Pharmaceutical Research, Vol. 8. No. 10, 1991
`
`Report
`
`Parenteral Peptide Formulations:
`Chemical and Physical Properties of
`Native Luteinizing Hormone-Releasing
`Hormone (LHRH) and Hydrophobic
`Analogues in Aqueous Solution
`
`Michael F. Powell,"2 Lynda M. Sanders,1
`Alan Rogerson,1 and Vicki Si1
`
`Received January 16, 1991; accepted May 6, I991
`
`The degradation of native LHRH in aqueous buffers of pH ~1—10
`obeyed the rate equation, kobs = kH+aH+ + k0 + kHOr(aHOr)",
`where x at 60—100°C was ~0.64 and temperature independent. Ex-
`trapolation to 25°C using the Arrhenius equation and secondary rate
`constants showed that native LHRH is reasonably stable at pH 5.4,
`giving a shelf life (190) of approximately 5 years. Regarding physical
`properties, hydrophobic LHRH analogues nafarelin and detirelix
`were found to be surface active as demonstrated by a decrease in
`apparent surface tension with increased peptide concentration. The
`CMC for detirelix at pH 7.4 was determined to be 5.3 X 10“ M
`(0.88 mg/ml), and that for nafarelin, >2 mg/ml. At higher concen-
`trations (~4—8 mg/ml), nafarelin and detirelix formed nematic liquid
`crystals of undulose extinction (birefringence, <0.001). The thermo—
`dynamic stability of these peptide liquid crystals was probed by
`determining their melting points (Tcm) in the presence of propylene
`glycol, a solvent which proved to be efficacious at suppressing ge-
`lation and at destabilizing liquid crystals as measured by a reduction
`in Tcm.
`KEY WORDS: peptide stability; liquid crystal formation; birefrin-
`gence; surface tension; luteinizing hormone-releasing hormone
`(LHRH); hydrolysis; aggregation; micelles; surface activity.
`
`properties of nafarelin and detirelix that are pertinent to their
`parenteral formulation, as well as some stability data on na-
`tive LHRH for comparison. We have determined the chem-
`ical stability of native LHRH with respect to temperature
`and pH, as well as probed the physical stability of hydro-
`phobic LHRH analogues, by measuring the surface activity
`of these amphiphilic peptides and by directly measuring the
`stability of peptide aggregates at higher temperatures. The
`second paper in this series will report on the effect of pH,
`added counterions and excipients on peptide aggregation and
`physical stability (10).
`
`EXPERIMENTAL
`
`Materials
`
`Nafarelin and detirelix (both as the diacetate salt) were
`synthesized and assayed by the Institute of Organic Chem-
`istry (Syntex Research). Native LHRH (as the acetate salt)
`was purchased from Sigma and used without further purifi-
`cation. Buffer solutions and added cosolvents were prepared
`from reagent grade or USP-grade chemicals. HPLC-grade
`acetonitrile and water purified by filtration and ion exchange
`(Nanopure) were used in the mobile phase.
`
`Apparatus
`
`Reversed-phase chromatography of native LHRH was
`carried out using an HPLC system consisting of a Waters
`Model 712 Wisp autoinjector, a Waters Model 45 pump, a
`Waters Model 730 data module, and a Waters Model 450
`spectrophotometric detector. A 250 X 4.6-mm Altex Ultra-
`sphere ODS column was used for analysis. pH’s were deter-
`mined using a Radiometer PHM 64 pH meter and a Radiom-
`eter GK2401C combination electrode calibrated at the solu-
`tion temperature.
