`
`The Biocompatibility of Parenteral Vehicles-In Vitro/In Vivo Screening
`comparison and the Effect of Excipients on Hemolysis
`
`ROGER CHERNG-CHYI FU"", DEBORAH M. LIDGATE, JOHN L. WHATLEY, and TIM McCUU.OUGH
`
`Institutes of Pharmaceutical Science and Toxicology, Syntex Research, Palo Alto, California
`
`ABSTRACT: The hemolytic potential for a series of intravenous solutions was determined by an in vitro
`testing procedure; the solutions were subsequently administered intravenously to rats and evaluated for in
`vivo biocompatability. Each test solution contained an excipientfrom one or more of the following categories:
`nonaqueous cosolvents; complexing agents; surfactants. The test results indicate that the in vitro hemolysis
`values closely predict the in vivo test results. Further, a commonly used parenteral cosolvent, propy_/ene
`glycol, was found to produce a large hemolytic response which can be alleviated by the addition of either a
`tonicifying agent or polyethylene glycol 400. These findings present useful information when formulating a
`parenteral vehicle utilizing an organic cosolvent.
`
`Introduction
`
`Drug compounds which display minimal solubility in an
`aqueous solution present a challenge when formulating a
`biocompatible intravenous injectable preparation. In this
`investigation, the compound of interest, a dihydropyridine
`compound, possesses very low intrinsic aqueous solubility
`of 12 µg/ mL (1). For clinical and toxicological testing,
`intravenous solutions containing up to 10 mg/mL are
`required. A series of formulations were prepared which
`solubilize the dihydropyridine compound in the desired
`concentration range. These solutions contain ex.cipients
`from one or more of the following categories: (i) nonaque(cid:173)
`ous cosolvents such as ethanol, propylene glycol (PG),
`polyethylene glycol 400 (PEG 400), dimethylisosorb~de
`(DMI), and dimethylacetamide (DMA); (ii) complex.mg
`agents such as nicotinamide; and (iii) surfactants such as
`polox.amer (pluronic L64) and polyox.yethylated vegeta(cid:173)
`ble oil (Emulphot4 EL-719).
`Incorporation of the various cosolvents and excipients
`can adversely impact the biological compatibility of the
`parenteral vehicle. Thus, in vivo biocompatibility of each
`solution was evaluated in rats after administration. Urine
`and blood samples were collected for examination, and the
`physical appearance at the site of injection as well as the
`general condition of the rat were monitored. In conjunc(cid:173)
`tion with the in vivo testing, in vitro tests were performed
`to determine the hemolytic potential of each solution.
`During the course of testing, propylene glycol proved to
`have a high degree of hemolytic potential as well as non(cid:173)
`biocompatible properties; these results coi~c:ide with pre-
`
`Received October 23, 1986. Accepted for publication August 8, 1987.
`" A~thor to whom inquiries should be directed.
`
`164
`
`viously reported finding~ (2-4). Because propylene glycol
`is a widely used cosolvent for parenteral administration
`(5, 6), various ex.cipients were evaluated in combination
`with PG to determine their tandem effect on hemolytic
`potential. These additional solutions were not designed to
`solubilize the dihydropyridine compound. Instead, the so(cid:173)
`lutions were selected to ex.amine whether a parenteral
`vehicle containing PG can exhibit biocompatibility.
`
`Experimental
`
`In vivo Biocompatibi/ity Testing
`
`Spraque-Dawley derived CD male rats were used. The
`rats were between 8-12 weeks of age at the time of treat(cid:173)
`ment. Twelve groups, each composed of five rats, were
`given a single intravenous bolus dose (2.5 mL/kg) of
`either test or control vehicle once daily for two weeks.
`Each dose was delivered over approximately l 0 sec via the
`tail vein. No attempt was made to inject the test vehicle at
`the same site each day. Clinical observations such as
`swelling and/ or bruising at the site of injection, pallor and
`activity level, were recorded at least once weekly. Body
`weights were recorded weekly and terminally. Urine sam(cid:173)
`ples were collected on study day l or 2 (for observation of
`an acute tox.icityreactfon) and on study day 13 ((or obser(cid:173)
`vation of a more chronic reaction). Blood samples for
`hematology and clinical chemistry evaluations were col(cid:173)
`lected on st:µdy day 15, the day after th~ final~ose.
