A Liquid Chromatographic Study of Stability of the Minor Determinants of Penicillin Allergy: A Stable Minor Determinant Mixture Skin Test Preparation

A Liquid Chromatographic Study of Stability of the Minor Determinants of Penicillin Allergy: A Stable Minor Determinant Mixture Skin Test Preparation CHARLOTTE RESSLER*’,PATRICIAM. NEAG*,A N D LOUISM. MENDELSON* Received April 18, 1983, from the Departments of *Pharmacology and “Pediatrics,University of Connecticut Health Center, Farmington, CT 06032. Accepted for publication November 9, 1984. Abstract 17 Various skin test reagents supplying minor determinants for detecting penicillin hypersensitivity have been examined by high-performance liquid chromatography (HPLC) for composition and stability. HPLC systems capable of separating and determining the four diastereoisomers of benZyl-D-peniCillOiC acid and the two benzyl-o-penilloic acids were developed for this purpose. The “simple skin test reagent,”consisting of an aged partial alkaline hydrolysate of penicillin, is possibly an adequate source of (5R,6R)-benzyl-~-penicilloatewhereas the “simple skin test reagent,”consisting of aged aqueous solution of penicillin, is a questionable source of this compound. A modified Levine, Voss, Redmond, and Zolov minor determinant mixture (MDM) reagent and the components (5R,6R)-benzyl-D-penicilloate and (5R)-benZyl-D-penillOate have been found to be highly labile in aqueous solution, giving rise to a mixture of diastereoisomers. The tendency to epimerize at C-5 was a prominent feature of (5R,6S)- and (5S,6R)-as well as (5R,6R)-benzyl-~-penicilloic acids. The MDM reagent has been prepared in single-dose ampules as a dried, lyophilized powder that can be stored without change and used as needed. Lyophilized MDM has served as a satisfactory substitute for freshly prepared MDM in several individuals with MDM-positive history and, in a recent clinical study, evaluating the question of penicillin skin test sensitization. This convenient, stable, single-doseform of the MDM reagent should facilitate skin testing for penicillin sensitivity.

A reasonably reliable skin test for detecting penicillin allergy was described some years ago in the literature based largely on the work of Levine and co-workers’.’ and Parker and coworkers.:’ Its clinical effectiveness has been amply confirmed since then by a series of investigators from independent laboratories who studied adverse reactions to penicillin* and skin tests to detect penicillin The skin test reagent currently considered satisfactory is essentially that of Levine, Voss, Redmond, and Zolov’. with the omission of n-benzyl-Dpenicilloyl-tu-amide. It consists of benzyl-D-penicillin, benzylD-penicilloylpolylysine (PPL),tu-benzyl-D-penicilloic acid,7and n-benzyl-o-penilloic acid.7 Penicillin and PPL, the major determinant, detect the majority of reactors, but a-penicilloic and a-penilloic acids, known as “minor determinants,” must be included to detect the minority of individuals who undergo lifethreatening reactions. Despite its usefulness, the test has not become routine. Probably the chief deterrent has been that, to date, it has not been possible to develop a stable minor determinant mixture (MDM) reagent.’ In skin testing with the MDM reagent, it has been good practice to make up MDM solutions just before use. However, such practice, in addition t o requiring laboratory facilities not always available to the physician, is time-consuming and generally inconvenient. Lacking the MDM reagent and its components, some investigators have substituted in the skin test “simple skin test reagents” (SSTR) consisting of certain aged solutions of penicillin or alkali-treated penicillin as a source of the minor An insufficient amount of documented data on the stability of MDM and components has been available. For this reason, 448

1 Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985

we undertook to examine the stability of MDM and its components with the aim of arriving at a suitably stable MDM preparation that could be made available to the patient as needed. To assess stability, we used high-performance liquid chromatography (HPLC),a technique that proved to be informative and convenient. Two “simple skin test reagents” derived from penicillin were also analyzed and their compositions were evaluated. The MDM reagent was found to be highly labile in solution, resulting in a mixture of diastereoisomers. Therefore, it was prepared in a lyophilized form in individual, sterile test units that were stored, without appreciable change, and that could be placed into solution as needed. Experimental details for the preparation and storage of the lyophilized MDM reagents are presented herein in addition to kinetic studies on stability.

