United States Patent [191
`Holthuis et al.
`
`||||||m|||||mumnlngtggygtwtunn||||||||||||||||||||||1
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,496,801
`Mar. 5, 1996
`
`[54] PARATHYROID HORMONE FORMULATION
`
`[75] Inventors: Josephus J. M. Holthuis, AJ Leiden;
`Albert Mekking, CR Woerden;
`Alwinus A. Voetman, DT Zwanenburg,
`all of Netherlands
`
`[73] Assignee: Allelix BioPharmaceuticals Inc.,
`Ontario, Canada
`
`[21] Appl. No.: 172,206
`[22] Filed:
`Dec. 23, 1993
`
`[51] Int. Cl.”S ......................... .. A61K 37/02; A61K 37/36
`[52] US. Cl. ................................ .. 514/12; 514/2; 514/970
`[58] Field of Search ................................. .. 514/2, 12, 970
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,768,177 10/1973 Thomas .............................. .. 273/146
`4,016,314 4/1977 Cowans et al.
`248/278
`4,105,602
`8/1978 Colescott et a1. ..
`.. 514/12
`4,199,060 4/1980 Howard ........... ..
`.. 206/231
`4,424,278
`l/1984 Bucovaz et a1.
`436/501
`4,690,952
`9/1987 Kagatanietal. ..
`. 514/808
`4,698,328 10/1987 Neer et al.
`.... .. 514/141
`4,812,311
`3/1989 Uchtman ..
`514/12
`4,833,125
`5/1989 Neer ................ ..
`5,059,587 10/1991 Yamamoto et a1. .................... .. 514/12
`
`FOREIGN PATENT DOCUMENTS
`
`2248550 4/1992 United Kingdom ......... .. A61K 37/24
`WO9311786 6/1993 WIPO .......................... .. A61K 37/24
`
`OTHER PUBLICATIONS
`
`Charbon et al Arch Int Pharmacodyn, 1968, 171(1):3
`“Diuretic and vaxcular action of parathyroid extracts in
`animals and man”.
`Hesch et al Calcif Tissue Int, 1989, 44:176 “Increase of
`vertebral density of combination therapy with pulsatile
`1-38hPTH and sequential addition of calcitonin nasal spray
`in osteoporotic patients”.
`Hock et a1 Endocrinology, 1990, 127:1804 “Anabolic effect
`of human synthetic parathyroid hormone-(l—34) depends on
`growth hormone”.
`Hodsman et a1 Bone and Mineral, 1991, 14:67 “Bone
`densitometric and histomorphometric responses to sequen
`tial human parathyroid hormone (1—38) and salmon calci
`tonin in osteoporotic patients”.
`Hodsman et al Bone Miner, 1990, 9(2):137 “Biochemical
`responses to sequential human parathyroid hormone (1—38)
`and calcitonin in osteoporotic patients”.
`Hulter et al J Clin Hypertens, 1986, 2(4):?160 “Chronic
`continuous PTH infusion results in hypertension in normal
`subjects”.
`Hulter et a1 Metabolism, 1984, 33(7):662 “Renal and sys
`temic magnesium metabolism during chronic continuous
`PI‘H infusion in normal subjects”.
`Isaac et a1 Horm Metab Res, 1980, 12(9):487 “Absence of
`effect of 1-34 hP'I‘H on plasma TSH, GH, FSH. LH, ACTH
`and cortisol in normal man”.
`Isaac et al J Clin Endocrinol and Metab, 1978, 47:18 “Elfect
`of parathyroid hormone on plasma prolactin in man”.
`
`Keutman et al Current Research on Calcium Regulating
`Hormones, Cooper, C. W. (ed), 1987, University of Texas
`Press, Austin, pp. 57-63.
`Kimmel et al Endocrinology, 1993, 32(4):1577 “The Elfect
`of recombinant human (1—84) or synthetic human (l—34)
`parathyroid hormone on the skeleton of adult osteopenic
`ovariectornized rats”.
`Kimura et a1 Biochem Biophys Res Comm, 114(2):493
`“Solution synthesis of [Asn76]—human parathyroid hormone
`(1-84)”.
