`
`AAPS PharmsciTech 2001
`PIiiiiiiISdtecIr
`http//www.pharmscitech.com
`Preparation Characterization and In Vivo Evaluation of Salmon
`Calcitonin Microspheres
`DcLuca2
`Dani and Patrick
`Bhas
`
`article 22
`
`Inhale Therapeutic Systems 150 Industrial Road San Carlos CA 94070
`2Faculty of Pharmaceutical Sciences University of Kentucky College of Pharmacy Lexington KY 40536
`
`Submitted June
`ABSTRACT
`
`2001 Accepted October
`
`2001
`
`Published October 17 2001
`
`INTRODUCTION
`
`Purpose
`
`characterize
`
`hydrophilic
`
`was
`This
`done
`prepare
`study
`and evaluate salmon calcitonin sCT
`microspheres ms in vivo using
`low molecular
`5050
`weight
`poly DL-lactide-co
`glycolide polymer PLGA Methods sCT ms were
`
`to
`
`prepared
`
`by
`
`particle
`
`dispersion/solvent
`process and characterized for
`extraction/evaporation
`drug content particle size surface morphology and
`integrity of encapsulated peptide Peptide
`structural
`stability and binding to the polymer was studied in 0.1
`phosphate buffer PB pH 7.4 and 0.1
`acetate
`buffer AB pH 4.0
`Serum sCT
`levels were
`monitored for weeks after subcutaneous injection of
`sCT ms to rats Results sCT ms were essentially free
`of discernible
`surface pores with
`size
`distribution in the range of 16 to 89 mm and mean
`particle size of 51 and 53 mm for
`batches Fourier
`Transform Matrix-assisted Laser Desorption mass
`spectrometry of the extracted peptide showed that the
`chemical
`did not
`alter
`its
`process
`encapsulation
`structure The peptide was substantially more stable in
`AB than in PB Peptide binding to the polymer was
`dependent on pH and was markedly higher in PB than
`in AB In vivo study proved that elevated serum sCT
`levels could be sustained for at
`least 10 days after
`administration of sCT ms to rats at
`dose of 1.0
`mg/kg Conclusions
`It was demonstrated that sCT
`into polymeric ms prepared
`could be incorporated
`low molecular weight hydrophilic PLGA
`from
`without
`
`using
`
`dispersion
`
`molecular
`
`structure
`
`tecbnique
`2-week
`
`altering
`
`formulation was
`
`dose of 1.0 mg/kg
`
`prepared at
`KEYWORDS
`Salmon
`calcitzzonin
`microspheres PLGA peptide interaction
`
`sCT
`
`Corresponcling Author Patrick
`of Pharmaceutical
`College
`Sciences
`Faculty
`Rose
`of Kentucky
`Pharmacy
`University
`Lexington KY 40536 USA Telephone 859-27- 1831
`859-323-0242 E-mail ppdelul pop.uky.edu
`Facsimi
`
`DeLuca Ph.D
`of
`
`Street
`
`Salmon
`
`calcitonin
`
`32
`
`amino
`
`acid
`
`sCT
`polypeptide has
`physiological role in the regulattin
`of calcium homeostasis and is
`inhibitor of
`
`potent
`from clinical
`
`trials
`
`in
`
`and
`
`bone
`
`early
`
`resorption Results
`animal studies have shown that sCT depresses
`and
`bone
`loss
`turnover
`prevents
`postmenopausal women
`sCT
`being
`
`bone
`
`is
`
`and ovariectomized rats
`
`used
`
`to
`
`treat
`
`clinically
`formulated
`
`It
`
`is currently
`
`or as
`
`as
`
`sterile
`
`osteoporosis
`solution for intramuscular or subcutaneous
`injection
`pulypeptide sCT is
`nasal spray Being
`vulnerable to digestive degradation and has
`very
`t2 after parenteral
`in the body
`short half- life
`administration is approximately 15 to 20 minutes
`The treatment of osteoporosis
`long-term
`requires
`therapy and because of these properties which are
`drawbacks to long-term therapy patient compliance
`limited especially when
`be
`can
`severely
`daily
`injection regimen of sCT is required Hence there is
`an ongoing effort to develop controlled-release dosage
`forms formulations that can effectively deliver sCT
`Poly DL lactide-co-glycolide copolymer PLGA
`has shown promise for the delivery of sCT
`This
`variety of
`commercially available in
`copolymer