`
`INTRODUCTION
`
`HPLC Conditions
`
`The low oral bioavailability of most peptides and pro-
`teins has provided a compelling driving force for parenteral
`delivery. Unfortunately, the parenteral formulation of pep-
`tides and proteins is far from trivial. For example, certain
`peptides, such as the hydrophobic LHRH analogues nafare-
`lin and detirelix (Fig. 1), adhere tenaciously to glass and
`other surfaces at low concentrations (1). At higher concen-
`trations, aqueous formulations of nafarelin or detirelix un-
`dergo peptide aggregation and solution gelation resulting in
`compromised physical stability (2). Furthermore, although
`the chemical stability of selected hydrophobic LHRH ana-
`logues is well understood (3—9), there exist only limited
`chemical stability data for the parent compound, native
`LHRH.
`
`A linear response (:1%) was obtained throughout the
`range of 0005—5 ug LHRH injected. The separation of
`LHRH from its degradation products was achieved using the
`following conditions: mobile phase, water: acetonitrile
`(82: 18, v/v) buffered with 0.2 M phosphate (pH 3); flow rate,
`1.2 mllmin; detection, 210 nm; injection volume, 50 iii; and
`typical LHRH retention time, 15 min. Stability specificity of
`this method was demonstrated by the following: (i) apparent
`pseudo-firstorder decay of LHRH to <5% drug remaining;
`(ii) the spectral similarly obtained for early, middle, and late
`peak splices; and (iii) the lack of degradation product peaks
`at or near the LHRH peak when the mobile phase was var-
`ied.
`
`Herein we report some of the chemical and physical
`
`Reaction Kinetics
`
`1 Institute of Pharmaceutical Sciences, Syntex Research, Palo Alto,
`California.
`2 To whom correspondence should be addressed at Genentech, Inc.,
`460 Pt. San Bruno Boulevard, South San Francisco, California
`94080
`
`The buffer concentration used in these experiments (ex-
`cept for HCl solutions) was held at 0.01 M to minimize buffer
`catalysis; the ionic strength was constant at pt = 0.15 (KCl).
`LHRH acetate was added to make the final drug concentra-
`tion 25 rig/ml (~2 X 10*5 M) and the pH’s of each solution
`were determined at 25, 60, and 80°C. The pH’s at 100°C were
`
`M24—8741/9l/1000«1258$06.50/0 © 1991 Plenum Publishing Corporation
`
`1258
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`

`Parental Peptide Formulations
`
`o H
`o H
`o H
`o H
`o H
`H
`II
`I
`[I
`I
`II
`I
`II
`I
`II
`I
`N
`0 CNCHCNC‘HCNCHCNCHCN
`CH2
`CH:
`CHon CH2
`
`i
`
`_
`
`I
`ll
`|[
`II
`I
`H
`II
`H 0
`H o
`o
`H o
`0
`HgN-C-CHz-N—C- N c-CH N c- eH- N- c- CH
`O (CHzls
`(EH2
`H
`CH
`“1”
`/ \
`c
`CHgCHg
`// \
`NH2
`
`HN
`
`OH
`III
`
`OH
`III
`
`OH
`III
`
`OH
`III
`
`OH
`III
`
`OH
`III
`
`CHg—CNCHCNCHCNCHCNCHCNCHCN
`CH2
`€le
`CH2
`CH2 OH cle
`
`6““
`
`0
`
`HO
`
`HO
`HO
`O
`I“
`III
`II
`N-C'- ?H-N-Cl--(iH- N-C-CH
`HEN-C-CfH-N-C-
`CH: O (CH2)3
`(EH2
`(CH2)4
`NH
`CH
`NH
`
`ll
`
`I
`|
`02H; C2“:
`
`1259
`
`ized light. Typically solutions were prepared at least 24 hr
`prior to examination. Liquid crystals, when observed
`through crossed polars, were detected as light-colored struc-
`tures of nondistinct crystal form against a black background
`(nematic liquid crystals of undulose extinction). Both nafare-
`[in and detirelix showed an observed optical retardation of
`~50—200 nm/0.3 mm, corresponding to a birefringence of
`~0.001 or less as calculated using the Michel—Levy chart
`(11,12). The extent of birefringence was verified by viewing
`through a “first-order red” plate compensator; by this
`method the liquid crystals were characterized by the second-
`order blue (addition) and first-order yellow-orange (subtrac-
`tion) colors. In most cases, photomicrographs were taken
`such that both the air (the concave side of the meniscus) and
`the solution regions were observed so as to provide an op-
`tical control for birefringence caused by imperfections in the
`microslide. The effect of temperature was determined by
`heating the microslides at 3°C/min until all traces of liquid
`crystallinity were lost, i.e. , until the background turned com-
`pletely black. This melting temperature is defined herein as
`the critical melting temperature (Tam).