`Law et al J Clin Invest, 1983, 72(3):1106 “Rapid develop
`ment of renal resistance to low doses of synthetic bovine
`parathyroid hormone fragment 1-34”.
`Lawoyin et al J Clin Endocrinol Metab, 1979, 49:783 “A
`patient with pseudohypoparathyroidism with increased
`serum calcium and 1ot,25—dihydroxyvitamin D after exog
`enous parathyroid hormone administration”.
`Leithner et al The Lancet, 1984, :367 “Parathyroid hormone
`does not inhibit platelet aggregation”.
`Martindale The Extra Pharmacoepia, The Pharmaceutical
`Press, London, 29th ed., 1989 at p. 1338 “Parathyroid
`calcitonin and biphosphonates”.
`Reeve et al Br Med J, 1980, 280:1340 “Anabolic effect of
`human parathyroid hormone fragment on trabecular bone in
`involutional osteoporosis: a multicentre trial”.
`Reeve et al Lancet, 1976, 1:1035 “Anabolic effect of low
`doses of a fragment of human parathyroid hormone on the
`skeleton in postmenopausal osteoporosis”.
`Reeve et al Calcif Tissue Res, 1976, 211469 “Preliminary
`trial of low doses of human parathyroid hormone peptide in
`treatment of osteoporosis”.
`Reeve et al Osteoporosis Int, 1991, 1:162 “hPTI-I 1-34
`treatment of osteoporosis with added hormone replacement
`therapy: biochemical, kinetic and histological responses”.
`Rabbani et al Endocrinology, 1988, 123:2709 “In?uence of
`the amino-terminus on in vitro and in vivo biological
`activity of synthetic parathyroid hormone—like peptides of
`malignancy”.
`Rodan et al J Clin Invest, 1983, 72:1511 “Factors associated
`with humoral hypercalcernia of malignancy stimulate ade
`nylate cyclase in osteoblastic cells”.
`Slovik et al J Bone and Mineral Res, 1986, 1(4):377 “Res
`toration of spinal bone in osteoporotic men by treatment
`with human parathyroid
`hormone
`(l—34)
`and
`1,25-dihydroxyvitamin D”.
`Tsai et al J Clin Endocrinol Metab, 1989, 69(5):1024 “Bone
`responsiveness to parathyroid hormone in normal and
`osteoporotic postmenopausal women”.
`Vincent H. L. Lee, “Peptide and Protein Drug Delivery”,
`published by Marcel Dekker, Inc., (N.Y.), pp. 514—516 and
`538.
`
`Primary Examiner—Ronald W. Gri?in
`Attorney, Agent, or Firm—Foley & Lardner
`[57]
`ABSTRACT
`Described herein are preparations containing parathyroid
`hormone that has been stablized with an excipient and
`buffering agent. Preferred preparations incorporate human
`PTH(1—84), mannitol as excipient and citrate as buffering
`agent, and are incorporated in vials as a freeze-dried powder
`for reconstitution to treat osteoporosis.
`
`24 Claims, 3 Drawing Sheets
`
`CFAD Exhibit 1005
`
`1
`
`

`

`US. Patent
`
`Mar. 5, 1996
`
`Sheet 1 of 3
`
`5,496,801
`
`F/G. 714
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`

`

`U.S. Patent
`
`Mar. 5, 1996
`Sheet 2 of 3
`FIG. 2A
`
`5,496,801
`
`% SDS PAGE PURITY
`
`mi
`
`8 5i 5
`8
`
`A
`
`pH4 ; 4°C
`
`*- 500ug m 1000ug
`- 250ug
`<>2500ug A 1000ug
`1
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`
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`
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`
`9
`
`H6125
`wage SDS PAGE PURITY
`‘5Q 25‘ Q
`AA 0 A A
`
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`
`95
`
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`
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`TIME (MONTHS)
`
`i
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`
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`
`9
`
`3
`
`

`

`US. Patent
`
`Mar. 5, 1996
`
`Sheet 3 of 3
`
`5,496,801
`
`HG. 20
`
`100 /0 SDS PAGE PURITY
`
` 95
`
`90
`
`85
`
`8O
`
`pH6 ; 4°C
`
`- 100ug,pH6
`D 500ug,pH6
`
`916 250ug,pH6
`<> 1000ug,pH6
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`TIME (MONTHS)
`
`HQ. 20
`
`0/0 SDS PAGE PURITY
`100
`
`95
`
`90
`
`85
`
`pH6 ; 37°C
`
`. 100ug,pH6
`U SOOug,pH6
`
`9K 250ug,pH6
`<> 1000ug,pH6
`
`80
`
`'
`0123456789
`
`TIME (MONTHS)
`
`4
`
`

`

`5,496,801
`
`1
`PARATHYROID HORMONE FORMULATION
`
`FIELD OF THE INVENTION
`
`This invention relates to pharmaceutical compositions
`containing parathyroid hormone. More particularly, the
`invention relates to parathyroid hormone formulations with
`improved storage stability.
