J. Pharm. Pharmacol. 1996,48: 1012-1015
`Received November 16, 1995
`Accepted February 27, 1996
`
`0 1996 J. Pharm. Pharmacoi.
`
`Local Tolerance of Subcutaneous Injections
`
`J. F R A N S S O N ' t A N D A . E S P A N D E R - J A N S S O N '
`*Pharmacia and Upjohn, S-1 12 87 Stockholm, ?Department of Pharmaceutics, Faculty of Pharmacy, Uppsala
`University, Box 570, S-751 23 Uppsala, Sweden
`
`Abstract
`Human insulin-like growth factor I (hIGF-I) has several possible clinical applications. Because subcutaneous
`administration of the drug can cause pain, local tolerance to injection of different formulations with or without
`hIGF-I has been investigated in man using isotonic saline solution as reference.
`The formulations, made isotonic with NaCI, ranged in pH from 6 to 7 with phosphate buffer concentrations
`of 5 to 50 mM. The local tolerance after injection was assessed as injection pain on a visual analogue scale, pain
`duration and local tolerance (redness, paleness and oedema). The discomfort at the injection site was lowest
`with 10 mM phosphate, pH 7. Injection of buffer at pH 6 (50 mM phosphate) caused significantly more pain
`than using 10 mM phosphate, whereas the pain at pH 6 using 10 mM phosphate did not differ significantly from
`that experienced on injection of the solution at pH 7 using either 10 or 50 mM phosphate. hIGF-I itself did not
`seem to cause pain.
`We concluded that for subcutaneous injections at non-physiological pH, the buffer strength should be kept as
`low as possible to avoid pain upon injection. We also hypothesize that when a non-physiological pH must be
`used for stability reasons, a lower buffer strength enables more rapid normalization of the pH at the injection
`site.
`
`Human insulin-like growth factor (hIGF-I) has insulin-like and
`growth-promoting effects and there are possibilities for future
`clinical use of recombinant hIGF-I in a wide variety of
`applications. hIGF-I is most stable at pH 6 (Fransson et a1
`1994) and a parenteral formulation at this non-physiological
`pH was developed for subcutaneous administration. The severe
`pain experienced by several patients in early clinical trials on
`injection with hIGF-I has been investigated in this study. In
`numerous studies with other drugs (Ipp et a1 1990; Zindel
`1989; Gazzaniga et a1 1993) subjects have reported pain upon
`subcutaneous injection. Factors that might cause pain include
`the injection volume and the speed of injection (Frenken et a1
`1994), osmolality (Doenicke et a1 1992), the pH of the
`formulation (Barnet & Kapp 1992), injection site (Ipp et a1
`1990), the size and quality of the injection needle (Coley et a1
`1987), the presence of irritating substances (Frenken et a1
`1993) and the temperature of the solution (Ross & Soltes
`1995).
`The purpose of buffers in pharmaceutical formulations is to
`maintain a stable pH, usually that at which the drug is most
`stable. The ability of a buffer to maintain a pH value is
`dependent on the pK, of the buffer, on the pH and on the
`concentration (Flynn 1980). We hypothesized that the injection
`pain could be reduced if a formulation with a lower buffer
`capacity was used for hIGF-I.
`Several clinical trials on analgesics have shown that pain
`scales can be a reliable and objective method of assessing the
`analgesic properties of drugs (Baiios et a1 1989). It is generally
`accepted that visual analogue scales are more sensitive and
`accurate than other measures, thus being generally advised in
`the evaluation of the intensity of pain (Langley & Sheppard
`1985).
`
`Correspondence: J. Fransson, Pharmacia and Upjohn, S-112 87
`Stockholm, Sweden.
`
`The aim of this study was to evaluate how pH, buffer con-
`centration and presence of hIGF-I affect local tolerance to
`subcutaneous injection of the solution.
`
`Materials and Methods
`
`Study design
`The investigation was designed as a double-blind, randomized,
`10 x 10 Graeco-Latin square with the three factors: subject
`(1-lo), site of injection (1-10) and injection order number (1-
`10). Eight formulations and two identical reference solutions
`containing 150 mM NaCl were compared and were balanced in
`respect of the site and of the injection number in the sequence
`of ten injections given to each subject. All injections were to
`be in 4 x 4 cm regions on the lower left and right arms, which
`are relatively sensitive to pain.
