United States Patent
`
`119]
`
`[11] Patent Number:
`
`5,731,291
`
`Mar. 24, 1998
`Sullivan et al.
`[45] Date of Patent:
`
`US00573 1291A
`
`“Esafosfina” sales brochure (Biomedica Foscama: Feren-
`tino, Italy.
`Farias, L.A., et al, “Elfects of fructose-1, 6—diphosphate,
`glucose and saline on cardiac resuscitation,” Anesthesiology
`65: 595-601 (1986).
`treatment of irreversible
`Granot, H., et a1, “Successful
`hemorrhagic shock in dogs with fruct0se—1,6 diphosphate
`and dichloroacetate,” Circ Shock 163-173 (1985).
`Haasinen, I.E., et al. “Mechanism of the effect of exogenous
`FDP on myocardial energy metabolism,”Ct‘rculation 83:
`584-593 (1991).
`Lazzarino G., et al, “Proctective effects of exogenously
`administered fructose—1,6-diphosphate from ischemia rep-
`erfusion damage induced on isolated rat heart.”Ital J Bio-
`chem 38: 251A-253A (1989).
`Lazzarino, G., et al, “Ischemia and reperfusion: effect of
`fructose-1,6—bisphosphate,”Free Radic Res Commun 16;
`325-339 (1992).
`Marchionni, N., et al, “Hemodynamic and electrocardio-
`graphic effects of fructose—1,6-diphosphate in acute myo-
`cardial infarction,” Am J Cardiol 56: 266-269 (1985).
`Markov, A.K., et al. “Prevention of arrhythmias with fruc-
`tose disphosphate in acute myocardial ischemia,” abstract,
`Circulation 62: III-143 (1980).
`Markov, A.K., et al, “Hemodynamic, electrocardiographic,
`and metabolic effects of fructose diphosphate on acute
`myocardial ischemia,” Am Heart J 100: 639-646 (1980).
`Markov, A.K., “Hemodynarnic, and metabolic efiects of
`fructose 1-6 diphosphate in inschemia and shock—experi-
`mental and clinical observations,” Ann Emerg Med 15:
`1470-7 (1986).
`Markov, A.K., et al. “Increasing survival of dogs subjected
`to hemorrhagic shock by administration of fructose 1-6
`diphosphate,” Surgery 102: 515-527 (1987).
`
`(List continued on next page.)
`
`Primary Examiner-Zohrey Fay
`Attorney, Agent, or Firm-Patrick D. Kelly
`
`[57]
`
`ABSTRACT
`
`A_ method is disclosed for preparing a partially lyophilized
`(freeze-dried) powder or solidified cake containing fructose-
`1,6-diphosphate (FDP), a naturally-occurring intermediate
`in glycolysis. Preferably, about 10% to 25% residual water
`(by weight) is left in the powder or cake. This high moisture
`content does not degrade or limit FDP’s stability or shelf
`life, and it provides for faster, less expensive processing. The
`methods disclosed herein also allow direct lyophilization
`inside a vial or other sealed container that will hold the
`lyophilized FDP, to avoid any need for milling, handling, or
`other treatment under conditions that might endanger its
`sterility. Lyophilized FDP can be used to create emergency
`injection kits which also contain aqueous solutions for
`mixing, and syringes and needles for injection. These kits
`can be carried in ambulances, police cars, firetrucks, etc.,
`and can be stored at nursing homes, swimming pools, and in
`the homes of people suifering from various conditions such
`as heart disease or sicle cell anemia. These kits will allow
`
`rapid (even pre-diagnostic) injection of FDP into people
`sulfering medical crises such as heart attacks, severe blood
`loss, suffocation, etc.
`
`18 Claims, 6 Drawing Sheets
`
`[54] PARTIALLY LYOPHILIZED FRUCTOSE-1,6-
`DIPHOSPHATE (FDP) FOR INJECTION
`INTO HUMANS
`
`[75]
`
`Inventors: Brian W. Sullivan. Escondido; Paul J.
`Marangos, Encinitas, both of Calif.
`
`[73] Assignees Cypros Pharmaceutical Corp.,
`Carlsbad, Calif.