`Vehicles i-12 listed below were chosen for evaluation
`based onth~ir abilitytosolubilize the subject drug unde
`development. As such, a systematic examination of al
`po5$ible combi11ations ofthe solvents used, was not unde
`take11. All vehicles prepared for testing had an appare
`pH in the range qf 5.0-7 .4 ..
`
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`
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`
`
`Composition
`
`Results and Discussion
`
`Formulation
`No.
`1
`2
`
`3
`
`4
`
`5
`
`6
`7
`
`8
`
`9
`
`Isotonic saline solution-control
`12% Ethanol, 15% PG, 20% PEG
`400 (in ABS)
`12% Ethanol, 15% PG, 20% PEG
`400 (in water)
`10% Ethanol, 40% PEG 400 (in
`ABS)
`10% Ethanol, 40% PEG 400 (in
`water)
`l 0% Ethanol, 40% PG (in ABS)
`40% Dimethylisosorbide (in acetate
`buffer)
`10% Ethanol, 20% nicotinamide (in
`water)
`10% Ethanol, 30% PG, l 0%
`nicotinamide (in water)
`15% DMA, 15% nicotinamide (in
`water)
`15% Ethanol, 6% pluronic L64 (in
`SAB)
`7% Emulphor EL 719 (in SAB)
`12
`ABS = acetate buffered saline (0.9% sodium chloride,
`0.012% glacial acetic acid, adjusted to a pH of 5.2
`± 0.2).
`SAB = sorbitol acetate buffer (5.0% sorbitol, 0.012% gla(cid:173)
`cial acetic acid, adjusted to a pH of 5.2 ± 0.2).
`
`11
`
`In vitro Hemolysis Testing
`
`Formulations were tested for hemolytic potential ac(cid:173)
`cording to the method developed by Reed and Y alkowsky
`(3). The method calls for mixing equal volumes of test
`vehicle with whole human citrated blood; the remaining
`intact red blood cells are washed several times with saline,
`then lysed with water. The hemoglobin concentration is
`determined by spectrophotometer and compared to a con(cid:173)
`trol sample (Formulation l) treated in the same manner.
`
`Excipient Effect on Propylene Glycol Hemolytic
`Activity-In vitro/In vivo Evaluation
`
`A 15% propylene glycol water solution was selected as
`the control. Various concentrations of sodium chloride,
`sorbitol, and polyethylene glycol 400 were added to the
`PG control solution. These solutions were tested for hemo(cid:173)
`lytic potential by the in vitro testing method described
`above. Based on the hemolysis data obtained, further test(cid:173)
`ing of certain solutions was performed in vivo for their
`biocompatibility, following the procedure outlined above.
`The solutions selected for this additional in vivo work are
`listed below.
`
`Formulation No.
`13
`14
`15
`16
`
`Composition
`15% PG in water
`15% PG, 20% PEG 400 in water
`15% PG, 1.8% NaCl in water
`15% PG, 9% sorbitol in water
`
`Formulations 2 through 12 were selected f
`
`.
`
`based ?n their ability to solu?ilize more than 10 ;r ;esting
`
`a sparingly water soluble dihydropyridine com g mLof
`pound A.
`.
`.
`1
`few formulations contam on y subtle modificat·
`·
`ions of e
`cipients such as water vs. buffered saline (Formul
`~-
`.
`ations 2
`d 5) h
`d
`and 3, an 4 an
`; t ese were compared for th b
`and salt effects on biocompatibility.
`e uffer
`A summary of in vivo test results are prese d .
`Tables I and IL With the exception of animals ~te
`in
`with vehicles 8 and 10, all other vehicle-treated
`r~ated
`. b
`1 .
`animals
`showed a norma mcrease m ody weight comparable
`the control group (Table I).
`to
`Anima~s in all g~?;.psd e:cept the saline control and
`~ormulationhl2, ex i 11~te c anges ~t the tail vein injec.