`comonomer
`ratios and molecular weights Previous
`work in this laboratory has shown that sCT could be
`successfully encapsulated with high loading efficiency
`in PLGA microspheres ms made from
`high
`molecular weight hydrophobic polymer that had end
`However wetting
`groups protected alkylated
`was difficult
`and required
`long time for bond
`and the release of sCT
`and biodegradation
`cleavage
`from these ms occurred in intermittent pulses through
`120 days followed by
`large peak between 120 and
`148
`The
`coincided with
`days
`latter
`solubilization of
`
`the polymer
`Recently because of the interest
`
`complete
`work
`in theft application as
`
`EXHIBIT _QOc-1
`WIT ________
`
`DATE QjL
`
`DAWN HILLIER RMR CRR
`
`ALKERMES Exh. 2027
`Luye v. Alkermes
`IPR2016-1096
`
`
`
`delivery matrices PLGA polymers with
`drug
`hydrophilic end groups have become available This
`focused
`and
`on
`study
`preparing
`characterizing
`sustained-release delivery system for sCT made from
`5050 low molecular weight hydrophilic PLGA
`and evaluating the system in vivo
`MATERIALS AND METHODS
`
`Materials
`
`Boehringer
`
`sCT was purchased from Bachem Inc Torrance
`CA PLGA RGSO2H polymer
`34035
`lot
`7800
`was obtained from
`molecular weight
`Ingelheim Inc Ingeiheim Gennany and
`was used for ms preparation Polyvinyl alcohol PVA
`average MW 30 000-70 000 was obtained from
`Sigma Chemical Co St Louis MO The solvents and
`grade and were
`other excipients were analytical
`from commercial sources Female Sprague
`purchased
`and
`Dawley rats that weighed approximately 250
`were 90
`days old were purchased from Harlan
`IN and
`in vivo
`the
`Indianapolis
`studies were conducted at the University of Kentucky
`College of Pharmacy Animal Research Facility in
`accordance with the institutional guidelines
`HPLC method for peptide assay
`
`Laboratories
`
`Bondclone
`
`The peptide was analyzed by reverse-phase high-
`performance liquid chromatography HPLC using
`LC-6A pumps an SIL-6B autoinjector an SPD-6AV
`and an SCL-6B system controller all
`from
`detector
`Instruments mc Columbia
`Shimadzu Scientific
`MID The column used was
`Bondclone
`10 C-18
`3.90 mm with
`column 150
`reversed-phase
`3.90 mm guard column
`10 C-l8 30
`Phenomenex Torrance CA Gradient elution was
`accomplished with 0.1% trifluoroacetic
`and acetonitrile 0.1% trifluoroacetic
`and
`acid
`from 30% to 50%
`increasing the amount of phase
`flow of 1.5 mL/min Standard
`over 10 minutes at
`curves of sCT ranging
`from 6.25
`to 200 j.tg/mL
`yielded linear responses over that concentration range
`with detection at 220 nm
`
`acid in water
`
`Polymer Characterization
`The molecular weight distribution of RG5O2H PLGA
`was
`determined
`by
`polymer
`permeation
`gel
`chromatography GPC using Waters M-45 solvent
`delivery system with Waters 990 Photodiode Array
`Detector and ultrastyragel columns connected
`in series
`
`Tg was determined
`calorimeter DSC equipped with
`refrigeration
`system Thermal Analysis Instruments DSC 2920
`New Castle DE From to 10 mg of the polymer
`in an aluminum pan Then under
`was
`sealed
`nitrogen purge the sample and reference empty pan
`rate of 5C mm
`from 20C to 80C
`were heated at
`to 20C then heated at 5C miii
`cooled at 10C miii
`to 65C
`Preparation of sCT ms
`dispersion method
`sCT ms were prepared
`and
`followed by solvent
`evaporation
`in methanol was
`solution of peptide
`Briefly
`solution of PLGA in methylene
`combined with
`chloride and stirred until clear The solution was then
`reactor with baffles Ace
`slowly injected into
`Glass mc Vineland NJ containing the continuous
`phase CP 0.35%
`solution of PVA pH 7.2
`L4R
`stirred at 3500 rpm with
`homogenizer Silverson Machines Ltd Waterside