`
`BACKGROUND TO THE INVENTION
`
`Parathyroid hormone (PTH) is a secreted, 84 amino acid
`product of the mammalian parathyroid gland that controls
`serum calcium levels through its action on various tissues,
`including bone. Studies in humans with certain forms of
`PTH have demonstrated an anabolic effect on bone, and
`have prompted signi?cant interest in its use for the treatment
`of osteoporosis and related bone disorders.
`Using the N-terminal 34 amino acids of the bovine and
`human hormone for example, which by all published
`accounts are deemed biologically equivalent to the full
`length hormone, it has been demonstrated in humans that
`parathyroid hormone enhances bone growth particularly
`when administered in pulsatile fashion by the sub-cutaneous
`and intravenous routes. A slightly di?erent form of PTH,
`human PTH(l-38) has shown similar results. Because of its
`only recent availability, the recombinant form of the full
`length, human hormone, i.e., human PTH(1—84) has not yet
`been studied in humans although studies in rats indicate an
`equipotent and in some respects somewhat improved ef?
`cacy in bone growth.
`PTH preparations used in these studies have been recon
`stituted from fresh or lyophilized hormone, and incorporate
`various forms of carrier, excipient and vehicle. Most are
`prepared in water-based vehicles such as saline, or water
`acidi?ed typically with acetic acid to solubilize the hor
`mone. The majority of reported formulations also incorpo
`rate albumin as a carrier (see for example Reeve et al, Br.
`Med. J., 1980, 280:6228; Reeve et al, Lancet, 1976, 111035;
`Reeve et al, Calcif Tissue Res, 1976, 21 :469; Hodsman et al,
`Bone Miner; 1990, 9(2):137; Tsai et al, J. Clin. Endocrinol
`Metab, 1989, 69(5):1024; Isaac et al, Horm Metab Res,
`1980, 12(9):487; Law et al, J Clin Invest, 1983, 72(3): 1106;
`and Hulter, J. Clin evpertens, 1986, 2(4):360). Other
`reported formulations have incorporated an excipient such
`as mannitol, which is present either with the lyophilized
`hormone or in the reconstitution vehicle. Formulations rep
`resentative of those employed for human studies include a
`human PTI-I(1—34) preparation consisting, upon reconstitu
`tion, of mannitol, heat inactivated human serum albumin,
`and caproic acid (a protease inhibitor) as absorption
`enhancer (see Reeve et a1, 1976, Calcif. Tissue Res., 21,
`Suppl, 469—477); a human PTH(1—38) preparation recon
`stituted into a saline vehicle (see Hodsman et al, 1991, 14(1),
`67—83); and a bovine PTH(1—34) preparation in aqueous
`vehicle pH adjusted with acetic acid and containing albu
`min. There is also an International Reference preparation
`which for human PTH consists of 100 rrg of hormone
`ampouled with 250 pg human serum albumin and 1.25 mg
`lactose (1981), and for bovine PTH consists of 10 pg
`lyophilized hormone in 0.01M acetic acid and 0.1% w/v
`mannitol (see Martindale, The Extra Phannacoepia, The
`Pharmaceutical Press, London, 29th Edition, 1989 at p.
`1338).