Results L i q u i d C h r o m a t o g r a p h y of D i a s t e r e o i s o m e r s (5R,6R93S)-,(5S,6R,3S)-,(5R96S,3S)-,a n d (5S,6S,3S)-, Benzylpenicilloic Acids, a n d Diastereoisomers (5R,3S)-, a n d (5S,3S)-Benzylpenilloic Acids-Standard curves relating the absorbancy a t 225-nm to the weights of sodium (5R,6R,3S)-benzylpenicilloatetrihydrate and (5R,3S)-benzylpenilloic acid hydrate demonstrated that the detector signal was linear between 2 and 8 pg for both compounds and that these substances can be quantitated by HPLC. Chromatographic systems consisting of p-Bondapak CI8 reversed-phase support and 15 or 20% MeOH:O.Ol M potassium phosphate buffer, pH 7, required only 5 min of chromatography time and were useful in assessing the stability of the original (5R,6R)-penicilloateand (5R)-penilloate in the MDM reagent, as seen in Fig. 1. They allowed the synthetic diastereoisomers (5R,6R)-(la),(5S,6R)- + (5R,6S)-( l b + I d ) and (5S,6S)-(lc) (Fig. 2) of benzyl-r>-penicilloic acid to be readily distinguished. However, (5S,6R)- and (5R,6S)-penicilloates co-chromatographed. Some resolution of these two developed in 5% MeOH:O.Ol M phosphate buffer, pH 7, as seen in Fig. 3. (5R,6R)- and (5S,6R)- separated with a ratio of retention volumes of N = 1.2; for (5S,6R)- and (5R,6S)-, a = 1.1; for (5R,6S)- and (5S,6S)-,(Y = 1.5. The one-component system 0.01 M potassium phosphate buffer, pH 7, which required 40 min, separated all four diastereoisomers completely, the (5S,GR,3S)-diastereoisomer now differing well in elution volume from (5R,6S,3S)-, as well as from (5R,6R,3S)- and (5S,GS,3S)-benzyIpenici~loates.The methyl esters of (5S,6R,3S)- and (5R,6S,3S)-benzylpenicilloates,obtained by treatment of the acids with diazomethane, were also readily distinguishable from each other by elution volume on HPLC. Diastereoisomeric (5R,3S)- and (5S,3S)-benzylpenilloates (2a and 2b) separated partially in 20% MeOH:O.Ol M buffer, pH 7 (Fig. l),and completely ( a = 1.1) in 15% MeOH:O.OI M buffer, pH 7. Table I gives the elution volumes in various 0022-3549/85/0400-0448$0 1.00/0

0 1985, American PharmaceuticalAssociation

A

B

1

OL

P (5-epi)

la, R' = COOH (5R,6R)

lb, R' = COOH (5S.6R)

2a, R' = H (5R)

2b, R' = H (5s)

y (5,6-diepi)

6 (6 - epi)

Ic, R' = COOH (5S,6S)

I d , R' = COOH (5R,6S)

Figure 2-Drastereorsomeric benzyl-ppenicillorcacids (la-d) and drastereoisomeric benzyl-o-penilloicacids (2a, b); R = C7H7CONH.

