Methods for the formulation and manufacture of artesunic acid for
United States Patent: 7,678,828
Issued: March 16, 2010
Inventors: Ellis; William Y.
(Laurel, MD), Lim; Peter (Palo Alto, CA), Maniar; Manaj (Freemont, CA)
Assignee: The United States
of America as represented by the Secretary of the Army (Washington, DC)
Appl. No.: 12/315,209
Filed: December 1, 2008
Pharm Bus Intell
& Healthcare Studies
A method for the manufacture of a sterile
intravenous or intramuscular formulation of artesunic acid and the
formulation are the subject of this invention. First the artesunic acid
powder is sterilized with ethylene oxide and placed into a sterile
container. The contained sterilized powder is then dissolved in sterile
sodium phosphate buffered solution to produce an injectable intravenous or
intramuscular formulation. The sodium phosphate dissolves and dilutes the
artesunic acid powder without caking or frothing resulting in an improved
drug product. The invention also relates to the formulation and a method
of treating a patient with either uncomplicated or severe and complicated
Description of the
BACKGROUND OF THE INVENTION
1. Field of the Invention
The methods of the present invention provide a unique and superior
formulation of artesunic acid for parenteral injection and for the
manufacture of the formulation under sterile conditions. The methods
described herein provide a demonstrably sterile, non-pyrogenic product
which dissolves rapidly with no frothing or caking, yielding a clear,
conveniently prepared solution the attending physician may administer with
confidence. The formulation that is prepared by the methods of the
invention is especially suitable for the treatment of severe and
2. Brief Description of Related Art
Although malaria affects about 250 million people and kills one to two
million children each year, the pharmaceutical industry has shown little
interest in developing new or manufacturing established antimalarial drugs
not only because risks are significant, but the returns on investment are
Currently, the most promising and most rapidly acting antimalarial drugs
are derivatives of artemisinin (qinghaosu) obtained from qinghao or sweet
wormwood (Artemesia annua); these drugs have been developed and
manufactured in China. Three compounds of the qinghao family have been
used: the parent artemisinin and two of its more-active derivatives: a
water-soluble hemisuccinate, artesunate (AS), and an oil-soluble ether,
artemether (AM). Both derivatives are metabolized to a common biologically
active metabolite, dihydroartemisinin (DHA). Although this facile
conversion (hydrolysis) to DHA contributes to the AS rapid antimalarial
activity, it also limits the choices of practical AS dosage formulations.
Artesunic acid is also known to be effective in the treatment of severe (neuropathic)
malaria, Artesunate versus quinine for treatment of severe falciparum
malaria, a randomized trial, Dondorp, et al; Lancet, vol. 366, pages
717-725, Aug. 27, 2005, incorporated herein in its entirely by reference.
However, Artesunic Acid is an intrinsically unstable compound, susceptible
to decomposition by heat, radiation, and virtually any aqueous solution.
Prior studies have confirmed the breakdown of artesunate in aqueous
AS has been used for injection with good results. However, there are
drawbacks of the current commercially available AS dosage form. It is a
two-component product consisting of a dry-fill powder of sterile artesunic
acid in a vial and a sterile 5% sodium bicarbonate solution in an ampoule.
This product, "Artesunate For Injection", is manufactured by Guilin
Pharmaceutical Factory, Guangxi, China. This presently used formulation,
when dissolved in the supplied bicarbonate buffer solution, results in
fizzing and incomplete solution so that the concentration (dose) to be
delivered may be uncertain.
The formulation of artesunic acid mentioned above is manufactured in
China, and prepared by an undivulged method which results in a product of
poor dissolution characteristics, and which froths and cakes upon
introduction of the dissolution medium (5% bicarbonate). As the AS
dissolves, carbon dioxide is evolved and trapped in the small volume of
the closed vial. The formed gas bubbles carry un-dissolved AS particles
throughout the vial, thereby reducing contact between these particles and
the dissolution medium and lengthening the time needed to completely
dissolve the AS. Moreover, this phenomenon reduces the investigator's
ability to see if the solution is complete so the next preparation step,
which is to dilute the AS/bicarbonate solution with 5 mL of sterile 5%
glucose solution, can begin. These delays can unduly lengthen the overall
solution preparation time, resulting in a shorter time period over which
the prepared solution can be administered.
Further and most importantly, the product coming from China is not
manufactured under the U.S. Food and Drug Administration's current Good
Manufacturing Practice (cGMP).