`A published 10 x 10 Graeco-Latin square (Peng 1967) was
`taken as a basis (Table 1). The ten rows of this square corre-
`spond to subjects, the ten columns correspond to injection
`sites, the ten Latin letters A-J correspond to formulations, and
`the ten numerical indices correspond to the injection number in
`the sequence of injections given to each subject. The assign-
`ments of subjects to rows, of injection sites to columns, and of
`letters A-J
`to formulations, were performed randomly. The
`resulting assignments were then transferred to personal maps
`specifying the site for each injection in each subject.
`
`Formulations
`Although phosphate is not ideal at pH 6, because of its low
`buffer capacity at this pH, sodium phosphate was chosen as
`buffer, because citrate buffer causes pain (Frenken et a1 1993).
`The compositions of the eight formulations and the reference
`solution are listed in Table 2. The formulations were prepared
`by mixing disodium phosphate, monosodium phosphate and
`
`MYLAN INST. EXHIBIT 1047 PAGE 1
`
`MYLAN INST. EXHIBIT 1047 PAGE 1
`
`

`

`LOCAL TOLERANCE OF SUBCUTANEOUS INJECTIONS
`The Graeco-Latin square experimental design. The columns correspond to the injection sites, the rows correspond
`Table 1.
`to the subjects and the indices correspond to the injection number.
`
`1013
`
`Row
`(subject no.)
`
`Column
`(site of injection)
`
`8
`
`5
`
`7
`
`3
`
`4
`
`10
`
`6
`
`9
`
`1
`
`2
`
`~
`
`6
`8
`10
`3
`7
`9
`5
`4
`2
`1
`
`sodium chloride in water for injection to give isotonic solu-
`tions of different pH and buffer concentration. hIGF-I was
`added to some of the solutions by ultrafiltration.
`
`Study population
`Ten healthy adult male volunteers, aged 18-40 and sensitive to
`injection pain, were recruited to the study. All potential par-
`ticipants were injected with 0.2 mL of a vehicle known to
`inflict pain on injection @H 6, 50 mM phosphate) over 20 s at
`a pre-study medical examination. At 30 s after injection each
`subject assessed the injection pain on a 100 mm horizontal
`visual analogue scale (0 mm =no pain, 100 mm = severe
`pain). Subjects that marked > 20 mm on this scale were eli-
`gible for inclusion in the study.
`
`Injection procedure
`After randomization, all subjects received a total of ten
`injections (0.2 mL injection- '). The ten injections, repre-
`senting the eight formulations and the two sodium chloride
`references, were injected subcutaneously using a 0.5 mL 28 G
`Microfine syringe (Beckton and Dickinson, USA) with a fixed
`cannula. The solutions were stored at 2 4 ° C until the day of
`use, when they were equilibrated to room temperature before
`injection. All injections were given subcutaneously over 20 s
`by one person.
`For each subject each of the ten injections was made every
`I5 min in a specified order and site on the lower left and right
`
`Table 2.
`The pH and concentrations of sodium phosphate buffer,
`sodium chloride and hlGF-I in the formulations.
`
`Formulation
`
`pH
`
`Phosphate Sodium chloride
`
`hlGF-I
`(mg mL-')
`
`A
`B
`C
`D
`E
`F
`G
`H
`I*
`J*
`
`6.0
`6.0
`7.0
`7.0
`6.0
`6.0
`6.0
`7.0
`8
`8
`
`50
`10
`50
`10
`5
`50
`10
`50
`0
`0
`
`112
`145
`112
`145
`150
`112
`145
`112
`I50
`150
`
`0
`0
`0
`0
`0
`5
`5
`5
`0
`0
`
`*Reference solutions
`
`arms. The subjects filled in assessment sheets after each
`injection. In order to estimate the local tolerance to injection,
`the variables evaluated were: injection pain 30 s after each
`injection, assessed on a 100 mm visual analogue scale
`(0 mm =no pain, 100 mm = severe pain); duration of injection
`pain, measured with a stop-watch given to each subject; local
`tolerance in terms of redness, paleness and oedema, assessed
`by one of the staff on a yes or no scale; and, for some subjects,
`description of the injection pain in words (e.g. burning, itch-
`ing).
`An erroneous exchange of injection 2 between subjects 7
`and 8 occurred during the study. Subject 7 did not receive
`formulation I and received formulation B twice, whereas
`subject 8 received formulation I twice and did not receive
`formulation B. All other subjects received all the formulations
`once. This imbalance was taken into account in the statistical
`handling of the data (see below).