`Freeze-fracture electron micrographs were produced by
`flash-freezing an aqueous solution of detirelix to —80°C in
`liquid freon and then cooling to —196°C in liquid nitrogen.
`Samples at — 196°C were then fractured by means of a
`cooled blade and etched and shadowed with platinum and
`carbon. Replicates were obtained by dissolving the sample in
`methanol and were then viewed by transmission electron
`microscopy.
`
`Fig. 1. Structure of (i) native LHRH (R = H), nafarelin (R =
`2-naphthylmethyl), and (ii) detirelix.
`
`RESULTS AND DISCUSSION
`
`Temperature and pH Dependence on Native LHRH
`Degradation. This was determined at 60, 80, and 100°C by
`reversed-phase HPLC analysis. All reactions obeyed
`pseudo—first—order kinetics over the time courses studied
`(Fig. 2). The observed pseudo-first-order rate constants
`shown in Table I and Fig. 3 indicate that the degradation of
`LHRH is both acid and base catalyzed. The slope of —1 in
`the acid region is indicative of specific acid catalysis by hy-
`dronium ion. In the base region, the observed rate is not
`stoichiometrically proportional to hydroxide ion concentra-
`tion but displays a slope of ~0.64. The pH rate profiles
`obeyed the semiempirical rate equation given by Eq. (1),
`where kH+, kc, and kHO‘ are the secondary rate constants for
`catalysis by hydronium ion, water (or a spontaneous
`
`
`extrapolated by linear least-squares analysis of 1/T(K’1)
`versus pH. The reaction solutions were then transferred to
`several 1-ml clear glass vials (treated, type A), flame—sealed,
`and incubated at 60, 80, or 100°C. At known time intervals
`samples were removed and immediately frozen at —20°C.
`Typically 8—10 samples were taken per kinetic run. Upon
`removal of the last sample the stored solutions were allowed
`to warm to room temperature, and then all samples were
`analyzed on the same day. Peak area integration values were
`used directly when fitting the data to first order kinetics;
`typically reactions were followed for two to five half—lives.
`
`Physical Study Methods
`
`The apparent surface tension (y) of LHRH and its ana-
`logues was measured using 21 Fisher Autotensiomat (Model
`215) and a 6-cm platinum ring. Control experiments demon-
`strated that at least 3 ml of solution was required for precise
`7 measurements when using a 6—cm ring. Sample solutions
`containing 0.01 M total buffer were allowed to equilibrate for
`several minutes before taking the final reading in triplicate.
`In experiments where 'y changed with time, 7 was taken at
`known time intervals using the point of ring release as the
`recorded time.
`Peptide liquid crystal melting temperatures (Tcm) were
`determined by loading peptide solutions into flat, glass cap-
`illary tubes (50 X 3 X 0.3 mm, Vitro Dynamics Inc., NY) and
`examining them microscopically (33X) under crossed polar-
`
`
`
`PercentRemaining
`
` 05O 4
`
`.3:O
`
` , 1 I
`
`
`10
`20
`30
`40
`50
`Time (days)
`Fig. 2. Degradation of native LHRH in pH 6.3 buffer at 60, 80, and
`100°C. The first order fit of the data was made by nonlinear least-
`squares analysis.