`tion area sue as: swe mg, encrustation, and/or disc 1
`1· h
`.
`Th
`h
`.
`o or-
`ese c anges were s ig t, or shght to moderat f
`ation.
`0
`vehicles 2, 3, 4, 5, 7, 9, and 10, and were slight to ma\
`~
`for vehicles 6, 8, and 11 (Table I).
`r e
`Hematological changes attributed to the experime t l
`n a
`·
`'f
`d
`d
`regimen were roam este as a ecrease in erythroc t
`hemoglobin, and hematocrit values; these changes WY e,
`"
`. 1
`ere
`most apparent 1or amma s treated with vehicles 8 and 9
`and were seen to a lesser extent with vehicles 6 and 12
`Treatment-related clinical chemistry changes showed
`slightly increased A/G:G (albumin to globulin) ratio
`following treatment "'.'ith vehi~les 3 and 5 and a slight!;
`decreased A/G:G ratio followmg treatment with vehicle
`8. Other cli~ical manifestations such as inactivity (vehi(cid:173)
`cles 6, 7, and 11) and pallor (vehicles 8, 9, and 11) were
`observed (Table II).
`The in vivo hemolysis testing results along with visual
`observations and dipstick testing results of urine samples
`from in vivo studies, are presented in Table III. Treat(cid:173)
`ment-related urinary changes consisted of discolored
`urine and occult blood present in urine. Discolored, red-
`
`T ABLE I. Body Weight Change and Vein Irritation Results
`After i.v. Administration of Test Vehicles
`Body Weight Change
`(gm)± SD
`
`Formulation No.
`
`Perivascular
`Irritation'
`
`31.4 ± 4.2
`I
`29.6 ± 2.3
`2
`32.6 ± 7.4
`3
`29.4 ± 10.5
`4
`30.8 ± 11.3
`5
`26.0 ± 9.7
`6
`27.4 ± 9.9
`7
`-4.8 ± 18.3
`8
`13.6 ± 20.7
`9
`-10.6 ± 12.3
`10
`26.2 ± 11.6
`11
`44.4 ± 10.0
`12
`41.0 ± 5.0
`13
`14
`42.6±8.l
`15
`40.0 ± 5.0
`45.8 ± 3.3
`16
`• Visual inspection of tail vein.
`I = slight.
`2 = moderate.
`3 = marked.
`
`0
`I
`2
`2
`1
`3
`I
`3
`I
`2
`3
`0
`I
`0
`0
`0
`
`Vol. 41, No. 5/September-October 1987
`
`165
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`TABLE II. Hematological Results Observed After i.v. Administration of Test Vehicles
`
`Formulation No.
`
`Red Blood Cell
`Count
`mil/cumm ± SD
`
`2
`3
`4
`5
`6
`7
`8
`9
`10
`II
`12
`13
`14
`15
`16
`
`8.16 ± 0.34
`8.24 ± 0.27
`8.16±0.29
`7 .67 ± 0.47
`7.75 ±0.43
`7.38 ± 0.49
`7.77±0.31
`6.82 ± 0.44
`6.71 ± 0.36
`8.14 ± 0.27
`7.83 ± 0.23
`7.46 ± 0.38
`7.97 ± 0.35
`7.73 ± 0.46
`7.96 ± 0.33
`7.78 ± 0.24
`
`Hemoglobin
`gm/lO0mL
`±SD
`
`14.7 ± 0.9
`15.0 ± 0.5
`14.8 ± 0.7
`14.3 ± 0.5
`14.5 ± 1.0
`13.6 ± 0.8
`14.5 ± 0.5
`12.0 ± 0.6
`12.1 ± 1.2
`14.9 ± 0.2
`14.l ±0.8
`12.9 ± 0.5
`14.7 ± 0.2
`14.4 ± 0.8
`14.7 ± 0.2
`14.5 ± 0.5
`
`Hematocrit
`%±SD
`
`39.8 ± 2.6
`40.0 ± 1.2
`39.4 ± 2.1
`38.0 ± 2.3
`38.6 ± 2.3
`35.8 ± 1.8
`38.6 ± 0.9
`32.4 ± 1.9
`33.2 ± 2.8
`39.5 ± 0.6
`37.0 ± 2.1
`34.8 ± 1.5
`39.5 ± 1.0
`37.4 ± 2.2
`39.8 ± 0.7
`39.8 ± 1.5
`
`A/G:G
`±SD
`
`1.5 ± 0.3
`1.5 ± 0.4
`2.1 ± 0.2
`1.9 ± 0.3
`2.1 ± 0.2
`1.3 ±0.1
`1.5 ± 0.2
`0.7 ± 0.1
`1.2 ± 0.5
`1.3 ± 0.3
`1.4 ± 0.4
`1.3 ± 0.2
`1.5 ± 0.3
`1.5 ± 0.3
`1.2 ± 0.l
`1.3 ± 0.2
`
`brown (bloody) urine was noted visually at each sampling
`interval and was primarily seen in animals given vehicle 6
`or 9. Red-brown urine was also noted at study day 1 or 2
`for several animals given vehicle 7 or 8. Occult blood was
`detected at each sampling interval and primarily observed
`for vehicles 6, 7, 8, 9, and 10.