2

Table I-Chromatographic Behavior of Diastereoisomeric Benzyl-D-PenilloicAcids and Benzyl-D-PenicilloicAcids and Some Methyl Esters on Reversed-Phase HPLC Elution Volumes, mL System"

I

1

I

I

I

1

I

1 2 3

o-Penilloic Acid

41.4 25.2

46.0 27.0

o-Penicilloic Acid

30.6 4.8 4.0

12

0

1

40.2 5.8

4.5

58.5 7.6 5.6

Methyl D-Penicilloateb 4

I

34.6

I

I

I

I

1

8 12 16 20 24 Elution Volume, mL

4

16.7

20.8

17

19.6

"System 1, 0.01 M potassium phosphate, pH 7; system 2, 15% MeOH:O.Ol M potassium phosphate, pH 7; system 3, 20% MeOH:O.Ol M potassium phosphate, pH 7; system 4, 50% MeOH:O.Ol M potassium phosphate, pH 7. The flow rate was 1 or 2 mL/min. bSeparations of methyl esters of (5R.6R)- and (5R,6S)- and of (5S.W)- and (5S,6R)penicilloates were incomplete and were not optimized.

Another SSTR, prepared by mixing penicillin with an aged solution of alkali-treated penicillin" contained the desired (5R,6R)-penicilloateat 4.8 mM or at about one-half its concentration in the MDM mixture in current use. Also present were 2 mM (55',6R)-/(5R,6S)-penicilloate and 23 mM penicillin. This reagent may be an adequate source of (5R,6R)-penicilloate for skin tests. Penilloic acid was not detected in either reagent. Stability of MDM Reagent and Components-Products of Decomposition of MDM Components in Solution-Figure 1A presents a chromatogram of the two components, (5R,6R)benzyl-D-penicilloic acid and (5R)-benzyl-~-benzylpenilloic acid, in a fresh solution of MDM reagent. After 24 h at room systems of the diastereoisomeric benzyl-D-penicilloic acids, temperature (Fig. lB), or 46 h in the refrigerator (Fig. lC), their methyl esters, and the diastereoisomeric benzyl-D-penil(5R,6R)- is seen to form two additional materials: (5S,6R)loic acids. (peak 2) and (5S,6S)-penicilloate (peak 3). At room temperaComposition of Two "Simple Skin Test Reagents" as ture, (5S, 6R)q~enicilloateis the predominant component. Determined by Liquid Chromatography-The SSTR (5R)-Penilloate (peak 4 ) gives rise to the additional material formed by allowing a solution of benzyl-D-penicillin a t p H 7.75 (5s)-penilloate (peak 5). These newly formed materials differed to age a t room temperature for 1 week' contained, in addition clearly in chromatographic behavior from known penicillin G to penicillin, a mixture of benzyl-D-penicilloates as (5R,6R)-, rearrangement and degradation products including benzyl-D(5S,6R)-/(5R,6S)- and (5S,6S)-stereoisomers in a combined concentration of 0.3 mM in which (5S,6R)-/(5R,GS)-penicil- penillic acid, benzyl-D-penicillenic acid, and penicillamine. Their diastereoisomeric structures were established by close loate predominated. The concentration of the desired (5R,6R)comparison by chromatography and co-chromatography by penicilloate was ~ 0 . mM, 1 or 1% of that in the MDM reagent HPLC in two solvent systems with authentic synthetic samples in current use. This reagent, therefore, would appear to be a of (5R,6R)-, (5S,6R)-, (5R,6S)-, and (5S,6S)-benzyl-~-penicilquestionable source of (5R,GR,3S)-penicilloate for skin tests.

Figure 1-HPLC chromatograms showing the decomposition at several temperatures of an MDM solution [a mixtureof 10 mM ~~-(SR,GR)-benzylPpenicilfoate(la) and 10 mM ~~-(5R)-benzyl-~penilioate (2a)l. Key: (A) the starting MDM solution; (6)after 24 h at 23-24 "C;(C) after 46 h at 3-4 "C. The solvent system was 20% Me0H:O.Ol M potassiumphosphate buffer,pH 7.0. The flow rate was 1 mL/min; the sensitivity was 0.7. Peaks 7, 2, and 3 represent C Y - ( ~ R , ~(la), R ) - P-(5S,6R)- (lb), and y-(5S,6S)benzyl-ppenicilloates (lc), respectively; peaks 4 and 5 represent 4 5 R ) - and p-(5S)-benzy/-~-peni/loates (2a and 2bJ, respectively.

Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985

/ 449

loic acids (see (see loic acids, acids, and and (5R)(5R)- and and (5S)-benzyl-D-penilloic (5S)-benzyl-D-penilloic acids above above and and Fig. Fig. 3). 3). on EpimerEpimerEffect Temperature and and Components Components of of MDM on Effect of Temperature ization-Figures like ization-Figures 44 and and 55 were were derived derived from from chromatograms chromatograms like of those 1. Figure Figure 44 shows shows the the rate rate of of conversion conversion of those in in Fig. Fig. 1. (5R,6R)- to (5S,6R)-/(5R,6S)- (5S,6S)-penicilloates in the absence and presence of added (5R)-penilloic acid at two temperatures. In MDM solution, (5R,6R)-penicilloate underwent underwent at +24"C within 1-24 h, 34-78% change; at +4"C in 24 h, 47% change. In the absence of penilloate, (5R,6R)-penicilloate underwent, within 1-24 h, 5-53% change; a t +4"C within 24 h, 9.4% change. Figure 5 shows the rate of conversion of (5R)- to to (5s)-penilloic acid in the absence and presence of added temperatures. In MDM (5R,6R)-penicilloic acid at the same two temperatures. solution, (5R)-penilloate underwent a t +24"C within 1-24 h, 30% change; change; in the absence 30-48% 30-48% change; at +4"c +4"C in 24 h, 30% of penicilloate at +24"C in 24 h, 44.5% 44.5% change; at +4"C, +4"c, 18.5% change. MDM solutions consisting of Of homogeneous (5R,6R)-

+

I

0

I

I

I

I

I

I

I

8

16

24

32

40

48

56

Elution Volume, mL diastereoFigure 3-HPLC chromatogram showing the separation of aa diastereoisomeric mixture of (5R,6R)-, (5S,6R)-, (5R,6S)-, and (5S,GS)-benzylM potassium potassium system was 5% Me0H:O.0 11 M o-penicilloates. The solvent system phosphate buffer, pH 7.0. The flow rate was 2 mL/min.

s -

'"i

24O C

a- 90-

c

m

2 80..-0 5 70a

-2. 6 0 -

*. -.-

50-

m

40'

. I -

0

g

.-c

30-

mN 20.-

&

.-E 10Cl

W

01, 0

1

2

3

4

5 Days

-.-

6

7

8

9

Figure 4-Kinetics of of epimerization of of (5R, (5R,6R)-benzyl-o-penicilloate 6R)-benzyl-o-penicilloateto to in an an MDM MDM solusolu(5S,6R)-/(5R,6S)-and (5S,6S)-benzyl-~-penicilloatesin tion, i.e., in the presence of of 10 10 mM mM (5R)-benzyl-~?-penilloate (5R)-benzyl-~?-penilloateat at 24 .)) and (-0-) and and 44O OC C ((.. and in in the the absence absence of of benzylpenilloafe benzylpenilloate 24 OC (-0-) at (-0-) and and 4OC 4OC (.-O-.). (.-O-.). For For terminology, terminology, structures, structures, and and at 24OC (-0-) HPLC and 2. 2. HPLC conditions, conditions,see see Figs. Figs. 71and 450 450

1/

Journal Journal of of Pharmaceutical Pharmaceutical Sciences Sciences Vol. Vol. 74, 74, No. No. 4, 4, April April 1985 1985

s

60 -

6 50-

ZTii 40.-c 4 c m a

.N

&

a

.E I

30-

- v

Y

.