`Chesham Buckinghamshire UK The temperature of
`at 25C for 30
`the reactor was maintained initially
`minutes and then at 35C for
`hours Once ms were
`formed and hardened the contents of the reactor were
`equipped with
`transferred to
`apparatus
`0.8 ji membrane filter Gelman Sciences Ann Arbor
`MI and the recovered product was rinsed with water
`and either lyophilized or dried under reduced pressure
`for 48 hours at room temperature Blank ms without
`sCT were prepared similarly
`
`by
`
`extraction
`
`filtration
`
`7.8
`
`300 mm each one with Jo4
`pores and one
`pores The polymer was dissolved in
`with i03
`tetrahydroftiran THF at 0.1% wtlvol eluted with
`THF at mL/min and analyzed at 230 nm Number
`weight Me weight
`average molecular
`average
`molecular weight Mw and polydispersity M/M
`
`were determined
`
`using
`
`differential
`
`scanning
`
`and
`
`Silverson
`
`Microsphere Characterization
`in ms was determined by HPLC after
`Peptide content
`the ms
`and
`chloride
`in methylene
`dissolving
`acetate buffer AB
`extracting the peptide with 0.1
`pH 4.0 Fourier Transform Matrix-assisted
`Laser
`Desorption FT-MALDI mass spectrometry with
`dihydrobenzene as the matrix and nitrogen laser at
`260 intensity was used to determine the molecular
`weight of sCT The integrity of encapsulated sCT was
`confirmed by comparing the mass spectrum of sCT
`extracted from the ms to that of standard sCT solution
`
`
`
`glass
`
`transition
`
`using
`
`Malvern 2600c Particle Sizer Malvern UK Surface
`morphology was analyzed
`scanning
`electron
`microscopy SEM with
`800 Tokyo
`on
`instrument after palladium/gold
`Japan
`coating
`aluminum stubs Total product yield was assessed
`gravimetrically on the basis of polymer/drug
`IN VITRO STUDIES
`
`of ms
`The
`was
`temperature
`determined by DSC Particle size distribution was
`determined
`
`laser
`
`diffraction
`
`technique
`
`by
`
`Hitachi
`
`recoveiy
`
`Peptide Stability
`
`Stock solutions of sCT were prepared by dissolving
`10 mg of sCT in 50 mL of 0.1
`phosphate buffer
`PB pH 7.4 or 0.1
`AB pH 4.0 All solutions were
`used immediately upon preparation Peptide stability
`temperatures 4C
`was determined in PB and AB at
`25C and 37C Approximately mL of fresh stock
`solutions were placed in mL glass scintillation vials
`and the vials were sealed Periodically aliquots were
`removed from each vial and the amount of intact
`peptide was assayed by HPLC
`Peptide Adsorption to PLGA ms
`Stock solutions of sCT were prepared by dissolving
`lsmgofsCTin30niLof0.l MPBpH7Aor0.l
`AB pH 4.0 AU solutions were used immediately
`preparation One milliliter
`sCT
`of
`stock
`upon
`solutions in PB and AB were incubated at 37C with
`10 mg of blank ms At each time point
`samples of
`each suspension were removed from the incubator
`The samples were centriffiged and the amount of
`peptide in the supernatant was determined by HPLC
`In Vivo Evaluation of sCT ms
`
`sCT ms were evaluated
`in vivo in female Sprague
`Dawley rats Three groups of rats were subjected to
`Free sCT was
`the treatments described in Table
`single injection was
`dissolved in 0.9% saline and
`administered
`dose of 71 ug/kg
`subcutaneously
`The microsphere lots were suspended
`in mixture of
`1% CMC and 2% mannitol The formulations were
`volume ie pL
`body weight
`body weight
`subcutaneously
`just
`single injection was given to
`below the neck region
`each animal
`
`at
`
`injected
`
`at point
`
`administered
`
`immediately after collecting
`
`predosing
`
`sample
`
`Blood samples were collected from the tail vein under
`light ether anesthesia
`Samples from the animals
`
`Treatment Regimens of Free sCT and sCT
`Table
`ms Administration in Rats
`
`Group
`Group