`
`[21] Appl. No.2 705,773
`
`[22] Filed:
`
`Aug. 30, 1996
`
`Related U.S. Application Data
`
`[63] Continuation-in-part of Ser. No. 646,600, May 8, 1996.
`
`[51]
`Int. Cl.5 ..................................................... A61K 31/70
`[52] U.S. Cl. .................................................... 514/23
`
`[58] Field of Search ................................................. 514/23
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,530,902
`.............................. 435/105
`7/1985 Perri et al.
`4,546,095
`
`..... 514/23 10/1985 Markov ... ......
`4,575,549
`3/1986 Diana et al.
`.. 536/117
`4,703,040 10/1987 Markov ......... ...
`.. ... 514/23
`4,870,057
`9/1989 Chiapparelli et al.
`514/23
`5,039,665
`8/1991 Markov ............
`..... 514/23
`5,094,947
`3/1992 Nakajima et al.
`.. 435/105
`5,434,255
`7/1995 Katayama et al.
`.. 536/117
`5,506,210
`4/1996 Parish et al.
`......
`516/23
`5,516,526
`5/1996 da la Torre .......
`.. 424/449
`5,571,906
`11/1996 Ceccardli et al. ...................... 536/117
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`1089615
`1089616
`1089654
`3323850
`
`7/1994 China .............................. C07H 1/06
`7/1994
`‘
`(I)7H 11/04
`
`7/1994
`......... Cl2N 1/18
`1/1985 Germany ....................... A61K 31/70
`
`OTHER PUBLICATIONS
`
`Chemical Abstracts 124: 56553 (1993). Ouyang et al.
`WPIDS 133439 (1989). Cavicchia et al.
`Angelos, M.G., et al, “Fructose—1, 6—diphosphate fails to
`limit early myocardial infarction size in a canine mode ,”
`Ann. Emerg. Med. 22: 171-177 (1993).
`Brunswick, R., et al, “Preservation of myocardium by infu-
`sion of fructose diphosphate following coronary occlusion,’’
`abstract, Am J Cardio 49: 1008 (1982).
`Cargnoni, A., et al, “Role of timing of administration in the
`cardioprotective effect of fructose—l, 6-bisphosphate,” Car-
`diovasc Drugs Ther 6: 209-217 (1992).
`Conti, V.R., et al, “Metabolic and functional effects of
`carbohydrate substrate with single-dose and multipledose
`potassium cardioplegia,” Ann. Thoracic Surg.36: 320-327
`(1983).
`de la Torre, J.C. ‘Treatment of head injury in mice, using a
`fructose 1,6—diphosphate and dimethyl sulfoxide combina-
`tion,” Nuerosurgery 37; 273-279 (1995).
`Eddy, L.J., et al, “Lack of a direct metabolic efiect of
`fructose, l,6—disphosphate in ischemic myocardium,” Am J
`Physiol 241: H576—583 (1995).
`
`FRESENIUS KABI 1010-0001
`
`

`
`5,731,291
`Page 2
`
`OI‘HER PUBLIC.’-‘£['IONS
`
`Mouchawar, A., et al, “A pathophysiological approach to the
`patient in shock, ” Int Anesthesiol. Clin. 31: 1-20 (1993).
`Pasque, M.K., et a, “Metabolic intervention to affect myo-
`cardial recovery following ischemia,”Annal of Surgery 200:
`1-12 (1984).
`Sernov, L.N., et al, ‘The characteristics of the cardioproc-
`tective action of fructose-,—1,6—d.iphosphate,” Biull Eksp Biol
`Med 111: 172-173 (1991) (abstract).
`Sernov, L.N., et al, ‘The antiacidotic and cardioprotective
`effects of fructose—1,6—diphosphate and dehydroascorbic
`acid,” Farmakol Toksikol 54: 24-26 (1991) (abstract).
`
`Sernov, L.N., et al, “A comparative evaluation of the car-
`dioprotective and antianginal actions of energy—providing
`agents,” Elcsp Klin Famzakol 55:13-15 (1992) (abstract).
`Stryer, L., Biochemistry, 2nd ed., pp. 266-267 (Freeman &
`Co., San Francisco, 1981).