`Commercial exploitation of parathyroid hormone requires
`the development of a formulation that is acceptable in terms
`of storage stability and ease of preparation and reconstitu
`
`l0
`
`15
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`25
`
`35
`
`40
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`2
`tion. Because it is a protein and thus far more labile than the
`traditionally small molecular weight drugs, however, the
`formulating of parathyroid hormone presents challenges not
`commonly encountered by the pharmaceutical industry. Fur
`thermore, and unlike other proteins that have been formu
`lated successfully, PTH is particularly sensitive to oxidation,
`and further requires that its N -terminal sequence remain
`intact in order to preserve bioactivity.
`It is an object of the present invention to provide a
`pharmaceutically useful PTH preparation, particularly one
`comprising, as active ingredient, the full length form of
`human PTH.
`
`SUMMARY OF THE INVENTION
`
`A pharmaceutically acceptable PTH preparation is now
`provided. According to one aspect of the present invention,
`the hormone preparation is in the form of a freeze dried
`composition, comprising a medically useful amount of par
`athyroid hormone, an excipient that will co-lyophilize with
`parathyroid hormone to form an amorphous cake, and a
`non-volatile buffering agent in an amount sufficient to adjust
`the pH of the preparation to a physiologically acceptable pH.
`In a preferred embodiment of the invention, the hormone in
`the preparation is human parathyroid hormone, the excipient
`is mannitol and the buffering agent is a citrate source.
`According to another aspect of the present invention,
`there is provided a process for obtaining a PT H preparation,
`which comprises the steps of combining in water the PTH,
`the buffering agent and the excipient, and then subjecting the
`resulting solution to a freeze-drying process that yields a
`product incorporating less than 2% water by weight.
`According to another aspect of the present invention,
`there is provided a method for obtaining a parathyroid
`hormone formulation for parenteral administration, which
`comprises the step of reconstituting a freeze-dried prepara
`tion of the present invention in sterile water.
`There is further provided in accordance with the invention
`a therapeutically useful kit, comprising a sterile vial con
`taining a freeze-dried preparation of the invention, a vehicle
`suitable for reconstitution thereof, and instructions for
`reconstitution, and optionally for administration. The kit
`may further comprise a device suitable for injection of the
`reconstituted preparation by the end user.
`The invention is now described in greater detail and with
`reference to the accompanying drawings in which:
`
`BRIEF REFERENCE TO THE DRAWINGS
`
`FIGS. 1 and 2 show the effect of storage at 4° C. and 37°
`C. on the stability of PTH preparations bulfered at pH 4 and
`pH6, where the stability is revealed by bioactivity assay
`(FIG. 1) and by SDS-PAGE analysis (FIG. 2).
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The invention relates to parathyroid hormone prepara
`tions that exhibit storage stability in terms of hormone
`composition and activity.
`As active ingredient, the preparation desirably incorpo
`rates the full length, 84 amino acid form of human parathy
`roid hormone, obtained either recombinantly, by peptide
`synthesis or by extraction from human ?uid. In this speci
`?cation, the human form of PTH is abbreviated
`hFl‘H(l-84), which has the amino acid sequence reported
`by Kimura et al, Biochem Biophys Res Comm, 114 (2):493.
`
`5
`
`

`

`5,496,801
`
`3 .
`As an alternative to the full length human form of PTH, the
`preparation may incorporate those homologues, fragments,
`or variants of human PTH that have human PTH activity as
`determined in the ovarectomized rat model of osteoporosis
`reported by Kimmel et al, Endocrinology, 1993, 32(4):1577
`and incorporated herein by reference.
`The parathyroid hormone may for example be the bovine
`or porcine forms of PTH (see Keutmann et a1, Current
`Research on Calcium Regulating Hormones, Cooper, C. W.
`(Ed), 1987, University of Texas Press, Austin, pp 57—63) or
`fragments or variants of the mature PTH homologues.
`Alternatives in the form of PTH fragments incorporate at
`least the ?rst 27 N-terminal residues of PTH, and desirably
`incorporate at least the ?rst 34 N-terrninal residues, such as
`PTH(l-34), PTH(1-37), PTH(1—38) and PI‘H(l-4l). Alter
`natives in the form of PTH variants incorporate from 1 to 5
`amino acid substitutions that improve PTH stability and
`half-life, such as the replacement of methionine residues at
`positions 8 and/or 18 with leucine or other hydrophobic
`amino acid that improves PTH stability against oxidation
`and the replacement of amino acids in the 25—27 region with
`trypsin-insensitive amino acids such as histidine or other
`amino acid that improves PTH stability against protease.