Therefore, it is an object of the present invention to provide an AS
product and a method for preparing an AS product that dissolves quickly,
thoroughly and does not cake or fizz upon dissolution.
It is another object of the present invention to prepare an AS product
that does not require an additional step of diluting with glucose and is
immediately usable upon dissolution.
Another object of the present invention is to develop a method for the
production of an artesunic acid solution for the intravenous or
intramuscular treatment of malaria that is sterile and manufactured under
current Good Manufacturing Practice (cGMP) as required by the U.S. Food
and Drug Administration.
Another object of the present invention is to sterilize artesunic acid
powder without decomposition.
Another object of the invention is to prepare an artesunic acid product
that has a shelf life of two years.
These and other objects will become apparent upon further reading of this
SUMMARY OF THE INVENTION
The invention is a method for the manufacture of an intravenous or
intramuscular formulation of artesunic acid. First the artesunic acid
powder is sterilized with ethylene oxide and placed into a sterile
container. Nitrogen is used to purge water vapor from the container, after
which the container is hermetically sealed. When used, the sterilized
powder is dissolved in sterile sodium phosphate buffered solution to
produce a solution suitable for intravenous or intramuscular
administration. The sodium phosphate buffered solution dissolves the
artesunic acid powder without caking or frothing, resulting in an improved
drug product. The invention also relates to the formulation and a method
of treating a patient with severe and complicated malaria.
The AS parenteral dosage form must be sterile and not produce CO.sub.2
when the AS dissolves. To avoid CO.sub.2 evolution, we used a
non-carbonate-containing, physiologically compatible basic medium. We also
manufactured our drug product under cGMP.
The dissolution medium is sodium phosphate buffered solution.
In addition to avoiding the production of gas, the dissolution medium must
rapidly dissolve the AS, produce a solution in which the dissolved AS is
sufficiently stable, and yields a solution of physiologically acceptable
pH and osmolality. After many trials and errors, we found that a
0.30.+-.0.05 M, pH 8.0.+-.0.3 sodium phosphate solution meets all of the
above requirements and is preferred. Slight variations from these values
The solute in the dissolution medium has been identified as sodium
phosphate by spectral and chromatographic evidence. The average phosphate
concentration is 0.30 plus or minus 0.05 M. The average solution volume is
11.0 plus or minus 0.5 mL. The average solution pH is 8.0 plus or minus
Preparation of the 0.30M, pH 8.0 sodium phosphate solution, following a
USP procedure, was straightforward and under cGMP. Sterile phosphate
solution, 0.30 M, pH 8.0, is manufactured by mixing appropriate weights of
monobasic and dibasic sodium phosphate in distilled water to a molarity of
0.30 M and pH of 8.0. The phosphate solution is then sterilized by
filtration through a 0.22.mu. filter into 20 mL vials (12.2 mL/vial). The
vials are sealed and then stored at room temperature.
Sterility of the product, achieved through sterile filtration of the
phosphate solution and autoclave of the filled, sealed vials, was
accomplished smoothly by Afton Scientific Corporation, Charlottesville,
Va. 22902. After having met USP requirements for identity of the product,
product sterility, endotoxin, solution concentration, volume, pH,
osmolality, and particulates, 10,900 vials of this medium were labeled
Afton Batch 57804, assigned WR135946; BR18064, and designated as Component
Two of our AS dosage form. The USP procedure is found in 2005 USP 28/NF
23, p2855; Composition of Standard Buffer Solutions, incorporated herein
The active component is Artesunic Acid (AS), 110 mg/vial, SRI Batch No.
14462-16, from SRI International, Menlo Park, Calif.
The Chemical Abstracts (CA) Index name for artesunic acid is: butanedioic
ester. The CA Registry Number is 88495-63-0, and the molecular formula is
C.sub.19H.sub.28O.sub.8. The formula weight of .alpha.-artesunic acid is
384.43 g/mol. This name also defines the stereochemistry at C-10 which,
according to the CIP convention, is based on the priority of groups
attached to C-10. The 10.alpha.- designation refers to the O-succinal
group oriented back or toward the peroxide bridge. The 10- designation
refers to the O-succinal group oriented away from the peroxide bridge. The
molecular formula, C.sub.19H.sub.28O.sub.8, corresponds to a molecular
composition of C, 59.36%; H, 7.34%; and 0, 33.29%; and a molecular weight
of 384.43. .alpha.-Artesunic Acid is shown in FIG. 2 (see Original Patent).