`The in vitro hemolytic data corresponds quite well with
`the results observed during in vivo testing. Generally,
`those solutions demonstrating a high hemolytic value by
`in vitro testing also produce negative physical changes
`with in vivo testing. The data indicate that the solutions
`most prone to elicit an in vivo hemolytic response contain
`excipients such as nicotinamide, propylene glycol, and
`dimethy lisosorbide.
`Because propylene glycol is a commonly used cosolvent
`
`for parenteral formulations, a diminishing of its toxicolog(cid:173)
`ical effects is desired. Using the same in vitro and in vivo
`techniques described above, two tonicifiers (sodium chlo(cid:173)
`ride and sorbitol) and polyethylene glycol 400 were evalu(cid:173)
`ated individually in 15% propylene glycol solution. The in
`vitro results for sodium chloride and sorbitol are shown in
`Figures 1 and 2, respectively. As expected with these
`compounds, hemolysis caused by the 15% propylene gly(cid:173)
`col solution was decreased with the addition of each of the
`tonicifying agents (2, 4).
`According to the osmotic calculation,.a 2.0% propylene
`glycol solution in water is iso-osmotic to human red blood
`cells. The fact that a 15% propylene glycol aqueous solu(cid:173)
`tion causes a high degree of hemolysis suggests the solu(cid:173)
`tion is hypotonic to red blood cells. The tonicifying agents,
`
`TABLE III. Comparison of In Vitro and In Vivo Testing Data:% Hemolysis vs. Urinary Observations
`Dal'. 1
`
`Formulation No.
`
`In Vitro
`% Hemolysis•
`
`Urine
`Color'
`
`Occult
`Blood<
`
`none-trace
`straw
`I
`0
`none-trace
`straw
`2
`2
`none
`straw
`7
`3
`none
`straw
`4
`0
`none
`straw
`3
`5
`marked
`red brown
`94
`6
`marked
`straw-red brown
`7
`23
`slight-marked
`straw-red brown
`63
`8
`marked
`straw-red brown
`78
`9
`none-marked
`straw
`10
`53
`none-trace
`1
`11
`straw
`none-trace
`straw
`0
`12
`yellow
`marked
`80
`13
`straw
`none
`14
`12
`none
`straw
`27
`15
`np_ne-trace
`straw
`27
`16
`• For the in vitro% hemolysis data, the standard;deviation for all samples is approximately ±5%.
`• Visual inspection.
`' Dipstick test.
`
`166
`
`Jou.rnal of Parenteral Science & Technol
`
`Dav 13
`
`Urine
`Color
`
`straw
`straw
`straw
`straw
`straw
`straw-red brown
`straw
`straw
`straw-red brown
`straw
`straw
`straw
`straw-pink
`straw
`straw
`straw
`
`Occult
`Blood
`
`none-trace
`none-slight
`none
`none
`none-moderate
`marked
`none-trace
`none-trace
`marked
`none-marked
`none-trace
`none
`none-marked
`none -
`none
`none
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`100
`
`81)
`
`60
`
`20
`
`100
`
`00
`
`60
`
`._
`
`l
`C . 40
`:
`
`20
`
`1.0
`
`Percent Sodium 0,lor1de (_,/v)
`Figure 1-Effect of sodium chloride on the hemolytic potential of a
`propylene glycol solution (15% w/v).