`Tortosa, A., et al, “Fructose—1,6—bisphosphate fails to ame-
`liorate delayed neuronal death in the CA1 area after transient
`forebrain ischaemia in gerbils,” Neuropharmacology 32:
`1367-1371 (1992).
`Zhang, J.N., et al, “Protective efl’ect of exogenous fruc-
`tose—1.6—disphosphate in cardiogenic shock,” Cardiovasc
`Res 22: 927-932 (1988).
`
`FRESENIUS KABI 1010-0002
`
`

`
`U.S. Patent
`
`Mar. 24, 1998
`
`Sheet 1 of 6
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`5,731,291
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`U.S. Patent
`
`Mar. 24, 1993
`
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`5,731,291
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`

`
`U.S. Patent
`
`Mar. 24, 1993
`
`Sheet 3 of 6
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`5,731,291
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`
`

`
`U.S. Patent
`
`Mar. 24, 1993
`
`Sheet 5 of 6
`
`5,731,291
`
`I FDP
`
`[] Placebo
`
`
`
`CreatineKinase
`
`3000
`
`2000
`
`1000
`
`Day 1(u/L)
`
`Day 2 (u/L) AUC (u/L.d)
`
`FIG. 5
`
`
`
`12 4
`
`8
`
`12
`
`16
`
`24
`
`BYPESS
`
`Hours after Bypass
`
`FIG. 6
`
`FRESENIUS KABI 1010-0007
`
`

`
`U.S. Patent
`
`Mar. 24, 1998
`
`Sheet 6 of 6
`
`5,731,291
`
`OFDP (250 mg/kg i.v., pre-op)
`
`O Placebo
`
`00 O
`
`AUCPAWP(mmHg.h) 3
`
`
`
`
`
`N.) O o
`
`1000
`
`2000
`
`3000
`
`4000
`
`5000
`
`A. U. C. CK(u/L.h)
`
`FIG. 7
`
`FRESENIUS KABI 1010-OOO8
`
`

`
`1
`
`2
`
`5,731,291
`
`PARTIALLY LYOPHILIZED FRUCTOSE-1,6-
`DIPHOSPHATE (FDP) FOR INJECTION
`INTO HUMANS
`
`RELATED APPLICATION
`
`This application is a continuation—in-part of U.S. patent
`application Ser. No. 08/646,600, filed on May 9, 1996.
`
`BACKGROUND OF THE INVENTION
`
`Glycolysis is a fundamental biological process which is
`essential to the generation and use of energy by cells. In this
`process. a molecule of glucose (a 6-carbon sugar) is broken
`apart to form 2 molecules of pyruvic acid, containing 3
`carbons each. in a series of ten distinct reactions which are
`controlled by enzymes. In subsequent reactions, pyruvate is
`converted into carbon dioxide and water, if enough oxygen
`is present in the cells, via the Krebs cycle. In cells that do not
`have enough oxygen, pyruvate is converted into lactic acid,
`via a diiferent pathway. Glycolysis is discussed in detafl in
`nearly any textbook on biochemistry, physiology, or cell
`biology; see, e.g., any edition of Stryer’s or Lehninger’s
`Biochemisny, Guyton’s Medical Physiology, or Alberts et
`al, Molecular Biology of the Cell.
`Fructose-1,6-diphosphate (FDP) is ,a naturally occurring
`sugar-phosphate molecule, which is created and then
`quickly consumed as an intermediate during the series of
`reactions that make up glycolysis. As a short-lived interme-
`diate that is quickly consumed, it normally is present in cells
`only at relatively low concentrations. It should be noted that
`some scientists refer to FDP as fructose-1,6-biphosphate, or
`fructose—l,6-bisphosphate.
`The 1,6-isomer of fructose diphosphate, which contains
`phosphate groups bonded to the #1 and #6 carbon atoms of
`the fructose molecule, is the only isomer of interest herein.
`Other isomers (such as fructose-2,6-diphosphate) are not
`relevant herein, and are excluded from any references herein
`to FDP or fructose diphosphate.