The formulation development of the active component AS requires
sterilization of the bulk drug. For a sterilization process to be
acceptable, not only sterility of the bulk chemical must be shown, but the
process must not alter the physical or chemical nature or the stability of
the material. The high purity AS bulk drug, a finely milled, white
crystalline powder manufactured by Knoll AG, Listal, Switzerland was used.
An acceptable EtO treatment cycle was developed and employed as follows:
Sterilization of Bulk Artesunic Acid
The bulk AS was sterilized before dry fill. Gas sterilization was used.
Below are the salient points of the method and the determinations for
sterility and pyrogenicity. Artesunic Acid is treated for one hour at 102
degrees Fahrenheit and 100% humidity. The chamber is evacuated and
ethylene oxide is introduced and maintained at constant pressure and 102
degrees Fahrenheit for four hours. The sterilant cycle is stopped; the
chamber is evacuated and washed twice with nitrogen and once with air, all
at 102 degrees Fahrenheit. Slight variations of this sterilization method
are possible. A sample of treated AS is chromatographed. Chromatograms for
both treated and untreated AS are identical. AS is stable under the
conditions of treatment. Samples are tested for residual ethylene oxide,
ethylene chlorohydrin and ethylene glycol. Neither ethylene chlorohydrin
nor ethylene glycol was detected. Ethylene oxide was detected but at
levels well below the FDA proposed limit. A microbial limits test was
performed and validated to determine the inhibitory properties of AS. The
test was negative. AS has no inhibitory properties in this test. (USP
27<61> & <71>). Sterility tests were performed to discover the possible
presence of bacteria, fungi, and spores. Samples were doped before
treatment with a spore strip, bacteria, and fungi. No colony forming units
were found in any test. The treated material is sterile. (USP 27<71>). The
Limulus Amebocyte Lysate test was performed to determine the endotoxin
levels in the treated AS. Endotoxin levels were below the detectable level
in the treated AS. (USP 27<85>)
Ethylene oxide is an effective sterilant for bulk artesunic acid. Results
from alidated sterility tests on sterilized artesunic acid meet USP
requirements for sterility testing. Sterilized artesunic acid also meets
USP requirements for endotoxins.
The EtO-treated AS was dry filled into sterile vials. The best mode for
this purpose was to use a portable, manually operated powder dispensing
machine was purchased from M&O Perry Industries, Corona, Calif. 92880.
Owing to the propensity of the AS bulk drug to clump and cling to the
metal surface of the machine, characteristics that prevent both complete
filling and complete discharge of the machine loads, the machine was
fitted with a plastic liner that reduced the clinging and enabled
quantitative discharges. The installation qualification (IQ)/operation
qualification (OQ)/and performance qualification (PQ) were performed to
qualify the filling machine for cGMP manufacturing. The Model LM-14 is a
compact, portable bench top unit complete with carrying handle. It is an
ideal machine for small fill weight, low volume applications. Other
filling machines exist which are suitable for large operations.
Pre-cleaned and sterilized 20-mL vials, sterilized gray butyl rubber
stoppers and flip-off aluminum seals were purchased. In a class 100 room,
under laminar flow, the vials were filled in a glove box with EtO-treated
AS. Scheduled weight checks were performed to ensure the filled weights
met specifications. The filled vials were stoppered, sealed, and tested
for release. After meeting requirements for sterility, identity, purity,
content uniformity, and after constitution in sodium phosphate buffer, for
solution pH, osmolality, and particulate counts, 5,500 of the filled vials
were labeled SRI Batch 14462-16, assigned WR256283:BR29487, and designated
as Component One of our AS dosage form.
Analytical Methods of Specifications for Sterile Intravenous Artesunate
(110 mg/Vial) -- see Original Patent.
The selection of a material for the AS placebo was based on a likeness in
appearance and physical characteristics to that of the AS dosage form, in
addition to being biologically inert. The placebo for the AS Dosage Form
was Mannitol, 200 mg/vial.
A large number of possible placebos were investigated. The two final
candidates were mannitol and glucose, with the former having a slight
edge. Because the particle size of the commercially available USP mannitol
was larger than that of the AS bulk drug, the mannitol was milled and
sieved to match the size and appearance of the AS powder prior to
sterilization. Sterilization by irradiation initially looked promising,
but after two weeks on the shelf the irradiated mannitol became
discolored. Ultimately, treatment with EtO proved successful, and the
sterilized mannitol was dry-filled into the same type of glass vials as
the active material and processed identically. Because the density of our
mannitol was nearly twice that of the AS bulk drug, the filled placebo
mass was nearly twice that of the active, to maintain comparable filled
volumes. After having met requirements on content uniformity, identity,
and purity, and after constitution with phosphate, for solution pH,
osmolality, and particulate counts, 2,500 vials of the placebo were
labeled SRI Batch 14462-28 and designated WR016506:BR29487. To maintain
anonymity, a common label, identifying both the AS and Placebo, was used
for vials of the active as well as vials of its placebo.