`
`Group
`
`sCT indicates
`
`Free sOT
`sCT ms
`sCT02
`sOT ms
`sOTO2
`salmon calcitonin
`
`0.071
`
`0.5
`
`1.0
`
`receiving free sCT were collected at
`from the
`
`hours after
`
`and 24
`
`animals
`
`Samples
`injection
`receiving the microsphere formulations at 0.5 mg/kg
`hour and
`and 1.0 mg/kg doses were collected at
`12 and 15 days after injection The samples
`in Microtainer
`tubes Becton
`were
`centrifuged
`Dickinson Franldin Lakes NJ to separate and collect
`the serum Serum samples were frozen and stored at
`radioimmunoassay RIA
`20C until analysis using
`Serum sCT analysis
`Serum sCT was measured by 125j RIA with
`commercially available kit Peninsula Laboratories
`San Carlos CA Serum samples were incubated with
`rabbit sCT antiserum for 24 hours at 4C followed by
`addition of 251-labeled sCT After an additional 24-
`hour
`second
`antirabbit
`antibody-goat
`incubation
`immunoglobulin G-was
`followed by 90-
`added
`minute incubation at room temperature to separate the
`and free sCT The antibody-bound
`antibody-bound
`then measured by MINAU
`radioactivity was
`counter Packard Downers Grove IL
`
`RESULTS
`
`The
`
`and
`
`Polymer Characterization
`of RG5O2H were approximately 7800
`1.8
`
`and
`4500
`RGS02H showed
`
`polydispersity
`respectively
`Tg of 36.4C
`
`Microsphere Characterization
`
`Thermal analysis of blank ms prepared from RGSO2H
`polymer showed an increase in Tg 39.5C compared
`to that of the raw polymer However
`the Tg of the
`lots of sCT ms sCTOl
`sCTO2 40.5C and
`and
`40.3C respectively was not much different
`than
`that of the blank ms Table
`
`
`
`Table
`
`Physical Properties of sCT and Blank ms
`
`Batch
`
`sCT Load
`wt
`
`Incorporation
`
`Efficiency
`
`wt%
`
`Blank ms
`sd mssCTO1
`sCT mssCTO2
`4.5
`90
`sCT indicates salmon cal citonin ms microspheres
`
`5.1
`
`102
`
`Yield
`wt
`
`80
`
`78
`
`80
`
`Ig
`
`39.5
`
`40.5
`
`40.3
`
`Particle Size jim
`90% 50% 10%
`
`86
`
`89
`
`89
`
`48
`
`53 ij
`nfl
`
`Figure
`
`Scanning electron microqraphs
`
`showing the surface morphology of blank and salmon calcitonin sCT
`
`of
`
`lots of sCT ms was
`The average particle size of the
`sCTO2 mm Incorporation
`53 sCTO1 and 51
`efficiencies of 102% and 90% revealed that most of
`within the ms
`sCT had
`been
`the
`encapsulated
`surface morphology by SEM
`Examination
`the
`revealed that both sCT as well as blank ms were free
`The mass
`of discernible
`surface pores Figure
`spectrum of sCT extracted from ms Figure
`was
`standard sCT solution figure
`similar to that of
`2figures should be called out in numerical order
`figures which
`rewrite senteuce or renumber
`integrity of the peptide
`
`confirmed that the structural
`was intact within the ms
`
`Pep tide Stability
`
`shows the effect of pH and temperature on
`Figure
`the stability of sCT in solution The peptide was
`substantially more stable in AB pH 4.0 than in PB
`pH 7.4 In PB at 37C 37% of the peptide was
`degraded within 24 hours and 100% after
`days In
`AB at 7C 95% of the initial amount still
`remained
`days 90% after
`days 71% after 15 days and
`after
`63% after 22 days Peptide degradation was slower at
`lower temperatures in both buffers and as much as
`82% and 91% of the initial amount still
`remained after
`in AB
`22 days at 22C and 4C respectively
`
`
`
`100
`
`80
`
`B6
`
`DC
`
`20
`
`t- 37C PB
`-25C PB
`t-4C PB
`37C AB
`25C AB
`--4C AB
`
`10
`
`15
`
`20
`
`25
`
`Days
`
`Figure
`
`Stability of salmon calcitonin sd at 37C
`25C and 4C in 0.1M phosphate buffer PB and
`acetate buffer AB
`
`30
`
`cc
`
`-J
`
`c20
`
`-D
`
`10
`
`10
`Time hours
`
`20
`
`Figure
`blank ms
`
`buffers
`
`Adsorption of salmon calcitonin 5CT to
`phosphate PB and acetate AB
`
`in 0.1
`
`In Vivo Study
`