`Numerous medical and scientific articles have suggested
`that FDP might potentially be useful as a medical treatment
`for patients and victims suifering from medical crises such
`as strokes, cardiac arrest, heart attack, suffocation, loss of
`blood due to injury, shooting, or stabbing, etc. Such articles
`include Markov et al 1980, 1986, and 1987, Brunswick et al
`1982, Marchionni et al 1985, Granot et al 1985, Farias et al
`1986 and 1989, Grandi et a1 1988, Zhang et al 1988, Cacioli
`et al 1988, Lazzarino et al 1989, Crescimanno et al 1990,
`Gregory et al 1990, Farias et al 1990, Janz et al 1991, Nakai
`et al 1991, Hassinen et al 1991, Bickler et al 1992, Lazzarino
`et al 1992, Cargnoni et al 1992, Kuluz et al 1993, Trimarchi
`et al 1993 and 1994. Gobbel et al 1994, Hardin et al 1994,
`Munger et al 1994, Kelleher et al 1994 and 1995, de la Torre
`1995, and Sano et al 1995. Relevant U.S. patents include
`U.S. Pat. No. 4,546,095 (Markov 1985), U.S. Pat. No.
`4,703,040 (Markov 1987), and U.S. Pat. No. 4,757,052
`(Markov 1988).
`Despite all of these published articles and patents, which
`stretch back to at least 1980, a high degree of skepticism and
`reluctance still exists regarding FDP use to treat ischemia or
`hypoxia. Except for a few small and very limited clinical
`trials, FDP simply is not used or prescribed by any practicing
`physicians, except possibly in a few foreign countries such
`as China and Italy.
`The absence of actual use of FDP on patients (many of
`whom desperately need the energy supplies that could be
`derived from FDP, as they are dying of massive heart
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`attacks, cardiac arrest, strokes, or blood loss) is believed to
`be due to a number of factors, including the following:
`(1) FDP is a diphosphate with a strong negative charge,
`and it is generally assumed by doctors and researchers
`that its highly—charged nature will prevent it from
`entering cells in substantial quantities. Since energy
`metabolism and glycolysis occur inside cells, it is
`generally assumed that FDP will not get to the relevant
`site in suflicient quantities to do any substantial good.
`(2) It is also believed that FDP has a very short half—life
`in the blood, and will effectively disappear from the
`blood within a few minutes after injection or infusion.
`(3) The amount of energy generated during glycolysis
`(i.e., the conversion of glucose to pyruvic acid) is only
`a small fraction of the energy generated by the aerobic
`(Krebs Cycle) oxidation of pyruvic acid to form carbon
`dioxide and water. Therefore, under conditions of tissue
`ischemia or hypoxia, where an oxygen deficit blocks
`aerobic conversion, it is generally assumed that FDP
`infusion would be insuflicient to supplementATP levels
`to a degree that can significantly aid cell survival.
`(4) As a corollary to the above, it is generally assumed that
`under conditions of ischemia or hypoxia, where inad-
`equate oxygen is present, an injection of FDP would
`likely lead to increases in lactic acid levels. This would
`be harmful, rather than beneficial.
`(5) It is also generally believed that FDP cannot penetrate
`the blood-brain barrier in significant quantities, and
`therefore oflers little or no benefit in protecting the
`brain or spinal cord against neuronal death and damage
`caused by stroke, head injuries, or other problems that
`generate ischemia in the brain (including stroke, car-
`diac arrest, hemorrhage or blood loss, etc). Since the
`brain is much more sensitive and vulnerable to damage
`by ischemia or hypoxia than any other organ, there is a
`general assumption that if the first people on the scene
`can’t help protect a patient’s brain against ischemic or
`hypoxic damage,
`then any benefits that might be
`offered by FDP injections to other organs, such as the
`heart, would merely be marginal and minor, and could
`not address or help solve the critical problem of rapid
`brain damage.
`(6) Drug intervention in acute ischemic trauma has proven
`to be extremely difficult and complex, for a large
`number of reasons. Among other things,
`it often
`requires 1 to 3 hours (or more) before a patient can be
`properly diagnosed in a manner that justifies the use of
`a specific drug. However, if that much time elapses
`before drug administration, it is often too late to inter-
`vene effectively because extensive cell death and per-
`manent tissue damage have already occurred.