In Phase I clinical trials the placebo was ethylene oxide treated mannitol,
exhibiting the same appearance and dissolution characteristics as the
Active Pharmaceutical Ingredient (API). The placebo was manufactured by
SRI International. All clinical materials are stored, maintained, and
shipped by the repository contractor (monitored and managed by The
Department of Chemical Information). The repository contractor also
prepares the double-blinded samples of artesunic acid or placebo for
clinical use under guidance from the Department of Chemical Information.
The placebo has provided an acceptable control for the recently completed
phase I clinical trials.
Analytical Methods and Specifications for Sterile Placebo for Injection
(200 mg/Vial) -- see Original Patent.
A typical dosage of .alpha.-artesunic acid for parenteral administration
is 10 mg/mL for a 10 mL injection. 110 mg is the unit dose for
manufacture. Typically, using a sterile syringe, 11 mL of sterile
Phosphate buffer for injection will be added to the 110 mg artesunate vial
and the vial swirled for about 4-6 minutes for full dissolution. Dosing is
1-4 mg/Kg body weight for intravenous administration with the possibility
of up to 8 mg/Kg in some cases. Preferred dosing is 2-3 mg/Kg body weight
for intravenous administration for three days. A drip bag is also suitable
for administration of the dose. A dosage of 50 mg/mL is suitable for IM
injection. IM treatment will be in the range of 1-5 mg/Kg body weight.
Give dosage one to two times per day for 3 days for IM. Because the
present inventors use a phosphate buffer solution, they are able to obtain
a higher concentration of AS for injection than that which can be obtained
with the 5% glucose dilution medium required by the Guilin formulation.
The cGMP-manufactured .alpha.-artesunic acid parenteral dosage form of the
invention offers several advantages over current, commercially available
version(s) of Artesunate drug. 1. The cGMP-manufactured sterile
dissolution medium, a 0.30 M, pH 8.0 solution of sodium phosphate,
completely dissolves the .alpha.-artesunic acid in 2-3 min, requiring only
gentle swirling. This rate of dissolution is several fold faster than that
found for the Guilin product, following its directions for preparation
given in its package insert. 2. Because the dissolution of AS in phosphate
is not accompanied by gaseous evolution, as in the case where bicarbonate
is used, determining solution completeness is readily achieved. 3. The
solution prepared in phosphate is ready for administration, as no further
preparation is required. The Guilin product, on the other hand, requires
an additional step of dilution of the AS/bicarbonate solution with 5 mL of
5% glucose, which also must be sterile. 4. The pH of our 10 mg AS/mL
solution in phosphate is 7.2, whereas that for 10 mg AS/mL solution in
bicarbonate/glucose is 7.9, a solution pH that is higher than ideal for
parenteral administration. 5. The osmolality of our 10 mg AS/mL solution
in phosphate is 320 and that for the 10 mg AS/mL solution in
bicarbonate/glucose is 410, a value also higher than ideal for parenteral
administration. 6. The phosphate buffer solution of the GMP manufactured
formulation allows AS concentrations high enough for effective IM
Although hydrolysis of AS in phosphate or bicarbonate/glucose begins
almost immediately upon dissolution, the rates of decomposition in the two
media are comparable. After two hrs at .about.24.degree. C. the solutions
were still visibly clear and therefore still can be administered.
In keeping with US FDA requirements, vials of the phosphate vehicle, the
AS, and the placebo are undergoing accelerated and shelf-life stability
Efficacy in Trials:
An Investigational New Drug Application (IND-64769) on this drug product
has been filed with the FDA and has been approved for use in clinical
trials. Phase Ia Safety and Tolerance single dose clinical trials hare
been concluded and were successful.
Phase Ia Safety and Tolerance of GMP Formulation
Phase Ia is a single dose double-blind placebo-controlled, randomized
study to evaluate the safety and tolerance of the GMP formulation of
intravenous artesunate. The study has been completed successfully as is
necessary to proceed to Phase IB and Phase II trials. Phase Ib and Phase
II trials are in progress.
