Pharmaceutical Development and Technology, 4(4), 475–480 (1999)
`
`RESEARCH ARTICLE
`
`Developing an Injectable Formula
`Containing an Oxygen-Sensitive Drug:
`A Case Study of Danofloxacin Injectable
`
`Kasra Kasraian, Anna A. Kuzniar, Gabrielle G. Wilson,
`and Julia A. Wood
`PfizerCentralResearch,Groton,Connecticut06340
`
`Received November 11, 1998; Accepted February 5, 1999
`
`ABSTRACT
`
`The purpose of this study was to assess the impact of impurities in formulation components, antioxi-
`dants, formulation pH, and processing/packaging on the extent of color change associated with oxida-
`tion of danofloxacin injectable. The methods used in this study include reversed-phase HPLC, UV–
`VIS spectrophotometry, atomic absorption spectroscopy, visual observation, and iodimetric titration
`for quantification of the antioxidant. The results from this study revealed that trace impurities from
`two different excipients significantly contributed to color change associated with oxidation. Polyvinyl
`pyrrolidone (PVP) introduced trace levels of peroxides into the solution. A second excipient also had
`a significant impact on stability because it introduced trace metal impurities into the product. The
`minimization of oxygen levels alone in the solution and headspace was not sufficient to completely
`eliminate the product instability. The addition of an antioxidant, monothioglycerol (MTG), resulted
`in a formulation less sensitive to processing variables. The impact of pH on the performance of MTG
`was also studied. At pH 7.5, MTG resulted in significant improvement in stability; however, at pH
`6.0 it was not effective as an antioxidant. Process modifications alone may not be sufficient to prevent
`oxidation. Chemical approaches, such as pH control, addition of an antioxidant, and control of compo-
`nents should be considered first as means of enhancing stability of oxygen-sensitive solutions.
`KEY WORDS: Antioxidants; Liquid formulation; Monothioglycerol; Oxidation; Trace impurities.
`
`INTRODUCTION
`
`Oxidative decomposition is among the most challeng-
`ing stability problems faced by formulation chemists.
`Oxidative decomposition in pharmaceutical preparations
`is typically referred to as autoxidation. Autoxidation of
`
`pharmaceuticals is described as a three-step process me-
`diated by free radicals: initiation, propagation, and termi-
`nation of the reaction to form byproducts (1). The initia-
`tion step can be produced by thermal or light-induced
`decomposition to form a free radical. The propagation of
`the free radical oxidation requires oxygen. If molecular
`
`Address correspondence to Kasra Kasraian, Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340. Fax:
`(860) 441-3972. E-mail: Kasra_Kasraian@groton.pfizer.com
`
`Copyright (cid:211)
`
`1999 by Marcel Dekker, Inc.
`
`www.dekker.com
`
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`oxygen, which is required for the propagation step, is re-
`moved or reduced significantly, one may be able to sub-
`stantially eliminate or reduce the oxidative process. Prop-
`agation will theoretically continue until no drug/active
`molecule remains to participate in the chain reaction;
`however, in practice this does not happen because free
`radicals may combine to form inactive compounds. This
`is referred to as the termination step. Termination occurs
`by combining two radicals or by scavenging them using
`a free radical inhibitor. Autoxidation is catalyzed by tem-
`perature, hydrogen ion concentration, trace metals, trace
`peroxides, or light (2–5).
`One approach to minimizing autoxidation of an oxy-
`gen-sensitive formulation is through oxygen control dur-
`ing the manufacture process. Possible steps taken to mini-
`mize oxygen in a liquid formulation include the control
`of manufacturing and packaging operations. The use of
`a nitrogen sparge during formulation compounding can
`reduce dissolved oxygen substantially. Exposure of the
`product to oxygen after packaging can be minimized by
`packaging the product with an inert gas headspace. In
`many cases, minimizing oxygen alone is not sufficient to
`prevent autoxidation, because trace levels of oxygen may
`be enough to initiate this reaction. A chemical approach
`to stabilization is the addition of an antioxidant to the
`formulation. Antioxidants protect oxygen-sensitive for-
`mulations by one of three mechanisms (6). First, antioxi-
`dants may undergo preferential degradation instead of the
`drug molecule because of higher oxidation potential. Sec-
`ond, antioxidants may inhibit the free radical chain reac-
`tion by serving as an acceptor of free radicals. Finally,
`antioxidants may inhibit the formation of free radicals
`(e.g., metal sequestering agents).
`This paper reviews some of the points to consider
`when developing an oxygen sensitive liquid formulation.