`(7) Contrary to the articles (cited above) which report that
`FDP may have beneficial effects in certain types of lab
`tests, a number of other articles have reported that FDP
`had no beneficial effects in other studies. Examples of
`these negative articles include Eddy et al 1981, Pasque
`et a1 1984, Tortosa et a1 1993, and Angelos et al 1993.
`For these and other reasons, it appears that little if any
`effort has been directed by the pharmaceutical industry
`toward developing FDP as a useful drug. Under the laws
`enforced by the U.S. Food and Drug Administration, FDP
`cannot be sold in the United States for administration to
`human patients by physicians. As noted above, with the
`possible exception of a few small clinical trials, FDP simply
`is not administered to any patients, on any sort of routine
`basis, regardless of how desperate their plight may be
`following a stroke, cardiac arrest, shooting, stabbing, etc.
`
`FRESENIUS KABI 1010-0009
`
`

`
`5,731,291
`
`3
`
`Currently_. there are only two known preparations of FDP
`which are commercially available anywhere in the world,
`other than research reagents that are sold in gram or milli-
`gram quantities by specialty chemical companies. One of
`these preparations is a non-sterile bulk powder, manufac-
`tured in Germany by Boehringer Mannheim. This material
`was purchased by the assignee (Cypros Pharmaceutical
`Corporation. of Carlsbad. Calif.) and used as the starting
`reagent for the lyophilized preparation described herein.
`The other commercially available FDP formulation is a
`lyophilized preparation that is manufactured in Italy by a
`company called Biochemica Foscama. To the best of the
`Applicant’s knowledge and belief. it is manufactured by
`steps that including the following: (1) pouring a large batch
`of an aqueous mixture of FDP into a large, flat tray; (2)
`freezing the mixture and subjecting it to a vacuum,
`to
`remove the water. thereby creating a large solidified cake;
`(3) grinding or milling the large cake into small particles; (4)
`loading the g;round—up particles into small vials; and, (5)
`sealing the vials.
`This process is not well suited for creating a sterile
`preparation. for injection into humans. For example, a
`manufacturing process which uses large machinery to
`handle and manipulate a large. flat cake. pass it through a
`device which grinds it up into small particles, pass the
`particles through various routing and funnelling devices in
`order to load those particles into small vials. and then seal
`the vials. creates numerous risks which seriously jeopardize
`the sterility of the resulting final product.
`It should also be noted that a lyophilized preparation of
`FDP in a sealed vial cannot be treated by a “terminal
`sterilization” process; i.e., non-sterile FDP cannot be placed
`in a vial which is then sealed, and subsequently subjected to
`a sterilizing step. such as autoclaving or ionizing radiation.
`Those types of terminal (post—sealing) sterilization treat-
`ments would seriously degrade the chemical quality of the
`FDP in the sealed vials.
`The final product of the lyophilized FDP preparation from
`Biochemica Foscama sutfers from several apparent short-
`comings. It is relatively inhomogeneous, and contains par-
`ticles of various different sizes; some appear to be small
`glass—like beads. while others appear to be relatively sticky,
`caramelized agglomerations. It is also relatively unstable;
`while pure FDP is a crystalline white powder, some of the
`particles in the Biochemica Foscama preparation (especially
`the larger particles) begin to turn a yellowish-brown color
`within a few days or weeks, when stored at room
`temperature, unopened. in the sealed vials.
`To the best of the Applicant’s knowledge and belief,
`neither the Boehringer Mannheim company (headquartered
`in Germany) nor the Biochernica Foscama company (located
`in Italy) have ever made any efiort to obtain permission from
`the U.S. Food and Drug Administration to sell their FDP
`preparations in the United States, for use on humans.
`Lyophilization
`Lyophilization is the technical name for a process often
`referred to as “freeze-drying”. In this process, an aqueous
`mixture or suspension is frozen into a solid, then it is
`subjected to a vacuum for a substantial period of time
`(usually a full day or more). The vacuum causes the water
`molecules to “sublima ”. i.e., to become gaseous and leave
`the solid. without going through a liquid state. This is
`comparable to the evaporation of carbon dioxide from “dry
`ice”, but it is much slower, and it requires high levels of
`vacuum. which can only be generated in a special and
`expensive type of lyophilization chamber which can gener-
`ate and sustain both (1) Very cold freezing temperatures,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`4
`which are much lower than the typical temperatures of
`conventional household freezers, and (2) very high levels of
`vacuum, measured in millitorr (thousandths of a tort; typical
`atmospheric pressure is 760 torr, or 760 millimeters of
`mercury column in a U-shaped device called a manometer).