`
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`

`1260
`
`Table 1. Observed Rate Constants for the Degradation of LHRH in
`Aqueous Solution
`60°C
`80°C
`100°C
`
`105 kobs
`106 kobs
`107 kobs
`(sec‘ 1)
`pH”
`(sec’ 1)
`pH"
`(sec’ 1)
`pH"
`Buffer“
`38
`1.12
`110
`1.12
`270
`1.11
`HCl
`0.16
`3.806
`0.30
`3.80C
`0.58
`3.80C
`HCl
`0.12
`4.53
`0.23
`4.52
`0.39
`4.45
`Acetate
`0.20
`5.53
`0.30
`5.51
`0.42
`5.43
`Acetate
`0.38
`6.30
`0.59
`6.30
`0.71
`6.27
`Phosphate
`2.8
`7.60
`3.2
`7.65
`2.8
`7.64
`Phosphate
`
`
`
`
`
`
`63 9.48 55 9.369.64Carbonate 24
`
`“ Total buffer concentration (except HCI solution), 0.01 M; ionic
`strength, u : 0.15.
`” pH’s were determined at the reaction temperature indicated ex-
`cept for 100°C, where pH’s were obtained from linear least-
`squares analysis of UT (K‘l) vs pH.
`‘ The pH before addition of LHRH acetate salt was ~3.02.
`
`kobs = kHtant + k0 + kHo‘(aHo’)x
`
`(1)
`
`reaction), and hydroxide ion, respectively. The terms aH+
`and (aHO—Y‘ are the hydronium ion and hydroxide ion activ—
`ities, where the exponent x represents the observed depen-
`dence of the reaction rate upon hydroxide ion activity. As
`suggested earlier for nafarelin (7) a slope of less than one in
`the basic region indicates that LHRH degrades by several
`different basic pathways, each with its own pH dependence
`and true kHO— catalytic coefficient. The rate data at 60—100°C
`were fitted by nonlinear least-squares analysis (13) to Eq. (1)
`using a four-parameter fit: kH+, k0, kHOA, and x. The best-fit
`value of x did not vary significantly over the temperature
`range studied (x = 0.65 i 0.05, 0.65 i 0.03, and 0.61 i 0.03
`at 60, 80, and 100°C, respectively), showing that a single
`value of x (x ~ 0.64) can be used without oversimplification
`to obtain the rate constants kH+, kc, and km), by Eq. (2).
`
`kobs = kHtaHt + k0 + kHo‘(aHo’)0‘64
`
`(2)
`
`The secondary rate constants obtained by this method
`are shown in Table II, as are the corresponding activation
`parameters. The rate constants at 25°C were estimated by
`
`logk(secr‘)
`
`
`
`Fig. 3. pH—rate profiles for the degradation of native LHRH at 60—
`100°C. The lower dashed line is the pH—rate profile for 25°C calcu-
`lated using Eq. (2) and the activation parameters obtained from the
`data at 60—100°C.
`
`Powell, Sanders, Rogerson, and Si
`
`linear least-squares extrapolation of the Arrhenius data; the
`lower line in Fig. 3 is the best fit of the calculated 25°C rate
`constants to Eq. (2).
`Two features are readily apparent: (i) the pH of maxi-
`mum stability for native LHRH (and probably structurally
`similar LHRH analogues as well) at 25°C is near pH 5.4, and
`(ii) native LHRH exhibits a shelf life of 2 years or more from
`pH ~4.2 to ~72. Comparison of these rate data with those
`reported earlier for nafarelin (7) and RS-26306 (8) shows that
`there is little difference in chemical stability between native
`LHRH and some of its hydrophobic analogues. This broad
`plateau region suggests that the spontaneous (or water-
`catalyzed) reaction for LHRH degradation largely deter-
`mines drug stability from pH 4 to pH 7. Additionally, reac-
`tion solutions of pH ~25 to ~95 show less than 10% deg-
`radation after 1 month, well within the time required to carry
`out the peptide liquid crystal studies herein.