`
`Blood glucose
`On treatment with hIGF-I there is a minor risk of hypogly-
`caemia. At the doses used in this study, this was considered to
`be very unlikely because the injections were given to non-
`fasting subjects. In order to minimize the possibilities of
`hypoglycaemia, non-fasting glucose was measured before the
`fist injection and after the last, and food was supplied during
`the study day.
`The concentration of glucose was measured with a Reflolux
`photometer (Boehringer Mannheim, Germany). Blood samples
`were taken from the finger tips from all subjects on the study
`day. The glucose levels were measured before the injections,
`and 0.5 and 3 h after the last injection.
`
`Statistics and data analysis
`The primary variable in the statistical analysis consisted of the
`visual analogue scale assessments of pain at each injection.
`Comparison of the eight formulations and two references was
`based on a graphical version of Tukey's T-procedure adapted
`to a 10 x 10 Graeco-Latin square design. Comparison between
`the effects of the formulation on the visual analogue scale
`responses could then be made in terms of so-called LSMEANS
`which, briefly, estimated what the means would have been if
`the design had been balanced. A slightly conservative variant
`of Tukey's T-procedure was used, namely to replace the
`
`MYLAN INST. EXHIBIT 1047 PAGE 2
`
`MYLAN INST. EXHIBIT 1047 PAGE 2
`
`

`

`1014
`J. FRANSSON AND A. ESPANDER-JANSSON
`standard error of the difference between any two LSMEANS
`by the maximum 7.237 of all such standard errors. The 5%
`upper quartile of the studentized range distribution with
`parameter k = 9 (formulations) and 64 degrees of freedom is
`equal to 4.54 (Hochberg & Tamhane 1987). The ‘uncertainty’
`intervals based on Tukey’s 2“-procedure to be used in the
`graphical display, each have endpoints of
`the
`form
`LSMEAN f 4 . 5 4 x 7.237/8, i.e. LSMEAN f 11.6. The for-
`mal comparisons of the formulation effects using Tukey’s T-
`procedure were considered to be approximate in view of the
`visual analogue scale used. The residual plot indicated, as
`expected, that the variability in the visual analogue scale
`scores was relatively small at low levels, although the statis-
`tical model used actually assumes constant variability. Tbis
`was compensated somewhat by the use of a slightly con-
`servative variant of Tukey’s 2“ procedure, with somewhat too
`large ‘uncertainty’ intervals.
`
`0
`
`n
`
`0
`
`o
`0 0 0
`
`0
`
`0
`
`0
`
`0 0 0
`
`0
`
`0
`
`60 -
`50 -
`z
`E 40-
`-
`I al
`m
`2 30-
`0 m -
`m
`3 20 -
`10 -
`
`v)
`
`m
`
`3
`5
`
`Results and Discussion
`As shown in Fig. 1 the different formulations caused different
`amounts of injection pain. The LSMEANS of the visual ana-
`logue scale scores and the end-points of the corresponding
`‘uncertainty’ intervals are shown in the figure. Non-over-
`lapping intervals indicate significant (at the 5% level) differ-
`ences between the effects of the formulations. Even though
`there were quite large inter-individual differences, as shown by
`Table 3, pH 6, 50 mM phosphate formulations clearly caused
`more injection pain than pH 6, 10 mM phosphate formulations.
`It was shown that injection of hIGF-I, (pH 6, 50 mM phos-
`phate), caused injection pain in at least 90% of the subjects in
`the study ( > 10 mm on visual analogue scale), whereas only
`30% of the subjects in the study marked more than 10 mm on
`the visual analogue scale on injection with a formulation of
`hIGF-I of the same pH but with only 10 mm phosphate. Fur-
`ther reduction in buffer concentration to 5 mm phosphate did
`not reduce pain further. Because formulations with or without
`hIGF-I caused similar amounts of injection pain, it is con-
`cluded that hIGF-I itself did not cause injection pain.
`
`FIG. I. Assessment of injection pain by visual analogue scale (0).
`Results from statistical evaluation of ‘uncertainty intervals’, upper (0)
`and lower limit (0) for simultaneous comparisons of formulations at
`an approximate significance level of 5% based on Tukey’s T-method.
`The formulations are described in Table 2.
`
`An hIGF-I preparation of pH 7 is not feasible because of the
`lower stability of hIGF-I. This pH caused less injection pain
`than pH 6, however. At pH 6, a lower buffer concentration
`resulted in less discomfort, possibly because a higher buffer
`concentration results in a slower change in solution pH at the
`injection site. The frequency and intensity of occurring red-
`ness, paleness and oedema at the injection site also decreased
`on reducing the buffer concentration or increasing the pH.
