the as parenteral dosage form must be sterile and not produce co 2 when the as dissolves . to avoid co 2 evolution , we used a non - carbonate - containing , physiologically compatible basic medium . we also manufactured our drug product under cgmp . 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 are acceptable . 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 0 . 3 . 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 . 22μ 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 by reference . 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 acid , [ 3r -( 3α , 5a , 6 , 8a , 9α , 10α , 12 , 12ar *)]- mono ( decahydro - 3 , 6 , 9 - trimethyl - 3 , 12 - epoxy - 12h - pyrano [ 4 , 3 - j ]- 1 , 2 - benzodioxepin - 10 - yl ) ester . the ca registry number is 88495 - 63 - 0 , and the molecular formula is c 19 h 28 o 8 . the formula weight of α - 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 10α - 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 19 h 28 o 8 , corresponds to a molecular composition of c , 59 . 36 %; h , 7 . 34 %; and 0 , 33 . 29 %; and a molecular weight of 384 . 43 . α - artesunic acid is shown in fig2 . 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 : 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 & lt ; 61 & gt ; & amp ; & lt ; 71 & gt ;). 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 & lt ; 71 & gt ;). 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 & lt ; 85 & gt ;) 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 & amp ; 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 . 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 . a typical dosage of α - 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 α - 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 α - 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 treatment . 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 ˜ 24 ° 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 studies . 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 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 . a phase ib 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 response . 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 patient . 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 ° 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 ° c . the stability studies are still ongoing . having generally described this invention , a further under - standing can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified . upon receipt of the accessible portions of the european drug master file ( dmf ) for artesunic acid from knoll , the inventors compared their analytical protocols for artesunic acid to those used in the dmf . the dmf method used by knoll is as follows : the reference solutions ( n = 5 each ) were prepared by accurately weighing between 3 . 472 to 15 . 977 mg of the reference and dissolving each in 1 . 00 ml of acetonitrile . a series of 30 - μl injections were made to deliver 104 . 2 to 479 . 3 μg of reference on column for assay . the mass of sample on column ( m x , μg ) was calculated using equation one ( eq . 1 ) where , w x is the sampled mass ( mg ) of the reference or sample ( s ) as weighed , v x is the volume of solvent ( 1 . 00 ml acetonitrile ) used , and v 1 is volume of solution injected ( 30 μl ). an area to mass on column response factor ( rf a ) vas calculated for the reference standard using equation two ( eq . 2 ) where , a r is the reference peak area , and p r is the reference purity (& gt ; 99 %) 3 . sample peak area data was used in equation three ( eq . 3 ) to calculate the mass ( m s ) of the sample , duplicating all the experimental conditions used by knoll , the inventors confirmed the results of its previously validated hplc assay . upon validation of the imported knoll assay , it was adopted as one of the assays to be used by the inventors to confirm the identity of artesunic acid samples and to test the purity of such samples . the major advantage of the knoll method was lowering the lod from 2 ug to 0 . 075 ug on column and decreasing the assay time from 16 minutes to 8 minutes . the major disadvantage is its inability to determine as in phosphate . precision , linearity , quantation , and accuracy were comparable for both methods . the inventors verified the identity and determined the purity of three samplings of wr256283 ; bq38641 , ( knoll lot 2 . 03 ). this was the milled sample of the bulk knoll drug substance used in formulation of the injectable artesunic acid for clinical trials . the three samples were taken to confirm the identity and uniformity of the received material ( sample a from the top of the container , sample b from the middle of the same container , and sample c from the bottom of the container ). they were compared to a reference sample received jun . 29 , 2001 ( wr256283 ; bp18288 ) using a number of analytical tests including , but not limited to , fourier transform infrared spectroscopy , proton nuclear magnetic resonance spectroscopy , elemental analysis , high performance liquid chromatography , thermogravimetric analysis , residual solvents by gas chromatography , and inductively coupled plasma . the samples were confirmed as being identical samples of artesunic acid . purity was determined with an hplc - based assay using the external standard method , with a known reference purity of & gt ; 99 %. hplc results confirmed sample purity was 99 . 