`Single administration of free sCT to rats at 71 pg/kg
`dose Figure 6A resulted in
`serum sCT peak
`hour
`later observed C0 760 pg/mL As expected the
`sCT levels returned rapidly to baseline levels at 24
`hours since sCT has
`very short half- life in vivo after
`Administration of 0.5
`parenteral administration
`mg/kg dose of sCT microspheres Figure 6B resulted
`in C11 of 791 pg/nt at
`hour Elevated serum sCT
`days and
`levels were sustained for at least
`
`LJiiL-
`
`Figure
`
`Fourier Transform Matrix-assisted
`FT-MALDI mass
`Desorption
`calcitonin sCT standard in 0.1
`
`Laser
`
`spectrum of salmon
`acetate buffer
`
`Figure
`
`Laser
`
`Fourier Transform Matrix-assisted
`FT-MALDI mass
`sCT
`Desorption
`spectrum of
`extracted from salmon calcitonin 5CT microspheres
`ms in 0.1
`acetate buffer
`Peptide Adsorption to PLGA ms
`As shown in Figure
`there was not much adsorption of
`AB pH 4.0 at 37C
`sCT to blank PLGA ms in 0.1
`and the amount of peptide remaining in the supematant
`24 hours was 97%
`2% of the imtial
`the amount of peptide adsorbed to
`In contrast
`PB pH 7.4 increased with time
`the blank ms in 0.1
`and approximately 19.7 ug of peptide was adsorbed to
`hours 41% of the initial amount
`mg of ms after
`Although no samples were analyzed between
`and 24
`hours saturation binding appeared to occur within 24
`hours at appmximately 20 jig/mg
`
`throughout
`amount
`
`
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`1200
`
`1000
`
`800
`
`I-
`
`600
`
`c0
`
`400
`
`200
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`Free sOT 71 pg/kg
`
`10
`
`15
`
`20
`
`scT ma 0.5 mg/kg
`
`10
`
`15
`
`20
`
`aCT ma 1.0 mg/kg
`
`10
`
`15
`
`20
`
`Time days
`
`Figure
`
`free sd
`
`Serum sCT levels after administration of
`and sCT ms to rats
`
`for
`
`detectable
`
`levels were observed
`days following
`administration Administration of 1.0 mg/kg dose of
`sCT microspheres produced C1 of 984 pg/nil at
`hour Figure 6C and elevated serum sCT levels were
`days after administration with the
`sustained beyond
`levels returning to baseline after 12 days
`
`.tnIt
`I..1j 001 LI IN
`
`UI
`
`elevated
`
`serum
`
`levels
`
`for
`
`shorter
`
`lithe
`
`GPC analysis of the raw 502H polymer showed that
`was 7800
`This was lower
`the
`than the typical
`502H lots which have
`in the range of 10 000
`account
`of
`This would
`the
`duration
`DSC
`desired
`than
`the blank ms prepared
`measurements indicated that
`slightly higher Tg 39.5C
`from 502H polymer had
`than did the raw polymer 36.4C This slight shift
`in
`T6 was most
`likely the result of the processing of the
`polymer during the microsphere preparation process
`On the other hand Ill of sCT ms 40.3C and 40.5C
`was comparable to blank ms
`marked shift
`in
`would have
`between
`indicated
`strong interaction
`sCT and the polymer in the dry state Thus the DSC
`in the dry state sCT did not
`that
`results suggest
`strongly with the hydrophilic
`low
`interact
`very
`molecular weight 502H PLGA polymer
`higher
`502H polymer would have
`molecular weight
`slightly higher T6 which would facilitate processing
`FT-MALDI mass spectmm of the extracted peptide
`showed that the encapsulation process did not alter its
`chemical structure Stability studies confirmed that the
`peptide was substantially more stable in solution at
`pH of 4.0 than at
`pH of 7.4 which was consistent
`pH of 3.3 for
`with previous studies that had reported
`maximum stability
`There was
`very
`adsorption of sCT to blank PLGA ms at
`pH of 4.0
`However
`the alsorption increased dramatically at
`pH of 7.4 These results indicate that the nonspecific
`adsorption of sCT to PLGA ms was greater at
`pH
`to the isoelectric point @j of sCT which is
`closer