`These forms of PTH are embraced by the term “parathyroid
`hormone” as used generically herein.
`The parathyroid hormone preparations of the present
`invention are provided in a powder form containing not
`more than 2% water by weight, that results from the freeze
`drying of a sterile, aqueous hormone solution prepared by
`mixing the selected parathyroid hormone, a non-volatile
`buffering agent and an excipient.
`The excipient incorporated in the preparation serves as a
`cryoprotectant during the freeze-drying process and also as
`a bulking agent to facilitate dosage formulation. Of the
`pharmaceutically acceptable excipients, the present inven
`tion avoids sugars such as lactose and maltose, and exploits
`only those excipients capable, when combined with the
`selected buffering agent, of forming a non-crystalline, amor
`phous cake when freeze-dried. In having selected the excipi
`ent on this basis, the cake resulting from the freeze-drying
`process is of the homogeneous quality desired for rapid
`reconstitution. Polyol-type excipients are preferred herein.
`An evaluation of caking properties of polyol-type excipients
`has revealed that mannitol is a particularly preferred excipi
`ent, not only for its ability to yield a quality cake, but also
`because mannitol itself confers some stability to the PTH in
`solution.
`The buffering agent incorporated in the preparation of the
`present invention, in addition to being acceptable pharma
`ceutically, is necessarily a non-volatile buffering agent, i.e.
`one that is not volatilized during the freeze-drying process to
`the extent that pH is reduced by more than 0.4 pH units.
`Buffering agents used previously in PTH preparations, such
`as acetic acid, were found to volatilize at differential rates
`during the freeze-drying process, leading not only to an
`inconsistent product but also to the loss of buifering agent,
`and hence inconsistent pH levels in the reconstituted prod
`uct. The non-volatile buffering agents incorporated in the
`present preparations are selected from those capable of
`buffering the preparation to a pH within a physiologically
`acceptable range. A pH that is physiologically acceptable is
`that which causes either no., or minimal, patient discomfort
`when the formulation is administered, and can thus vary
`depending on the mode of administration. For preparations
`that will be diluted prior to administration, such as by
`dissolution in a stock infusion solution, the pH of the
`preparation per se can vary widely, e.g., from about pH 3 to
`
`25
`
`30
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`about pH 9. Where the preparation is to be administered
`directly after reconstitution, the PTH preparation is buffered
`desirably to within the pH range from 3.5 to 7.5. Suitable
`non-volatile buffers are accordingly those pharrnaceutically
`acceptable agents that can buffer the pH of the preparation
`to within the target pH range, and include phosphate-based
`buifers and, preferably, citrate-based buffers such as sodium
`citrate/citric acid.
`To provide storage stable preparations of parathyroid
`hormone in accordance with the invention, the selected
`non-volatile buffering agent is incorporated to yield a ?nal
`pH within the range from 3.5 to 6.5, and the excipient is
`incorporated to yield a ?nal concentration in the range from
`2% to 10% (w/v). In embodiments of the invention, the pH
`rendered by the buffering agent is in the range from 3.8 to
`6.2, and the ?nal concentration of the excipient is from 3 to
`7%, e.g. 4 to 6% (w/v). Most preferably, the buffering agent
`is a citrate source such as monosodium citrate/citric acid and
`the excipient is 5% mannitol (w/v).
`The PTH preparations of the present invention incorpo
`rate PTH in a medically effective amount, a term used with
`reference to amounts useful either therapeutically or in
`medical diagnosis. The particular amount of parathyroid
`hormone incorporated in the preparation can be pre-deter
`mined based on the type of PTH selected and on the intended
`end-use of the preparation. In one application, the prepara
`tions are exploited for therapeutic purposes, and particularly
`for the treatment of osteoporosis. Osteoporosis therapy
`entails administration of the reconstituted preparation by
`injection, desirably sub-cutaneous injection, in unit doses
`that re?ect the prescribed treatment regimen but are, for
`human P'I‘H(l—84), within the range from 25 pg PTH/ml. of
`injected solution to 500 pg/mL of injected solution per
`patient, with injection volumes being desirably from 0.3 to
`1.3 mL. Accordingly, the puri?ed and sterile-?ltered PTH is
`desirably incorporated with the buffering agent and excipi
`ent to form an aqueous solution containing PTH in a
`concentration range from 25 ug/mL to 250 ug/mL, prefer
`ably 50 ug/mL to 150 ug/mL. Molar equivalents of the
`substantially equipotent forms of PTH, such as the
`PTH(1—84) variants and fragments, can be similarly incor
`porated in place of the human PTH(l-84), if desired.