`Surfactant Nature ofNafarelin and Detirelix. This was
`probed by determining their apparent surface tension ('y) of
`aqueous peptide solutions at 25°C. A cursory glance at the
`structures in Fig. 1 shows that detirelix and nafarelin should
`be more surface active than native LHRH. At neutral pH
`detirelix has two protonated aminoacids (arginine and di-
`ethyl homoarginine) which are close to the N terminus, mak-
`ing this end hydrophilic. Near the C terminus, detirelix has
`several large organic aminoacids (naphthylalanine, p-chlo-
`rophenylalanine, tryptophan, and tyrosine), making this end
`lipophilic (14). The aggregation of amphiphiles is known to
`be driven by “opposing forces” of attraction of the lipophilic
`regions and geometrical packing constraints, the balance of
`which is responsible for the characteristics and maintenance
`of subsequent structures (15). Detirelix and nafarelin have
`such lipophilic and hydrophobic regions, making them both
`surface active and highly susceptible to aggregation in aque-
`ous solution.
`
`The apparent surface tension of detirelix in pH 7.4 phos-
`phate buffer showed a classical (15) 'y versus log[peptide]
`profile (Fig. 4), giving from the sharp change in slope a crit-
`ical micelle concentration (CMC) of 5.3 X 10"4 M (0.88
`mg/ml). The limiting slope below the CMC, 6y/6log[peptide],
`is ~6.6 mN m‘ ‘, providing via the Gibbs equation [Eq. (3)]
`a surface excess concentration (Y) of 1.16 X 10‘6 mol m2.
`
`By
`1
`A = 2.303 RT Slog[peptide]
`
`(3)
`
`Using Eq. (4), where NA is Avagadros number, the calcu-
`lated surface area/molecule (A) is 1.43 nmz.
`
`A = 1/(NAY)
`
`(4)
`
`Inspection of Fig. 4 shows that 'y for nafarelin also de-
`creases with increasing nafarelin concentration at pH 7.4.
`The slope ofthis plot is 9.8 mN m”, giving Y = 1.7 x 10’6
`mol m2 and a surface area/molecule of 0.97 nmz. The surface
`area per molecule for nafarelin is slightly smaller than for
`detirelix, presumably because of the different head group
`size. It is known that the size of the hydrophilic head group,
`rather than the size or the length of the lipophilic chain,
`affects the value of Y (16—19). Nafarelin has a hydrophilic
`region consisting of the sequence Gly—Pro—Arg, whereas de-
`tarelix has a slightly greater hydrophilic region, consisting of
`
`
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`

`Parental Peptide Formulations
`
`1261
`
`Table 11. Secondary Rate Constants and Activation Parameters for the Degradation of LHRH in Aqueous Solution“
`
`Activation parameters
`Rate
`(AHi, kcal mol‘ 1)
`constant
`25°C
`60°C
`80°C
`100°C
`(ASi, cal K’l mol")
`
`
`Temperature
`
`kH— (M7l sec”)
`
`1.4 X 1075b
`
`3.0 X 1074
`
`1.3 X 10'3
`
`5.0 X 10’3
`
`16.6
`AHle =
`ASi = —25.0
`
`k0 (sec‘l)
`
`5.3 X 10—10b
`
`2.5 X 10—8
`
`1.4 X 10—7
`
`8.3 X 10’7
`
`20.8
`AHi =
`ASi = -30.9
`AH? =
`19.3
`2.2 X 10—2
`5.0 X 10’3
`8.5 X 10’4
`2.5 X 10’”
`kHo‘ (M‘O‘64 sec'l)C
`A51 2 —14.8
`
`
`“ Calculated using Eq. (2) (see text). The values of Kw used in calculating 0140’ from the pH’s at 25, 60, 80, and 100°C were 14.0, 13.02,
`12.60,and 12.29 M’ ‘, respectively.
`1’ Calculated from the activation parameters shown in the last column.