`
`sodium chloride and sorbitol, achieve substantial or maxi(cid:173)
`mal protection in the propylene glycol vehicle at concen(cid:173)
`trations of 1.8% for sodium chloride and 20% for sorbitol.
`These concentrations are also greater than the concentra(cid:173)
`tion needed to make an isotonic aqueous solution (that is,
`0.9% for sodium chloride and 5.0% for sorbitol). These
`tonicifying agents are effective in preventing hemolysis of
`the propylene glycol-water vehicle through their colliga(cid:173)
`tive properties. In general, colligative properties can be
`assessed by freezing point depression ( using an osmome(cid:173)
`ter ); but because 15% propylene glycol itself does not
`freeze by this method, the effect of the tonicifying agent
`on this property cannot be evaluated.
`Figure 3 shows hemolysis as a function of the addition
`of polyethylene glycol 400 (PEG 400) to a 15% propylene
`glycol solution. The graph again shows a decline in hemo-
`
`100
`
`C j
`
`40
`
`20
`
`5.0
`
`10.0
`
`15.0
`
`20,0
`
`25.0
`
`ll.O
`
`Percent Sorb1tol (w/v)
`Figure 2-Effect of sorbitol on the hemolytic potential of a propylene
`glycol solution (15% w/v).
`
`10
`
`20
`
`40
`Percent Polyethylene Glycol 400 h,/v)
`Figure 3-Effect of polyethylene glycol 400 on the hemolytic
`tial of a propylene glycol solution (15% w/v).
`
`JO
`
`l-0
`
`poten-
`
`lysis_ with the_ a~dition o~ P~G 400. For reference, PEG
`400 m water, 1s 1so-osmot1c with 0.9% sodium chloride t
`concentration of 11.6% (7). PEG 400 exhibits the gre/t a
`.
`f"
`est
`. 1
`b
`su stant1a protective e 1ect at a concentration of about
`20%. As o?served with the tonicifyi~g agents, a larger
`concentration than that needed for an 1so-osmotic soluti
`is required for red blood cell (RBC) protection in prop;~
`ene glycol.
`The reduction of hemolysis by PEG 400 in the propyl(cid:173)
`ene gl_ycol-water system may be attributed to: (J) an
`osmotic effect of PEG 400; and (2) a potential complex
`formation, through hydrogen bonding, between the pro(cid:173)
`pylene glycol and PEG 400. This complex formation
`thereby modifies the hemolytic property of propylene gly(cid:173)
`col.
`The finding that PEG 400 demonstrates protective ef(cid:173)
`fects against PG is useful in formulating a cosolvent sys(cid:173)
`tem for lipophilic drugs because it: (i) provides a more
`biocompatible solution through its protective effect on
`RBCs against PG; and (ii) increases the solubility power
`of the cosolvent system.
`To evaluate the correlation of the in vitro hemolytic
`values to an in vivo response, solutions 13-16 were pre(cid:173)
`pared and tested in vivo following the procedure outlined
`above. All the samples chosen gave approximately 60%
`decrease for in vitro hemolysis. For sodium chloride and
`PEG 400 this represented the greatest protective effect
`achievable. For sorbitol, a 9% concentration level was
`chosen to compare with the two solutions prepared above;
`maximum in vitro protection, however, was found to occur
`at 20%. Animals given vehicle 13 (15% PG, 85% water)
`exhibited occult blood in their urine on study days I and
`13. One of the samples collected on study day 13 was pink
`in color (presumably due to blood in the urine). Animals
`given vehicles 14-16 did not exhibit any form of urinary
`changes. For all vehicles, no other treatment-related
`changes were seen for any other parameter measured:
`clinical condition, body weights, hematology or clinical
`chemistr:r (Tables I, II and III). The results confirm that
`
`Vol. 41, No. 5/September-October 1987
`
`167
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`.