`In one embodiment of the invention, the preparations are
`provided in a form that yields a unit dose of 50-150 pg
`human PTH(1—84) upon reconstitution into about 1 mL
`(0.84.2 mL) of the reconstitution vehicle, and the vials are
`accordingly loaded with about 1 mL of the aqueous PTH
`preparation, for subsequent freeze-drying.
`In a preferred embodiment of the invention, the PTH
`preparation subjected to freeze-drying comprises from 25 to
`250 pg/mL of human PTH(1-84), from 2 to 8% by weight
`of mannitol, and a citrate source in an amount capable of
`buffering the preparation to within the range from 3.5 to 6.5
`upon reconstitution in sterile water. In speci?c embodiments
`of the invention, the citrate buffering agent is incorporated
`in an amount su?icient to buffer the pH to 6.0i0.4.
`Once the preparation is obtained as an aqueous solution
`containing desired amounts and concentrations of the bu?i
`ering agent, excipient and PTH, individual vials are ?lled
`with the solution to the desired volume, and the vials are
`then subjected collectively to the freeze-drying process.
`As is conventional in the art of formulation, the freeze
`drying, or lyophilization, process entails a temperature
`cycling process that is controlled carefully to ensure that
`drying proceeds uniformly and to substantial completion,
`i.e. to yield a powder containing not more than 2% water by
`
`6
`
`

`

`5,496,801
`
`15
`
`25
`
`5
`weight, and preferably not more than 1.5% water by weight.
`A protocol suitable for obtaining the present freeze-dried
`PTH preparations entails subjecting vials ?lled with the
`aqueous PTH preparation to a drying process having at least
`two diiferent drying stages, the ?rst being performed to drive
`unbound water from the aqueous preparation without caus
`ing collapse of the cake. This is achieved by ?rst cooling the
`vialled aqueous PTH preparation to a product-ice tempera
`ture of lower than —30° C., preferably about —50° C., and
`then increasing shelf temperature to, and holding at, about
`~10° C. under reduced pressure of not more than 350 ubar,
`e.g. 260 ubar, until substantially all unbound water is driven
`off. Under the conditions speci?ed in the examples herein, a
`drying time of 16 hours is appropriate. The second drying
`cycle is designed to liberate bound water from the cake,
`while again avoiding collapse of the cake and using a
`temperature below that deleterious to PTH bioactivity. This
`second drying step can be achieved under further reduced
`pressure (<50 ubar) at —10° C. for 3 hours, then warming to
`and holding at 25° C. until substantially all (<2%) of the
`bound water is driven oif, e.g., for at least 12 hours but
`preferably for 16 hours or more. On completion, the vials
`can be sealed, for example by automated stoppering, and
`then removed from the freeze-drier and capped.
`The PT H preparations of the present invention are com
`plete in the sense that the end-user need reconstitute the
`preparation solely in sterile water to generate an adminis
`trable formulation. For this purpose, and in accordance with
`another aspect of the present invention, there is provided a
`medically useful kit, comprising at least one vial containing
`a freeze~dried PTH preparation of the invention, at least one
`vial containing sterile water for reconstitution of the prepa~
`ration, and a sheet of instructions directing reconstitution of
`the freeze-dried PTH. The kit may further comprise an
`injection device for administration of the reconstituted for
`mulation by the end-user. In one embodiment of the inven
`tion, the injection device is a hypodermic needle, for
`example a 25 gauge needle and a syringe capable of receiv
`ing a solution volume of about 0.5-5 mL, e.g. l or 2 mL.