`A non-physiological pH changes the chemical equilibrium
`between physiological buffers in subcutis and cutis tissue. This
`change causes transport of potassium ions which depolarize the
`nerve endings, causing pain (Guyton 1991). The magnitude
`
`Summary of the assessments of pain on a visual analogue scale (mm) for each
`Table 3.
`formulation versus subject number. The formulations are described in Table I .
`
`~
`
`~~~~
`
`~
`
`Subject
`number
`
`A
`
`29.0
`2.0
`1.0
`45.0
`56.0
`26.0
`18,O
`64.0
`20.0
`47.0
`
`30.9
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`Mean
`
`~
`
`Formulation
`
`B
`
`C
`
`D
`
`E
`
`F
`
`G
`
`H
`
`I + J
`
`0.0
`0.0
`16.0
`0.0
`7.0
`11.0
`0.0 10.0
`1.0
`19.0
`30.0
`28.0
`0.0
`7.0
`15.0
`2.0
`6.0
`7.0
`5.0
`2.0
`10.5
`-*
`0.0 28.0
`0.0 12.0
`0.0
`0.0
`73.0
`12.0
`
`9.7
`
`6.5
`
`16.3
`
`14.0
`14.0
`3.0
`33.0
`38.0
`23.0
`2.0
`17.0
`3.0
`4.0
`
`15.1
`
`13.0
`57.0
`0.0
`33.0
`78.0
`21.0
`13.0
`75.0
`41.0
`53.0
`
`2.0
`21.0
`5.0
`17.0
`60.0
`8.0
`0.0
`2.0
`7.0
`0.0
`
`11.0
`0.0
`7.0
`15.0
`0.0
`9.0
`3.0
`2.0
`27.0
`9.0
`
`0.0
`0.0
`1.0
`20.5
`8.5
`2.0
`0.0
`10.3
`29.5
`1.5
`
`38.4
`
`12.2
`
`8.3
`
`7.2
`
`*Subject number 8 did not receive formulation B.
`
`MYLAN INST. EXHIBIT 1047 PAGE 3
`
`MYLAN INST. EXHIBIT 1047 PAGE 3
`
`

`

`1015
`
`Acknowledgements
`We thank Associate Professor Bertil Karlmark and Dr Marta
`Ryde for their expertise, Dr Ebba Florin-Robertson for valu-
`able comments during the preparation of the manuscript, and
`Dr OIivier Gilbaud for the statistical analysis.
`
`LOCAL TOLERANCE OF SUBCUTANEOUS MJECTIONS
`and duration of the depolarization would be dependent on the
`pH and buffer concentration. In other words, the induced
`depolarization of the nerve endings is more quickly normalized
`at lower buffer concentrations.
`Pain is difficult to evaluate, partly because of its subjective
`character and the complex feelings that pain evokes. The
`subjective sensation of pain differs greatly within individuals,
`as is obvious from the large inter-individual differences in
`visual analogue scale scores in Table 3.
`The Graeco-Latin square design used in this study was
`chosen in order to obtain as much information as possible from
`a limited number of subjects. This design requires complete
`balance in order to achieve convincing results on completion
`of the study. In this study, however, imbalance occurred as
`stated at the end of the section on injection procedure. It was,
`therefore, necessary to modify the statistics somewhat on
`calculation of the results.
`There is a significant difference in pain, measured on a
`visual analogue scale, caused by injections at pH 6 and pH 7
`when the buffer concentration is high (50 mM). The pain
`experienced at pH 6 can, however, be reduced substantially by
`reducing the buffer concentration. It is the buffer capacity of
`the solution (i.e. resistance to pH changes) that determines how
`stable the pH value will be when the solution is injected into
`the tissue. The results from this study show that because the
`hIGF-I formulation of pH 6 in 10 mM phosphate results in less
`pain at the injection site it is a more suitable hIGF-I for-
`mulation of than that of pH 6 in 50 mM phosphate. As the
`formulations with hIGF-I and the corresponding vehicles cause
`a similar degree of injection pain, it is shown that the pain is
`not caused by the hIGF-I itself.
`
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`MYLAN INST. EXHIBIT 1047 PAGE 4
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`MYLAN INST. EXHIBIT 1047 PAGE 4
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`