`Although many of the variables involved in oxidative
`degradation have been reported by others, this paper re-
`lates the significance of these variables to practical expe-
`riences and specific examples that were encountered dur-
`ing the development of such a product. The examples
`were derived from a recently developed injectable formu-
`lation of danofloxacin (see structure in Fig. 1). During
`the development of this liquid injectable formulation, a
`color change subsequently determined to be associated
`with oxidation was observed during long-term product
`storage. The factors contributing to this color change
`and approaches used to prevent this color change are
`discussed in this paper. During the development of
`danofloxacin injectable, the impact of impurities in for-
`mulation components (excipients/packaging), antioxi-
`dants, formulation pH, and processing/packaging on the
`
`Figure 1. Structure of danofloxacin.
`
`extent of color change was studied. Each of these vari-
`ables is discussed in this review using specific examples,
`with the hope that it will serve as ‘‘best practice’’ guid-
`ance to formulation chemists developing oxygen-sensi-
`tive liquid formulations.
`
`FORMULATION COMPONENT
`IMPURITIES
`
`Often in the development of pharmaceutical dosage
`forms there is the need for the use of functional additives.
`These additives may serve as antioxidants, buffers, bulk-
`ing agents, chelating agents, antimicrobial agents, solubi-
`lizing agents, surfactants, or tonicity-adjusting agents (7).
`Additives usually provide safe, efficacious, and elegant
`dosage forms; however, these excipients should be scruti-
`nized because they may in some cases actually contrib-
`ute to product instability. During the development of
`danofloxacin injectable, two cases were encountered in
`which trace impurities in the excipients contributed to the
`autoxidation of danofloxacin.
`The first case involved the excipient polyvinyl pyrroli-
`done (PVP). During development, it was determined that
`the presence of PVP in the formulation contributed to the
`color changes of the product on storage. Further investi-
`gation revealed that the stabilization of product color
`with the removal of PVP was due to trace peroxides pres-
`ent in this component. Polymers, such as PVP, often
`carry low levels of peroxides. When the peroxide level
`was controlled in the PVP, the product color change on
`stability was significantly minimized. Table 1 illustrates
`how the formulation color stability was impacted when
`two different sources of PVP were used. Formulations
`prepared with PVP containing higher levels of peroxides
`exhibited more color change on stability than those for-
`mulations prepared with PVP containing lower levels of
`peroxides. The significance of trace peroxides on this
`degradation, apparent by product color changes, was fur-
`ther demonstrated by challenge of the product to varied
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`Table 1
`
`The Effect of Trace Peroxides in PVP and Headspace Gas on
`Product Color Stability (2 Week Storage at 70(cid:176) C)
`Final Color (2 Weeks at 70(cid:176) C)
`(Visual Inspection)
`
`Initial Color
`(Visual Inspection)
`
`Formulation Containing
`
`PVP (peroxide levels not controlled):
`Air headspace
`PVP (peroxide levels not controlled): N2
`headspace
`PVP (peroxide levels controlled to
`,400 ppm): Air headspace
`PVP (peroxide levels controlled to
`,400 ppm): N2 headspace
`
`Medium yellow
`
`Medium yellow
`
`Medium yellow
`
`Medium yellow
`
`Dark amber (significant color
`change)
`Amber (significant color
`change)
`Medium yellow (no color
`change)
`Medium yellow (no color
`change)
`
`headspace (air versus nitrogen purged). The trace perox-
`ides introduced into the formulation through this excipi-
`ent resulted in more color change than did the presence
`of an air headspace in the vial. This experience empha-
`sizes the relatively significant effect of trace peroxides
`on the autoxidation pathway.
`The second case involved an additive used as a solubi-
`lizing agent. The danofloxacin formulation exhibited var-
`ied color stability depending on the supplier of the solubi-
`lizing agent used in the formulation. Three different
`suppliers of the solubilizing agent were included in the
`study. As shown in Table 2, the product color changed
`dramatically when the solubilizing agent from vendor A
`was used in the formulation during a 2-week stress study,
`whereas product color was not effected when the excipi-
`ents from vendors B and C were used. It was determined
`that this variability in color stability was due to trace iron
`in the excipient. The solubilizing agent obtained from
`vendor A contained 140 ppm of iron, whereas the product
`from vendors B and C had iron levels of 13 ppm and 0
`ppm, respectively.
`As with trace peroxide, minimization of trace iron in
`the ingoing excipients resulted in an improvement in
`
`product stability. Autoxidation, which is a chain reaction
`that begins with the formation of a free radical, is cata-
`lyzed by trace metals and peroxides (6). Most often for-
`mulators use chelating agents to prevent catalysis of
`autoxidation by trace metals; however, in this case the
`use of a chelating agent was prohibited by the nature of
`the formulation. Therefore, control of these trace impuri-
`ties in materials used in formulations that are prone to
`oxidation is suggested. Furthermore, packaging compo-
`nents (container/closure systems) should also be consid-
`ered as potential sources of trace metals.