`Alternatively, the kit may comprise a vial containing mul
`tiple doses of PTH, and a companion vial containing enough
`sterile water to reconstitute that multiple dose formulation.
`In use, the end-user draws from the water-?lled vial into
`the injection device, and transfers that water to the PTH
`?lled vial to cause mixing and reconstitution of the freeze
`dried PTH powder, if necessary using the needle to draw and
`eject the mixture until the powder is visibly dissolved. The
`present FI‘H preparation has the advantage, however, that
`mixing is rapid, being complete without mixing within one
`minute and more usually within 30 seconds. After mixing,
`the end-user injects the PTH formulation in the manner and
`amount prescribed by the physician. In the case-where a
`multi-dose vial is provided, a bacteriostatic agent should be
`incorporated, and the formulation remaining after adminis
`tration of each dose can be refrigerated for subsequent use
`within a time frame of several days.
`In addition to their therapeutic use, the present PTH
`preparations can be formulated and administered to aid in
`medical diagnosis, and particularly to assist in establishing
`the diagnosis of hypoparathyroidism and pseudohypopar~
`athyoidism in hypocalcernic patients. Except for the dose of
`PTH, the composition of the PTH preparation will remain as
`described herein for therapeutic use. An intravenously
`infused, single dose of human PTH(l-84) or PTH
`bioequivalent that is equal to 200 International Units of PTH
`activity is appropriate for this diagnostic purpose. Diagnosis
`is then made by determining the effect of administered PTH
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`on urinary cAMP levels, with cAMP elevation being indica
`tive of the hypoparathyroidism condition, rather than its
`pseudoform.
`
`EXAMPLES
`
`Aqueous PTH preparations were ?rst prepared for sub
`sequent freeze-drying by mixing human PTH(1—84), as
`hormone; mannitol, as excipient; and a citrate source, as
`buffering agent.
`As a ?rst step in generating the preparations, two aqueous
`admixtures were prepared from a sterile 20% (w/v) mannitol
`injectable solution (British Pharmacopeia). The 20% man
`nitol solution was blended (l) with an aqueous citric acid
`solution to yield a ?rst aqueous admixture of 10 mM citric
`acid and 5% mannitol, and (2) with an aqueous sodium
`citrate monohydrate solution to yield a second aqueous
`admixture of 10 mM citrate and 5% mannitol. pH adjusted
`solutions of 5% mannitol were then obtained by blending the
`admixtures, in volumes appropriate to yield a 5% mannitol
`solution at about pH 4 (i0.2) solution and a 5% mannitol
`solution at about pH 6 (i0.2).
`The 5% mannitol solutions (pH 4 and pH 6) then received
`measured amounts of freeze-dried human PTH(l—84),
`which was rnicrobially produced, puri?ed and then sterile
`?ltered before being freeze-dried. Measured amounts of the
`PTH were then added to the pH 4 and pH 6 solutions of 5%
`mannitol, to generate stock solutions that, when vialled at a
`volume of 1.1 mL, gave vials containing PTH in the fol
`lowing pg amounts: 100, 250, 500, 1,000 and 2,500.
`For freeze-drying, solutions containing PTH at each of the
`prepared concentrations were aseptically ?lled either by
`hand or by an automated dispenser in 1.1 mL volumes into
`5 mL glass vials (USP Type I) and then loaded in trays into
`a sterilized, nitrogen-purged freeze-dryer pre-cooled to —50°
`C. After loading, and a pre-freezing period of 4 hours, the
`freeze-drying chamber was evacuated by reducing pressure
`to 0.26 mbar for one hour. The primary drying cycle was
`then implemented, consisting of gradual warming over thirty
`minutes from —50° C. to —10° C. at which the vials were held
`for 16 hours. The second drying cycle was then imple
`mented, consisting of further warming from —10° C. to 25°
`C. at further reduced pressure of 0.05 mbar over three hours
`and then holding at this temperature and pressure for 16
`hours. At the end of the second drying cycle, the chamber
`was purged with nitrogen and brought to 0.85—0.95 bar. The
`tray was then raised to engage rubber stoppers in the mouths
`of the vials and the vial trays removed and over~capped with
`an aluminum seal following pressure equilibration.