`‘ The secondary rate constants for hydroxide ion catalysis demonstrated a 0.64 exponent dependence on hydroxide ion concentration, and
`so the value of kHOr has the units of M ’0'“ sec‘ 1. Fortunately, this does not detract from its usefulness in calculating the pH—rate profile
`at 25°C.
`
`the CMC
`Gly—Pro—Arg—Leu—(homodiethyl Arg). Further,
`for nafarelin is greater than 2 mg/ml, somewhat higher than
`for detirelix, presumably because nafarelin has a weaker
`lipophilic attraction than detirelix, or because the larger head
`group size for detirelix prevents close packing of the peptide
`monomers to give micelles and higher ordered structures.
`Liquid Crystal Formation. The liquid crystal formation
`of nafarelin and detirelix was studied by freeze—fracture elec-
`tron microscopy and by examination under crossed polar-
`ized light using optical microscopy. Upon freeze-fracture
`analysis, concentrated solutions of detirelix (for example, 15
`mg/ml as shown in Fig. 5) formed lyotropic mesophases,
`indicating a marked ordering of drug in solution. Similar
`freeze-fracture replicates were also observed for nafarelin
`solutions but were absent in control solutions containing
`only buffer (data not shown). Structural ordering of this type
`has been observed previously, for example,
`in freeze-
`fracture replicates of emulsions and creams (20). Peptide
`aggregation in other peptides and proteins has also been re-
`ported, for example, for pentagastrin (21) and insulin (22).
`Birefringence due to liquid crystal formation in aqueous
`nafarelin and detirelix solutions was determined by sample
`observation under crossed polars. Although measurement of
`birefringence is a sensitive method for the detection of liquid
`crystals, the extent of birefringence is difficult to quantify,
`especially because of its time-dependent nature and un-
`known relation to the extent or thermodynamic stability of
`
`
`
`
`
`ApparentSurfaceTension
`
`(mNm”) 0')a:\lO01O
`
`— 3
`- 4
`— 5
`— 6
`Log Concentration (M)
`
`
`
`the peptide liquid crystal structures. Herein we have chosen
`the critical melting temperature (Tom) as an indicator for
`characterizing peptide liquid crystals. In general, Tcm was
`independent of liquid crystal maturation (Fig. 6) and in-
`creased only slightly with increasing peptide concentration
`(Figs. 7 and 8). In the absence of added electrolyte, detirelix
`and nafarelin did not form liquid crystals at concentrations
`below 4 and 8 mg/ml, respectively. That detirelix formed
`liquid crystals at a lower concentration than nafarelin is in
`keeping with Tanford’s suggestion (15) that hydrophobic
`self-association is the driving force behind aggregate forma-
`tion—by this criterion detirelix is more hydrophobic than
`nafarelin (Fig. 1). This is also supported by our observation
`that the hydrophilic parent peptide, native LHRH, did not
`exhibit birefringence or anisotropicity, even at 30 mg/ml
`LHRH in 1 M NaCl solution. Under these extreme condi-
`tions both nafarelin and detirelix gel immediately, indicative
`of their hydrophobic nature as compared with native LHRH.
`Thus, both indirect (surface activity and determination
`
`
`
`<—— 300 nm
`
`
`
`>
`
`Fig. 4. Effect of detirelix (O) and nafarelin (0) concentration on the
`apparent surface tension (7) at 25°C. The sharp break at 5.3 X 10'4
`M is the critical micelle concentration for detirelix in 0.01 M phos-
`phate buffer (pH 7.4).
`
`Fig. 5. A freeze—fracture replicate of detirelix in aqueous solution
`(15 mg/ml) as viewed by transmission electron microscopy. Control
`electron photomicrographs (not shown) without added peptide did
`not exhibit the ordered array in this figure.
`
`
`
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`

`

`Powell, Sanders, Rogerson, and Si
`
`70
`
`,
`
`,
`
`77777g
`
`60 ,
`
`W
`
`i
`
`.
`
`, l
`5
`
`W/
`K
`A
`\Ci
`0
`2 MM
`i—“
`o—o/Ofl/T
`.