`
`ANTIOXIDANTS
`
`Minimizing oxygen concentration alone is often not
`sufficient to eliminate completely the possibility of deg-
`radation because only trace levels of oxygen can propa-
`gate autoxidation. For the danofloxacin formulation,
`control of processing operations (e.g., use of nitrogen
`sparging to reduce dissolved oxygen, control of filled vial
`headspace composition, and thermal effect of terminal
`sterilization) alone did not completely eliminate the prod-
`
`Table 2
`The Impact of Trace Iron on Color Stability (2 Week Stress Study at 30(cid:176) C/Partially Filled Vials)
`
`Vendor (Iron Content)
`
`Vendor A (140 ppm Fe)
`Vendor B (13 ppm Fe)
`Vendor C (0 ppm)
`
`Initial Color
`(Absorbance at 450 nm)
`
`Color After 2 Weeks
`(Absorbance at 450 nm)
`
`0.13 au
`0.13 au
`0.12 au
`
`0.39 au
`0.13 au
`0.12 au
`
`Comment
`
`Significant color change
`No color change
`No color change
`
`Excipient from vendor A contained 140 ppm iron impurity, excipient from vendor B contained 13 ppm iron, and excipient from
`vendor C contained 0 ppm (none detected). Note: solution color monitored at 450 nm.
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`Table 3
`
`Effect of Formulation pH on Product Color
`Stability (2 Week Storage at 70(cid:176) C)
`
`Formulation pH
`
`Color Change (Visual)
`
`6.6
`7.2
`7.7
`8.0
`
`No color change
`Slight color change
`Significant color change
`Very significant color change
`
`challenge. Because the breakdown product of the antioxi-
`dant may have an adverse effect on the stability of the
`active ingredient, quantitation of the antioxidant and/or
`its breakdown product(s) during stability storage is essen-
`tial to the full understanding of the product (3).
`
`FORMULATION pH
`
`Oxidation of most compounds is minimized at acidic
`pH (6,9). For this reason, oxygen-sensitive compounds
`are typically formulated at a lower pH to increase their
`resistance to oxidation during shelf storage. Development
`of the danofloxacin injectable formulation followed this
`pattern. The data in Table 3 demonstrate that as the pH
`of the formulation is lowered, less color change over sta-
`bility is exhibited.
`Although pH can directly influence the extent of drug
`oxidation, it may also impact the performance of the anti-
`oxidant. Antioxidants are effective in stabilizing oxygen-
`sensitive drugs when they are preferentially oxidized in
`place of the drug (6). For the danofloxacin formulation,
`MTG was ultimately selected as the antioxidant. The im-
`pact of formulation pH on the performance of MTG as
`an antioxidant for danofloxacin was assessed during pre-
`formulation. The preformulation studies were done at a
`concentration of 0.1 mg/ml of danofloxacin, a much
`lower concentration than the commercial formulation,
`which is 180 mg/ml.1 Figures 3 and 4 represent the per-
`cent loss of danofloxacin at pH 7.5 and 6.0, respectively.
`At pH 7.5, MTG has a significant effect on the stability
`of danofloxacin. At
`this pH, formulations containing
`MTG had improved stability relative to formulations not
`
`1 The percent loss of danofloxacin illustrated in Figs. 3 and 4 is
`much greater than that observed in the commercial formulation,
`because at 0.1 mg/ml of danofloxacin, the ratio of drug to oxy-
`gen molecules in the headspace is less favorable relative to a
`180-mg/ml formulation packaged in the same vial.
`
`Figure 2. The effect of headspace gas, storage temperature,
`and terminal sterilization on color stability of formulations (a)
`without MTG; (b) with MTG as an antioxidant through 6 weeks
`of storage.
`
`uct oxidation/color change on stability, as exhibited in
`Fig. 2(a). When the product was manufactured without
`an antioxidant, the formulation color varied as a function
`of headspace composition,
`terminal sterilization, and
`storage temperature. With the appropriate antioxidant
`and selection of the antioxidant level, autoxidation of the
`drug can be inhibited until all of the antioxidant is prefer-
`entially consumed. The addition of an antioxidant, in this
`case monothioglycerol (MTG), resulted in a formulation
`less sensitive to processing variables, as shown in Fig.
`2(b). MTG was selected following a comprehensive
`screening of potential antioxidants, whereby it provided
`the desired stability for this multidose product both dur-
`ing shelf-life and the proposed in-use period.
`Although it is difficult to accurately predict the effi-
`ciency of an antioxidant, initial selection can be based
`on the difference in redox potential between the drug and
`antioxidant (8). The best way to assess the effectiveness
`of an antioxidant is to subject the formulation containing
`the antioxidant to standard oxidative stress conditions
`(i.e., addition of peroxides and purging with oxygen).