3 plus or minus 0 . 3 %. residual solvents in the knoll material include heptanes ( 0 . 09 %) and ethyl acetate ( 0 . 04 %), plus trace amounts (& lt ; 0 . 01 %) of methanol and ethanol . lead was not found . sri verified that an ethylene oxide sterilization treatment ( 4 hours at 102 degrees f .) does not degrade artesunate ; the treated material meets usp requirements for sterility . the eto treated sample was purged with nitrogen to remove residual ethylene oxide . subsequently , bioburden , bacteriostasis , fungistasis , and endotoxin tests were performed to validate the sterility treatment method . tests for ethylene oxide derivatives were negative and the residual eto was found to be well below the fda recommended levels . tests for artesunate breakdown products , including dihydroartemisinin , were similarly negative . results from validated bioburden and lal tests on sterilized artesunate met usp requirements for sterility and endotoxins . the average chromatographic purity after ethylene oxide treatment was found to be 99 . 9 plus or minus 0 . 4 % relative to the reference standard . qualitative and quantitative assay results verified the chemical integrity of the ethylene oxide - treated artesunate . these results establish the time zero data point for future ethylene oxide - treated artesunate stability studies . six thousand dry - filled vials of formulated artesunic acid 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 systems . as packaged for clinical use , 20 ml vials have been dry - filled with 110 mg of ethylene oxide sterilized artesunic acid , stoppered , and sealed . stability studies at knoll have shown at least two years stability for bulk artesunic acid stored under nitrogen @ 25 ° c . tests of the dry - filled artesunate formulation were used in the glp 14 - day dog toxicity study . a concentrated formulation of 50 mg as / ml was developed and manufactured for a 14 - day cglp toxicity study in dogs . the dry - filled artesunic acid formulation used in the glp 14 - day dog toxicity study was confirmed to be of high purity by independent analysis . the artesunic acid content weights , calculated from determining the mg of artesunic acid / ml in reconstituted samples , met the requirements set forth in usp article & lt ; 905 & gt ; and ranged between 501 to 519 mg / vial . the potential toxicity of gmp artesunate of the invention was tested in beagle dogs . the artesunate was administered daily by rapid intravenous infusion ( over 4 to 6 minutes ) for 14 days . four groups consisting of 4 dogs / sex / group were treated daily with doses of artesunate at 10 , 20 , 35 , or 50 mg / kg / day at dose volumes of 1 ml / kg . one group of 4 dogs / sex received sterile 0 . 3 m phosphate buffer ( control article ) and served as the control group . the study was divided into two parts . after 14 doses , 2 dogs / sex / group were necropsied on study day ( sd ) 15 . the remaining two dogs / sex / group were allowed a 2 - week treatment - free recovery period and were necropsied on study day 29 . measurements included survival , clinical observations , body weights , electrocardiography , hematology , clinical chemistry , coagulation parameters , gross pathology , organ weights , and histopathology ( wu and senate , 2004 ). intravenous doses of artesunate up to and including 50 mg / kg / day did not result in test article - related effects on mortality , clinical observations , body weights , body weight gains , food consumption , electrocardiographic output , clinical chemistry and coagulation , gross pathology , organ weights , and histopathology . during the course of the study , erythema , diarrhea , emesis , mucoid feces , and soft feces were observed sporadically in both control and test article - treated groups , and were not considered to be test article - related . intravenous administration of artesunate at doses of 20 , 35 , or 50 mg / kg / day for 14 days in beagle dogs resulted in lowered red blood cell parameters ( rbc , hgb , hct , and retic ) measured on study day 15 . the lower reticulocyte counts suggested that there was not a regenerative response to the lower rbcs . the lowered red blood cell parameters found on study day 15 were not present on study day 29 . based on the results of this study , artesunate , when administered intravenously for 14 days at doses up to and including 50 mg / kg / day , did not result in any other test article - related adverse effects except on the measure hematology . at doses of 20 mg / kg / day and above , intravenous administration of artesunate for 14 days resulted in a transient test article - related effect on red blood cell parameters , including rbc , hgb , hct , and retic . 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 forth herein .
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 malaria .