`40 ,
`1.5% O 20% El
`5% A 30% o .
`.
`l
`l
`,
`l
`l
`15% v
`20
`25
`15
`10
`Concentration (mg/mL)
`Fig. 8. Effect of PG (‘70, w/v) as added cosolvent on the critical
`melting temperature (Tm) of detirelix liquid crystals.
`
`
`
`‘
`30
`
`and (iii) detirelix forms liquid crystals at a lower concentra-
`tion (~4 mg/ml) than nafarelin (~8 mg/ml). The higher Tcm’s
`for nafarelin demonstrates that there exists a balance be-
`tween kinetic and thermodynamic forces affecting the for-
`mation and stability of peptide liquid crystals. These results
`indicate that, even though nafarelin forms liquid crystals
`slowly, once formed they are more stable. Such an observa—
`tion is a breakdown of the Hammond postulate (a positive
`correlation between AG" and AG+), a tenet of chemistry
`that, unfortunately, has many exceptions.
`
`CONCLUSIONS
`
`Comparison of the pH—rate data herein for native
`LHRH with data reported earlier for nafarelin (7) and RS-
`26306 (8) demonstrates that hydrophobic modification of
`LHRH has little effect on chemical stability. In contrast, the
`hydrophobic nature of certain LHRH decapeptides such as
`nafarelin and detirelix gives rise to unusual solution dynam-
`ics, including peptide aggregation and liquid crystal forma-
`tion. These liquid crystals eventually result in solution gela-
`tion, with the ramification of compromised formulation ele-
`gance and utility. We have demonstrated herein that a
`pharmaceutically acceptable cosolvent, propylene glycol,
`raises the minimum concentration for peptide aggregation,
`and thus, many of the potential problems associated with
`aggregate formation may be minimized or eradicated by co-
`solvent addition.
`
`ACKNOWLEDGMENTS
`
`We are indebted to Nancy Benson, Department of Elec-
`tron Microscopy, University of California, Berkeley, for the
`production of freeze-fracture replicates and to Dave
`Johnson, Jeff Fleitman, Peter Mishky, Katrina Herb, and
`John Nestor for their help and comments.
`
`REFERENCES
`
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`
`65
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`”W
`\1or
`
`\IO
`
`' l/L/ ‘
`Detirelix
`.
`A
`‘
`—1___l—l—l—;J
`3
`6
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`12
`15
`Time (days)
`
`Fig. 6. Effect of reaction time on the critical melting temperature
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`
`of CMC) and direct measurements (optical observation)
`demonstrate that hydrophobic LHRH analogues aggregate
`readily in aqueous solution. Because the aggregation of de-
`tirelix and nafarelin has important pharmaceutical conse—
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`crystal formation may be suppressed by added cosolvent.
`Peptide Liquid Crystals. These were disrupted by add-
`ing organic cosolvent where, in general, increasing amounts
`of propylene glycol (PG) and higher temperatures sup-
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`of Fig. 7 shows that added PG reduced Tom, particularly
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`
`Detirelix exhibited lower Tom’s than nafarelin (Fig. 8),
`indicating that detirelix liquid crystals are less stable than
`nafarelin liquid crystals at elevated temperatures (40—80°C).
`This is somewhat surprising inasmuch as (i) detirelix exhibits
`a lower CMC (0.88 mg/ml) than does nafarelin, (ii) detirelix
`at 10 mg/ml or greater readily forms liquid crystals in less
`than 1 hr, whereas nafarelin often takes more than 1 week,
`
`80 W70
`
`so
`
`5
`
`1.5% 0
`5% A
`15% D
`L.M-_‘p_‘_l—
`15
`20
`25
`10
`Concentration (mg/mL)
`Fig. 7. Effect of PG (%, w/v) as added cosolvent on the critical
`melting temperature (Tom) of nafarelin liquid crystals.
`
`53
`
`E'
`
`—
`
`
`
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