"

L .-.-.*----

,r

,-__,

0 ,R . 0

/.+'

/'

0

0

]

24OC

1

4oc

w" g 20a

m I 8

10-

-

Figure 5-Kinetics of epimerization of (5R)-benzyl-~peni//oate to (5s)benzyl-o-penilloate in an MDM solution, i.e., in the presence of 10 mM (5R,6R)-benzy/-~-penici//oateat 24OC (-O-) and 4OC (.-W-.) and in the absence of benzylpenicilloate at 24 " C (-0-) and 4 OC (. -0- .). For terminology, structures, and HPLC conditions, see Figs. 1 and 2.

penicilloate and (5R)-penilloate, or those containing 6% l b and 14% 2b, were frozen rapidly and stored a t -17°C. They remained unchanged in each component for 9 d, as did separate frozen solutions of l a and 2a. All aged solutions of (SR,GR)-penicilloate, except those maintained frozen, contained, in addition to (5S,6R)-/(5R,6S)penicilloate, varying amounts of (5S,6S)-penicilloate ( l c ) .The kinetics indicated that l c arose after (5S,6R)-penicilloate had formed. The aged MDM mixtures of (5R,6R)-penicilloate and (5R)penilloate contained larger amounts of l c than did an aged solution of (5R,GR)-penicilloate. Maintained for 2 d at room temperatures an MDM mixture contained (5R,6R)-,(5S,6R)/(5R,6S)-, and (5S,6S)-penicilloate in the proportion 1:5.6:2. After 1 d at +4"C, the proportion was 1:0.57:0.24. Of the decomposition products, (5S,6S)- constituted 26-29%. By contrast, after having been allowed to stand alone at room temperature or a t +4"C, comparable penicilloate solutions contained (5S,6S)- as only 14-17% of the product. This difference in extent of formation of (5S,6S)-penicilloate held even when the total degree of decomposition of (5R,6R)-penicilloate (87%) was similar in the two solutions. The presence of penilloate thus appears to catalyze epimerization to (5S,6S)-penicilloate. Whether this effect is an instance of base catalysis by the penilloate anion remains t o be established. At +4"C the catalytic effect of penilloate on the epimerization of (5R,6R)-penicilloate was substantial. At this temperature, penicilloate similarly had a catalytic effect on the epimerization of (5R,3S)penilloate. At room temperature, when epimerization was rapid, the catalytic effect appeared to be less significant. Comparison of Penicilloate and Penilloate in Extent of Epirnerization-The initial rates of decomposition of (5R,6R)penicilloate and (5R)-penilloate were rapid and comparable, especially in MDM solutions. However, (5R,6R) -penicilloate ultimately underwent a considerably greater degree of change (90%) than did (5R)-penilloate which reached an equilibrium mixture consisting of 50% each of (5R)- and (5s)- diastereoisomers. Further Studies of Epimerization, with (5R,6R)-, (5S,6R)-, and (5R,6S)-Benzyl-~-Penicilloic AcidsWhen aqueous solutions of these three diastereoisomers were allowed to stand separately a t pH 3 under mild conditions and were analyzed by HPLC, each of them showed a tendency for its C-5 proton to exchange readily. Thus, (5R,6R)- formed (5S, 6R)-; (5S,6R)- formed (5R,6R)-; (5R,6S)- formed (5S,6S)-. The results are included in Table I1 and are summarized in the following scheme, in which those reactions examined and observed experimentally are indicated by unbroken lines. The broken line represents an overall reaction. In each case, the

Table Il-Epimerization Reactions of Three Diastereoisomeric Benzyl-o-PenicilloicAcids

Conc., mg/mL Buffer'

Compound

Conditions pH

Time,

Chromatographic Temp., Systemb "C

Product Composition, Yoc (W6N

(5S.6Wd (5S,6S)

E :,

(5R,W)

