautomated mnicrodilution technique of
Desjardins, et al. (1979) Antimicrob. Agents Chemther. 16:710-718
and Chulay et al. (1983) Exp. Parasitol. 55:138-146. Two strains of
Plasmodium falciparum clones, from CDC Indochina III (W-2), CDC
Sierra Leone I (D-6). The W-2 elone is susceptible to mefloquine
but resistant to chloroquine, sulfadoxine, pyrimethamine, and
quinine. The D-6 clone is resistant to mefloquine but susceptible
to chloroquine, sulfadoxine, pyrimethamine, and quinine. These
clones were derived by direct visualization and micromanipulation
from patient isolates. Test compounds were initially dissolved in
DMSO and diluted 400-fold in RPMI 1640 culture medium supplemented
with 25 mM HEPES, 32 mM HaHCO.sub.3, and 10% Albumax I (GIBCO BRL,
Grand Island, N.Y.). These solutions were subsequently serially
diluted 2-fold with a Biomek 1000 (Beckrnan, Fullerton, Calif.)
over 11 different concentrations. The parasites were exposed to
serial dilutions of each compound for 48 h and incubated at
37.degree. C. with 5% O.sub.2, 5% CO.sub.2, and 90% N.sub.2 prior
to the addition of [.sup.3H]hypoxanthine. After a fuirther
incubation of 18 h, parasite DNA was harvested from each microtiter
well using Packard Filtermate 196 Harvester (Meriden, Conn.) onto
glass filters. Uptake of [.sup.3H]hypoxanthine was measured with a
Packard Topcount scintillation counter. Concentration-response data
were analyzed by a nonlinear regression logistic dose-response
model, and the IC.sub.50 values (50% inhibitory concentrations) for
each compound were determined.
[0104] FIG. 28 indicates that both cyclodextrin formulations of
artelinic acid .beta.-cyclodextrin and
hydroxypropyl-.beta.-cyclodextrin) yielded very similar in vitro
activity against multi-drug resistant strains of malaria as
indicated. All data indicated IC.sub.50 concentrations within 4
ng/mL of the uncomplexed artelinate salt (artelinic acid control).
Therefore, complexation of the artemisinin molecule was not found
to inhibit antimalarial efficacy.
Advantages
[0105] The complexed cyclodextrin-artemisinins formulation does not
precipitate or degrade over time. Formulations of artemisinins and
cyclodextrin have been observed to remain completely soluble for up
to seven weeks at elevated physiological temperatures (40 degrees
C.) without any degradation and up to 6 months at room temperature.
The complexed cyclodextrin formulation of the artemisinins does not
change color over time. Formulations of artemisinins and
cyclodextrin have been observed to remain colorless for several
weeks at elevated physiological temperatures of 40 degrees C.
EXAMPLE
Example 1
Formation of Artelinic Acid/Cyclodextrin Complex
[0106] Measure 2 moles of cyclodextrin and pre-issolve in buffer,
deionized water, or saline. Sonicate the mixture to completely
dissolve the cyclodextrin. Add 1 mole equivalent of artelinic acid
and sonicate. Incubate at 40.degree. C. for 2-3 hours. Higher
concentrations of artelinic acid require longer incubation times,
such as overnight, to promote complexation.
Example 2
Formation of Artesunic Acid/Cyclodextrin Complex
[0107] Measure 1 mole of cyclodextrin and pre-dissolve in buffer,
deionized water, or saline. Sonicate the mixture to completely
dissolve the cyclodextrin. Add 1 mole equivalent of artesunic acid
and sonicate. Incubate at 40.degree. C. for 2-3 hours. Higher
concentrations of artesunic acid require longer incubation times to
promote complexation.
[0108] The use of the complexed cyclodextrin formulation of the
artemisinins described provides a shielding effect to protect the
body from local toxic effects from the antimalarial agent until the
drug is diluted sufficiently into the system.
[0109] The process of making the complexed artemisinins of the
invention can be performed on a large scale using similar
conditions.
[0110] Having now fully described the invention, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention as set for the herein.
* * * * *