`Because of the extremely high requirements for sterility
`and quality control, lyophilization of pharmaceuticals is a
`very expensive process. It requires a lot of energy to sustain
`the proper freezing and vacuum conditions in a lyophiliza-
`tion chamber, and each batch of drugs must sit
`in an
`expensive chamber, absolutely motionless, for many hours
`or days. For these and other reasons. any processing
`improvement that can speed up a lyophilization protocol
`without compromising the quality or sterility of the product
`is regarded as an improvement over longer, slower pro-
`cesses.
`Residual Moisture Levels
`One of the most important factors in lyophilizing a drug
`product is the final water content. Most lyophilization pro-
`tocols include both a “primary drying” stage, which reduces
`moisture content to a level of about 7% to 10%, by weight,
`followed by a “secondary drying” stage, which usually
`reduces the water content to less than 2%. Reducing residual
`water content to less than about 2% helps keep hydrolysis
`(or other chemical reactions that might alter the chemical
`bonds in the drug molecule) to a
`Relevant prior art which discusses residual moisture con-
`tent includes the following:
`(1) U.S. Pat. No. 4,537,883, which states: “Final moisture
`content of the dried product is generally below 1.0%,
`although some products, mainly biologicals, may have
`a final moisture content which could range as high as
`about 10%. Usually, the improvement in stability of the
`lyophilizate, compared to the solution, is due to the
`absence of water in the pharmaceutical composition.”
`(2) Adams, G. D. J., Freeze—Dryz'ng of Biological
`Materials, Drying Technology, 9(4), (1991), pages
`891-925, which states: “The prolonged drying stage is
`called secondary drying, water being removed by des-
`orption. During secondary drying the water content is
`reduced to 2% or less.”
`
`(3) Greiif, D._. “Freeze-Drying Cycles,” in International
`Symposium on Freeze Drying of Biological Products
`(published by S. Karger, Basel. Switzerland, 1977, pp.
`105-115): “During the drying process, suspensions will
`contain biologic materials in at least three states: (1)
`dried materials containing low contents of residual
`moisture (3—5%), (2) dried material containing less
`than 3% residual moisture, and (3) non-dried, frozen
`material.”
`
`(4) A section entitled, “Formulation and Process
`Guidelines,” by Michael J. Pikal and Steven L. Nail, in
`the printed course notes from Lyophilization: A Short
`Course, sponsored by Parenteral Drug Association, Inc.
`(Mar. 13-14, 1996), states as follows: “Secondary
`Drying: .
`.
`. The first question is, ‘What is the desired
`level of residual water?’ This issue should be addressed
`during fonnulation development studies.
`In many
`cases, the lower the residual moisture, the more stable
`is the product. However. there may not be a significant
`diflerence in stability between zero water. and some
`moderate moisture level, such as 1% .
`.
`. An optimum
`level of water which is relatively high (5%) would be
`diflicult, and formulation approaches should be
`explored in an attempt to circumvent this requirement.”
`(5) Guidelinesfor the Determination ofResidual Moisture
`in Dried Biological Products, Docket No. 89D-0140,
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`January 1990, issued by CBER (the Center for Bio-
`logicals Evaluation and Research), which is part of the
`U.S. Food and Drug Administration: “Residual mois-
`ture has been the term used to describe the low level of
`surface water, usually from less than 1% to 5%, remain-
`ing in a freeze-dried vaccine or other biological product
`after the bulk of the aqueous solvent has been removed
`during the freeze-drying (vacuum sublimation) pro-
`cess.”
`
`The Applicant is not aware of any commercially available
`lyophilized drug with a residual moisture greater than 10%.
`Accordingly, the Applicant’s discovery that FDP can be
`“partially lyophilized”, allowing residual water contents to
`remain at least 10%, and up to about 25%, while retaining
`excellent stability, would have been very surprising for any
`type of drug. It was even more surprising for fructose
`diphosphate, since its two phosphate bonds were expected to
`be quite vulnerable to hydrolysis.