`•rro test results are predictive of in vivo findings,
`the ,nhvz ddition of either 9% sorbitol, 1.8% sodium chlo(cid:173)
`a~d t ez~o/o PEG 400 improves the in vivo acceptability of
`ride or
`.
`!So/o pG solution.
`.
`the h eneral finding that in vivo toxicology data (hemo-
`T eg
`b
`·
`·
`.
`.
`f l
`tential) can be gauge y zn vitro testing 1s use u
`.
`lyuc p:Ormulating a parenteral preparation. An ideal for-
`h. h
`.
`when
`.
`. .
`I t·on would contain excip1ents w ic are nontoxic,
`B
`"
`h
`.
`l
`mua t
`. . •
`• ·tating and nonsensitizing.
`ut 1or p ysica or
`non1rn
`'
`.
`.
`1
`·cal reasons, these cntena cannot a ways be met; the
`cben11
`. .
`d
`be
`dd't' on of solvents an excipients may
`necessary to
`ah~ ~ea desired drug solubility. This study has demon(cid:173)
`ac ,e
`d
`· ·
`h
`PG
`t d that various solvents an excipients sue as
`,
`stra e
`.
`.
`DMl, and nicotinamid~ are detn~ent~l to red blood cells
`d exhibit negative side effects 1n vwo; the adverse ef(cid:173)
`:;cts produced by propJlene glyco_l ~a~ be diminished by
`the incorporation of_ either a tomcifying agent or PEG
`400. This latter finding suggests that no~aqueous. cosol-
`ts in general, might be made more biocompatible by
`f
`ven
`'
`·
`d · .
`F
`h
`l
`.
`mbining with certain a ditives. urt er ormu ation
`·1
`co
`d
`development with a nonaq_ueous _cosolve_nt shoul. ~nta1 an
`• vitro hemolysis screening using various additives (for
`h
`,n
`• •
`example, tonicifying agents, or alternate exciptents, sue
`as PEG 400) to determine suitability. Then a concentra(cid:173)
`tion vs. hemolysis profile should be generated for the
`
`excipient-cosolvent combination to determine optimal in
`vitro biocompatibility.
`It should be noted that although in vitro hemolysis data
`successfully predicts in vivo hemolysis, additional unrelat(cid:173)
`ed adverse effects may become apparent during in vivo
`testing (as was seen with vehicle 3, 5, and 11). Therefore,
`after initial in vitro screening for hemolysis, the proposed
`vehicles should ultimately be screened in vivo prior to
`being judged acceptable for parenteral use.
`
`References
`
`I. Unpublished data from the Preformulation Department of Syntex
`Research. Private communication with Leo Gu.
`2. Fort, F. L., Heyman, I. A., and Kesterson, J. W. "Hemolysis study of
`aqueous polyethylene glycol 400, propylene glycol, and ethanol com(cid:173)
`binations in vivo and in vitro," J. Parenter. Sci. Technol., 38, 82
`(1984).
`3. Reed, K. W. and Yalkowsky, S. H., "Lysisofhuman red blood cells in
`the presence of various cosolvents," J. Parenter. Sci. Techno/., 39, 64
`( 1985).
`4. Cadwallader, D. E., "Behavior of erythrocytes in various solvent
`systems," J. Pharm. Sci., 52, No. 12, 1175 (1963).
`5. Spiegel, A. J. and Noseworthy, M. M., "Use of nonaqueous solvents
`in parenteral products," J. Pharm. Sci., 52, 10 (1963).
`6. Wang, Y-C. J. and Kowal, R. R., "Review of excipients and pH's for
`parenteral products used in the United States," J. Parenter. Drug.
`Assoc., 34, 6 (1980).
`7. Smith. B. L. and Cadwallader, D. E., "Behavior of erythrocytes in
`various solvent systems," J. Pharm. Sci., 56, No. 3, 351 ( 1967).
`
`168
`
`Journal of Parenteral Science & Technology
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