`The formulation stability (active ingredient and the anti-
`oxidant content) may be assessed over some stability
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`Figure 3. The effect of MTG on danofloxacin stability at pH
`7.5 (drug concentration of 0.1 mg/ml; storage temperature of
`70(cid:176) C).
`
`containing MTG (Fig. 3). However, at pH 6.0 there was
`no apparent effect of MTG on the stability of danofloxa-
`cin. Therefore, MTG is more effective as an antioxidant
`at pH 7.5 than at pH 6.0. This is believed to be because
`the oxidation potential for the MTG changes as the pH
`is lowered, making MTG less likely to be oxidized at
`lower pH. A cyclic voltammogram of MTG as a function
`of pH is provided in Fig. 5. As illustrated in this volta-
`mmogram, MTG is oxidized at a much lower potential
`at pH 7.5 than at pH 6.0. MTG consumes any residual
`dissolved oxygen and headspace oxygen much faster at
`pH 7.5 and hence prevents the oxidation of danofloxacin.
`In summary, pH may have a direct effect on the oxidation
`rate of the drug molecule and may impact the oxidation
`potential of an added antioxidant.
`
`Figure 4. The effect of MTG on danofloxacin stability at pH
`6.0 (drug concentration of 0.1 mg/ml; storage temperature of
`70(cid:176) C).
`
`Figure 5. Cyclic voltammogram of MTG as a function of pH.
`
`PACKAGING
`
`The selection of the appropriate container and closure
`is critical in the development of any dosage form. Aside
`from the general compatibility concerns and container/
`closure integrity issues, the package headspace-to-prod-
`uct ratio plays a significant role in the stability of an oxy-
`gen-sensitive (liquid) product. For an oxygen-sensitive
`formulation, several other factors must be taken into
`consideration. These may include (a) light-protected
`container or package to prevent light-induced catalysis
`of autoxidation; (b) acceptable resealability of closures
`to prevent
`leakage and contamination, especially for
`multiuse vials; and (c) maintenance of inert atmosphere
`(e.g., nitrogen purge of headspace).
`All of the above factors played a role in the develop-
`ment of danofloxacin. Most notably, however, the vial
`size/configuration had a significant impact on the rate
`of product oxidation. Generally, larger vials provide a
`lower headspace-to-volume ratio than smaller vials, thus
`the extent of oxidation is typically less in larger vials
`(10). Early stability studies on danofloxacin packaged in
`smaller vials revealed a greater degree of color change
`than when the same solution was placed in larger vials.
`An example of the effect of vial size on oxidation is illus-
`trated in Fig. 6. Product packaged with an air headspace
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`ACKNOWLEDGMENTS
`
`The authors would like to acknowledge Drs. William
`Lambert, Robert Lloyd, Imran Ahmed, Michael Cohen,
`Wayne Boettner, and Joseph Taylor for their technical
`suggestions and support during the development of this
`product. The authors would also like to acknowledge Ms.
`Beth Schapira, Mr. Curtis McCracken, Mr. James Arther-
`holt, and Mr. Mike Graziosi for analytical and manufac-
`turing support throughout this project, and Dr. Joseph
`Stemple for determining the oxidation potentials of the
`selected antioxidant.
`
`Figure 6. The effect of vial size (headspace-to-volume ratio)
`on the extent of oxidation.
`
`REFERENCES
`
`(approximately 20% oxygen) demonstrated a 5% loss of
`the MTG in the 250-ml vial, whereas as much as 15%
`of the MTG was consumed when the formulation was
`packaged in 50- and 100-ml vials.
`
`SUMMARY
`
`Once oxidative decomposition reactions have been
`identified as a potential product stability problem, several
`approaches can be utilized by the formulator to enhance
`chemical stability. Process modification, such as purging
`the solution and headspace with an inert gas such as nitro-
`gen, is a straightforward approach. However, it is not al-
`ways possible or even practical to remove all oxygen
`from the solution, and remaining trace amounts of oxy-
`gen can destabilize the product. Chemical approaches,
`such as lowering formulation pH and the addition of an
`antioxidant, are usually the first choice to enhance prod-
`uct stability. Trace impurities, namely peroxides and trace
`metals, carried into a formulation through ingoing com-
`ponents, may also have a catalyzing effect on the autoxi-
`dation pathway. Aside from processing or chemical ap-
`proaches to control oxidation, the importance of product
`packaging must also be considered. Well-protected, prop-
`erly sealed packages that provide an acceptable headspace-
`to-product ratio can also provide some robustness to the
`product, thus making it less sensitive to oxidation.
`
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