`Stability: The 5% Total and 1% Single Impurity Tests
`Another important issue in FDP lyophilization is chemical
`stability, which is required to sustain chemical purity after
`periods of storage such as a month or more. The goal of
`lyophilization is to create FDP preparations that have suf-
`ficient chemical stability and shelf life to be medically
`useful, without suffering serious levels of hydrolysis or other
`chemical degradation, when stored and handled in sealed
`vials without refrigeration for periods such as a month or
`longer. This goal is quantified by establishing “benchrnark”
`tests, so that compliance or noncompliance of any given
`batch (or any particular manufacturing process) can be
`evaluated by impartial and objective standards. One such
`benchmark test that is commonly applied to drugs intended
`for human use is as follows: a preparation of a candidate
`drug must not contain more than 5% total impurities, and it
`also must not contain more than 1% of any single impurity.
`This standard of purity and stability is referred to herein as
`the “5% total, 1% single impurity rule”.
`This standard has been applied, by the U.S. Food and
`Drug Administration, to all human clinical trials which
`involve fructose-1,6-diphosphate that have been conducted
`to date by the Applicant, Cypros Pharmaceutical Corpora-
`tion. It is used and applied herein,
`in the text of this
`application, and in the claims; any reference herein to a
`“chemically stable” lyophilized preparation of FDP refers to
`a lyophilized preparation of FD_P which meets the “5% total,
`1% single impurity rule” after storage for a month (or
`longer) at room temperature.
`As used herein, “chemical degradation” of FDP refers to
`breakage or rearrangement of any carbon-oxygen or oxygen-
`phosphorous bond; this includes any loss of a phosphate
`group from the molecule, and it also includes the process
`called “caramelization,” in which separate molecules
`become bonded to each other, to fonn agglomerated mol-
`ecules.
`However, it should be noted that FDP (which is acidic,
`and which is most stable in a salt form, in any aqueous
`solution) may ionize or be converted into any particular salt,
`without losing its stucture (or its metabolic activity as an
`intermediate in glycolysis). Accordingly, ionization and salt
`formation are not regarded as chemical degradation; they do
`not
`involve breakage or rearrangement of any carbon-
`oxygen or oxygen-phosphorous bond.
`In practice, the 1% requirement (for any single impurity)
`is the crucial standard. That part of the two-prong impurity
`standard is almost always violated long before the 5% total
`impurity requirement is even approached In FDP, the two
`phosphate bonds are highly susceptible to hydrolysis (i.e.,
`
`water-induced cleavage). Accordingly, either or both of the
`phosphate groups will leave the FDP, generating fructose or
`fructose monophosphate. The remaining phosphate groups
`are attached to either the #1 or #6 carbon atoms, usually in
`roughly equal proportions. Either form of fructose mono-
`phosphate can be easily distinguished from FDP, using high
`performance liquid chromatography (HPLC).
`This technical factor also strongly supports the assertion
`that partial lyophilization of FDP, which leaves in a water
`content higher than 10% to give a stable preparation, would
`not be obvious to anyone with ordinary sldll in the art. Since
`hydrolysis of the phosphate bonds of FDP is the crucial
`problem that must be overcome to provide a preparation
`with adequate chemical stability, then anyone with ordinary
`skill in lyophilization chemistry would normally assume that
`the water content must be reduced as low as possible, even
`if extreme measures are required, to reduce the risk of
`hydrolysis. The result discovered by the Inventor herein
`pointed in the exact opposite direction from what was
`expected.
`As noted above, the 1-month stability test period is merely
`a benchmark standard, for analytical and comparative pur-
`poses. Many of the FDP preparations disclosed herein have
`aged for a year or more and still satisfy the impurity
`standards by a comfortable margin. Samples that were
`analyzed shortly before this application was filed showed
`less than 1% total impurities after 425 days of storage at
`room temperature.
`It should also be noted that samples of the Biochemica
`Foscama lyophilized preparation have been analyzed, by the
`Applicant, for purity, using HPLC analysis (which detects
`hydrolysis, but
`is less likely to reliably detect
`caramelization). The samples tested contained more than 1%
`of single impurities, and therefore did not meet the FDA
`standard which was applied to the Applicant herein.