`This is consistent with earlier work
`around 10.2
`sCT adsorption
`PLGA Mw 34
`alkylated
`maximum
`end
`where
`hydrophobic
`groups
`adsorption occurred near the p1 of sCT and almost no
`pH less than
`adsorption was observed at
`was thought
`higher net charge on the peptide
`that
`pH below p1 could result
`molecules at
`in repulsion
`within and between peptide molecules At the p1 of
`the peptide molecules with
`zero net charge could
`approach each other more closely and form more
`compact conformation resulting in more effective
`adsorption The results of that study suggested that
`interactions played an important role in
`hydrophobic
`the adsorption Interestingly at pH 7.4 the amount of
`sCT
`adsorbed
`low molecular
`weight
`the
`hydrophilic PLGA 502H was much lower 19.7 jig
`
`on
`
`to
`
`higher molecular weight
`
`000
`
`containing
`
`It
`
`to
`
`
`
`elevated
`
`sCT/mg PLGA compared with the high molecular
`weight hydrophobic PLGA 80 jig sCT/mg PLGA
`serum sCT levels could be
`
`Because
`sustained for about
`II days following administration
`of sCT ms to rats at
`dose of 1.0 mg/kg this was
`2-week formulation
`to be promising as
`thought
`502H
`higher molecular weight
`especially with
`PLGA
`
`CONCLUSIONS
`
`the
`
`It was demonstrated that sCT could be incorporated
`into polymeric ms prepared from
`low molecular
`hydrophilic PLGA with
`weight
`dispersion
`technique without altering
`its molecular structure
`DSC measurements
`lack of strong
`indicated
`interaction between sCT and the polymer in the dry
`state Peptide stability was greater at lower pH while
`at higher pH
`binding was
`higher
`significantly
`Elevated serum sCT levels could be sustained for at
`least 10 days following administration of 1.0 mg/kg of
`sCT ms to rats
`
`REFERENCES
`
`Wronski TJ Yen CF Burton KW et al Skeletal
`
`effects
`
`of
`
`calcitonin
`
`in
`
`ovariectomized
`
`rats
`
`Endocrinology 1991 1292246-2250
`Ohnhaus EE Fischer JA
`Born
`Huwyler
`Plasma kinetics and urinary excretion of exogenous
`human and salmon calcitonin in man Am Physiol
`l979236E15-E19
`
`Millest AJ Evans JR Young JJ Johnstone
`release of salmon calcitonin in vivo from
`Sustained
`
`lactideglycolide copolymer depots Calcif Tissue Tnt
`19935236l-364
`Mehta RC Thanoo BC DeLuca PP
`Jeyanthi
`Effect of processing parameters on the properties of
`PLGA
`microspheres
`1997 142 163-174
`10 Lee KC Lee YJ Song HM Chun CJ DeLuca PP
`Degradation of synthetic salmon calcitonin in aqueous
`
`peptide-containing
`
`Microencapsulation
`
`solution Pharm Res 1992
`
`11 Tsai
`
`Mehta RC DeLuca PP Adsorption of
`Effect
`peptides to poly DL-lactide-co-glycolide
`of solution properties on the adsorption Tnt Pharm
`1996 12743-52
`
`Montagnani
`Agnusdei
`An effective
`regimen of
`salmon calcitonin in early postmenopausal bone loss
`
`Gennari
`
`Civitelli
`
`Calcif Tissue mt 1992
`
`Gonnelli
`
`intranasal
`
`Bigi
`
`et al Effects of
`
`ovariectomy Calcif Tissue Tnt 990 14
`
`JC Whitehead MI
`Stevenson
`Maclntyre
`Wimalawansa SJ Banks LM Healy MJR Calcitonin
`for prevention of postmcnopausal bone loss Lancet
`1988 l8591900-902
`Mazzuoli GF Tabolli
`salmon
`on the
`
`calcitonin
`
`bone loss
`
`induced
`
`by
`
`Ballanti
`
`in rats after
`
`11
`
`Ramires PA Richardson JL
`Bonucci
`Benedetti LM Prevention of ovariectomy
`osteopenia
`administration of hyaff
`vaginal
`salmon
`calcitonin Calcif
`
`microspheres
`containing
`Tissue Tnt 199556274-279
`McSheehy PM Farina
`evaluation of the calcitonin
`analogue
`Pharmacologic
`SB 205614 in models of osteoclastic bone resorption
`in vitro and
`vivo
`comparison with salmon
`calcitonin and elcatonin Bone 1995l6435-444
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