`Vials containing the freezed~dried PTH preparations, at
`various concentrations and at pH 4 or pH 6, were then stored
`at 4° C. and 37° C. for subsequent analysis at various time
`points of l, 2, 3, 6 and 9 months. Analysis of stability was
`performed by reconstituting the vialed preparation into 1.1
`mL of sterile water. This was achieved by injecting the water
`through the rubber stopper, then, after allowing up to one
`minute for reconstitution, removing the solution for analysis.
`Results of the stability trials are reported below in the
`context of the various tests employed to evaluate the prepa
`ration:
`Bioactivity of the PTH was measured using the estab
`lished rat osteosarcoma cell (UMR l06)-based assay of
`PTH-stimulated adenylate cyclase production. Protocols for
`this PTH assay are reported by Rodan et al in J. Clin. Invest,
`1983, 72:1511 and by Rabbani et al in Endocrinol. 1988,
`123:2709. After up to nine months in storage, no signi?cant
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`decrease in PTH bioactivity was observed at either 4° C. or
`37° C., at any PTH concentration, or at either pH 4 or pH 6.
`FIG. 1 illustrates, with a line of best ?t between averaged
`results, the analytical results for a PTH preparation contain
`ing a PTH dose of 1,000 pg.
`SDS-PAGE analysis of the reconstituted PTH prepara
`tions, performed in the conventional manner, similarly
`revealed no signi?cant decrease of purity during storage at
`either pH, temperature and storage temperatures examined,
`as shown in FIG. 2. Some decrease in purity was revealed by
`RP-HPLC analysis of the reconstituted formulation, but only
`at the higher 37° C. storage temperature (0.7% decrease in
`purity per month of storage), with 4° C. storage showing no
`signi?cant purity decrease by reversed phase-HPLC analy
`sis. The stability of the intact PTH was also revealed by
`immunoassay (Allegro) to be constant throughout the stor
`age period at all concentrations, pHs and temperatures
`evaluated.
`Residual moisture in the PTH preparation was determined
`by the standard Karl-Fischer technique and indicated that the
`water content of all freeze-dried preparations remained
`below 2% by weight, and typically at about 1% by weight,
`throughout the storage period.
`pH upon reconstitution revealed no signi?cant pH alter
`ation throughout the freeze-drying and storage process,
`con?rming that the buifering agent had not volatilized
`during lyophilization. Preparations buffered to pH 4
`remained at pH 4i0.2, and those buifered to pH 6 remained
`at pH 6i0.4.
`The rate and extent of dissolution of the freeze-dried
`preparations were examined. All batches dissolved in 1.1 mL
`sterile water within one minute at room temperature. The
`maximum dissolution time observed was 0.5 minutes for the
`pH 4 preparations, and 0.4 minutes for the pH 6 prepara
`tions. Furthermore, no particles were observed upon recon
`stitution of the freeze-dried powder at either pH and at either
`storage temperature.
`We claim:
`1. A parathyroid hormone preparation, comprising:
`a medically useful amount of parathyroid hormone
`(1—84);
`a polyol excipient that co-lyophilizes with said hormone
`to yield an amorphous cake;
`a non-volatile buffering agent in an amount su?icient to
`adjust the pH of the preparation to within a physiologi
`cally acceptable pH range; and
`water.
`2. A parathyroid hormone preparation according to claim
`1, wherein said excipient is mannitol.
`3. A parathyroid hormone formulation according to claim
`1, wherein the buifering agent is a citrate source.
`4. A parathyroid hormone formulation according to claim
`3, wherein said excipient is mannitol.
`5. A parathyroid hormone preparation according to claim
`1, in the form of a freeze-dried powder containing not more
`than 2% water by weight.
`6. A parathyroid hormone preparation according to claim
`5, wherein the hormone is human PTH(l-84).
`7. A parathyroid hormone preparation according to claim
`6, wherein said excipient is mannitol.
`8. A parathyroid hormone preparation according to claim
`7, wherein the buffering agent is a citrate source.
`9. A parathyroid hormone preparation according to claim
`8, wherein said excipient is mannitol.
`10. A parathyroid hormone preparation comprising
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`parathyroid hormone (l—84) in a concentration within the
`range from 25