Phase Ib Safety, Tolerance and Pharmacokinetics/Pharmacodynamics of GMP
A Phase 1b is a double-blind, placebo-controlled, randomized multiple dose
escalation study to evaluate the safety, tolerance, and pharmacokinetics/pharmacodynamics
of GMP formulation of intravenous artesunate in healthy human subjects in
3 doses using a dose escalation format using a placebo control. An
objective is to determine the safety of multiple dose administration of
escalating doses of artesunate that bracket the anticipated compassionate
use dose of 2.4 mg/kg by measuring adverse events (AE) and cardiovascular
responses (heart rate (HR), blood pressure (BP), and electrocardiogram (ECG)).
Another objective is to determine the safety and tolerability of the
compassionate use of 3 doses of artesunate in escalating doses of 0.5,
1.0, 2.0, 4.0, and 8.0 mg/kg with placebo control. The primary and
secondary outcomes are to assess AE and hemodynamic and cardiac responses
(BP,HR, ECG) and to determine pharmacokinetic parameters of artesunate and
its major metabolite DHA as well as to assess preliminary dose-toxic
The study design is as follows: Phase I, randomized, double-blind,
placebo-controlled trial using multiple ascending doses of intravenous
artesunate to determine its safety, tolerability and pharmacokinetics in
healthy male and female subjects. Subjects will be screened within 21 days
of dosing. At the screening visit, subjects will undergo baseline VS, PE,
CBC with smear, differential and indices, reticulocyte count measured by
flow cytometry, haptoglobin, COAGs, Chem, UA, urine drug screen, urine HCG
and medical and medication history. Eligible subjects will be scheduled
for a 6-hour outpatient visit for pre-dose ECGs and VS done to
approximately match dosing schedule on Day 1. On Day 0, subjects will be
admitted to the CPU to begin the inpatient phase of the study. Subjects
will have a brief physical and review all procedures for the inpatient
stay. On Day 1, pre-dose, VS and ECG will be performed. Subjects then will
receive IV study drug or placebo. Subjects will be closely monitored by
evaluating hemodynamic measurements, periodic ECGs, and assessment of
spontaneously reported AEs. Blood will be drawn for blood count and
chemistry analysis within 12 hours of the first and last doses. PK will be
drawn at designated times after each dose administered. On Days 2 and 3
subjects will receive their second and third doses, respectively, of study
drug or placebo followed by close clinical monitoring and laboratory
measurements as described for the first doses given. Subjects will be
discharged 24 hours after the third dose of drug or placebo and followed
as outpatients on Days 7, 10, and 15. The study population will consist of
40 healthy male and non-pregnant female adults given artesunate GMP
manufactured for injection intravenously.
The duration of the study will be a screening of up to 21 days; 5 days
(four nights) inpatient and 3 outpatient visits (last visit day 15) per
Phase II Trials:
In Phase II trials, the artesunic acid parenteral dosage form of the
invention vas given intravenously to human subjects in Africa to treat
malaria. In trials in Africa, COL Peter Weina, Chief, Department of
Pharmacology, Walter Reed Army Institute of Research has reported 30 adult
male and female volunteer patients with uncomplicated malaria have been
successfully treated using the treatment regimen as outlined in this
application. Successfully treated is defined as safely clearing P.
falciparum malaria parasites from the blood. Patients were given a single
dose of 1-4 milligrams per kilogram body weight in the form of an
injection through an IV catheter (a tube with a needle attached) once a
day for 3 days in a row. There were no adverse effects from the GMP IV
treatment of the artesunate of the invention. The single adverse effect
was with the standard-of-care positive control drug Malarone.
Six thousand dry-filled vials of formulated artesunate for clinical use
have been packaged. One thousand of the vials have been reserved for
long-term stability testing under various conditions, including elevated
temperatures and humidities, to test the integrity and durability of the
packaging system. As packaged for clinical use, 20 ml vials have been
dry-filled with 110 mg of ethylene oxide sterilized artesunate, stoppered,
and sealed. Stability studies at Knoll have shown at least two years
stability for bulk artesunic acid stored under nitrogen @ 25.degree. C.
The sterilized bulk drug of the invention has been tested and is still
undergoing stability studies. The sterilized bulk drug has shown no
evidence of degradation for 20 months at 25.degree. C. The stability
studies are still ongoing.
Claim 1 of 18 Claims
1. A method for the manufacture of an
injectable intravenous or intramuscular formulation of artesunic acid
comprising the steps of: a. sterilizing bulk artesunic acid powder; b.
dissolving said sterilized bulk artesunic acid in sterile sodium phosphate
buffered solution to produce said injectable formulation.
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