`Accordingly, one object of this invention is to disclose a
`method of partially lyophilizing FDP, to create a sterile
`sealed product that contains at least 10% and up to about
`25% residual water content, by weight.
`Another object of this invention is to disclose that par-
`tially lyophilized FDP which contains 10% to 25% residual
`water, by weight, is chemically stable and has good shelf life
`when stored for months at room temperature without refrig-
`eration.
`
`Another object of this invention is to disclose that sec-
`ondary vaporizing agents, such as tertiary butyl alcohol, can
`be used during the lyophilization of FDP to provide
`improved final products.
`Another object of this invention is to disclose that par-
`tially lyophilized FDP offers a stable preparation that
`enables emergency injection of FDP into patients or victims
`who are suffering medical crises (such as a heart attack,
`suflocation, loss of blood due to injury, etc.) and who need
`immediate medical assistance. Such treatment can be admin-
`istered by any person (such as an ambulance attendant,
`policeman, or nursing home attendant) who is trained to
`administer an intravenous injection, without requiring any
`delays while waiting for transportation or access to a doctor
`or diagnostic equipment.
`Another object of this invention is to disclose pre-
`packaged injection kits for use in medical emergencies.
`These injection kits contain partially lyophilized FDP, as
`disclosed herein, along with a sterile aqueous solution, to
`reconstitute the FDP into an injectable liquid, and a syringe
`and needle. Preferably, all of these components should be
`contained within a durable case, made of a material such as
`hard molded plastic. This would render such emergency
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`injection kits suitable for being carried and kept in places
`where they can facilitate rapid emergency use, such as in
`ambulances, trunks of police cars or firetrucks, and first aid
`kits in nursing homes and at swimming pools and beaches.
`These and other objects of the invention will become
`more apparent through the following summary, drawings,
`and description of the preferred embodiments.
`SUMMARY OF THE INVENTION
`
`A practical and economic method is disclosed for prepar-
`ing a partially lyophilized (freeze-dried) powder or solidified
`cake containing fructose—l.6-diphosphate (FDP), a
`naturally-occurring chemical which is an intermediate in
`glycolysis. The preferred methods leave at least 10%, and up
`to about 25%, residual water (by weight) in the powder or
`cake. This surprisingly high moisture content does not
`degrade or limit FDP’s stability or shelf life, and it provides
`for faster. less expensive processing. The methods disclosed
`herein also allow direct lyophilization inside a vial or other
`sealed container that will hold the lyophilized FDP during
`storage, shipping and handling. This avoids any need for
`milling, handling. or other treatment under conditions that
`might endanger its sterility. Among other benefits,
`this
`invention provides for emergency injection kits containing
`lyophilized FDP, sterile aqueous solutions, and syringes, that
`can be carried in ambulances, police cars, etc. These emer-
`gency kits will allow rapid injection of FDP into victims of
`medical emergencies such as heart attacks, severe blood
`loss. suffocation. etc.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a graph showing temperatures, as a function of
`time, for a preferred partial lyophilization procedure for FDP
`as described herein.
`
`FIG. 2 is a graph showing vacuum pressures. as a function
`of time, for a preferred FDP partial lyophilization procedure
`as described herein.
`
`FIG. 3 is a cutaway side view of a dual—compartment
`sealed sterile vial containing a partially lyophilized FDP
`powder or cake in one compartment, and a sterile aqueous
`solution in a second compartment. The septum that separates
`the two compartments can be pushed out of its sealed
`blocldng position. to reconstitute an aqueous solution of
`FDP for injection into a human patient.
`FIG. 4 is a perspective View of a hard—shell plastic case
`which encloses and protects a hypodermic syringe and
`needle, several sealed vials containing partially lyophilized
`FDP, and a sealed container of an aqueous solution (such as
`a dextrose solution, Ringer’s lactate, etc.) for reconstituting
`the FDP into aqueous solution. This injection kit is designed
`for emergency use. so that someone who is not a physician
`but who is trained to deliver injections (such as ambulance
`attendants, police, military personnel, nursing hom