(5S,6R)-Penicilloic acid 3.3

A

8

24

B

3

24

room 4 room 4

3, 5 3 3

9 3 46 34

86 94 47 63

5 3 7 3

room 4 room 4

2, 5 2 2 2

47 90 43 72

47 10 57 28

6

room room 4 room

5

3 3

31

27 37 13 16 17 40

(SR,6R)-Penicilloic acid 3.5

A

8

24

C

3

24

A A

7.5 8

24 22

3

7d 4 22

3, 5

(5R,GS)-Penicilloic acid 3

1.7

3.3 1.7

50

2

1

19 20

39 60 86 65 63 59

room 2 1 4 2 0.5 16 84 a Buffer A, 0.1 M sodium phosphate; 6 ,0.1 M sodium citrate; C, 70% EtOH incorporated into buffer A. Chromatographic system 2, 15% MeOH:O.Ol M potassium phosphate, pH 7.0; system 3, 20% MeOH:O.Ol M potassium phosphate, pH 7.0; system 5, 5% MeOH:O.Ol M potassium phosphate, pH 7.0. Corrected for 6.8% (5R,6R)- in starting (5S,6R)-penicilloicacid; corrected for 4.3% (5R,6R)- and 12.9% (5S,6S)- in starting (5R,6S)-penicilloicacid. The absence of (5R,6S)-penicilloicacid is unconfirmed in samples analyzed in systems other than system 5. ' Essentially similar results were obtained with buffer B. A" A"

degree of epimerization at C-5 within 1d at room temperature was at least 40-57%. Epimerization at C-5 frequently took place at pH 7.5 or 8 as well. Thus, within 1 d at room temperature, (5R,6R)- formed 47% (5S,6R)-, and (5R,6S)- formed 58% (5S,6S)- (5S,6R)-. However, the (5S,6R)-diastereoisomer remained chromatographicallyunchanged under the mildly alkaline condition. Lyophilized M i n o r D e t e r m i n a n t Mixture-Stablility Characteristics-The ampules of lyophilized MDM reagent contained a white fluffy powder. On addition of 0.2 mL of sterile water it dissolved completely and easily to give the MDM reagent containing (5R,6R)-benzyl-~-penicilloate and (5R)benzyl-D-penilloate in the desired 10 mM concentration as well as the desired buffer and pH. Typical starting materials used in early preparations of lyophilized MDM had been stored for 1 year and contained (5R,6R)-penicilloate acid with <2% (5S,GR)-isomerand (5R)-penilloatewith 9% (5s)-isomer. Randomly selected ampules of lyophilized MDM prepared from such materials typically contained (5R,GR)-penicilloate with 3% (5S,GR)-isorner and (5R)-penilloate with 11%(55')-isomer. Homogeneous starting materials yielded lyophilized MDM with 4 . 7 % l b and <5% 2b. Storage of the ampuled lyophilized MDM reagent for 1week at +3"C or at room temperature resulted in no significant change in composition. After 1 year at -25"C, the lyophilized material retained >97% of the (SR,GR)-penicilloate and >91% of the (5R)-penilloate originally present. However, it is noted that an occasional batch of sealed ampules showed some transformation of both components after standing a t room temperature for 3 weeks. This was attributed to less cautious desic-

+

cation conditions before the ampules were sealed. Therefore, lyophilized MDM powders are maintained under extremely rigorous anhydrous conditions until sealed, and then are stored at freezer temperature. Biological Properties-In skin tests of three individuals with MDM-positive history, the responses to University of Connecticut (UConn) lyophilized MDM and lyophilized penicilloate were highly positive and comparable to each other in two of the three individuals. Johns Hopkins University (JHU) MDM and (5R,6R)-penicilloate reagents gave similar results that, moreover, were comparable to the UConn products. The third individual, MT, failed to react to the two lyophilized preparations, although he gave some response to the JHU materials. In the first two individuals, responses to MDM and penicilloate from both laboratories were 2-3 times that to penicillin alone, whereas the third individual, MT, gave only low and equal response to all J H U materials including MDM, penicillin, penicilloate, and penilloate. This could suggest a nonspecific response of M T to JHU materials, although the negative response of M T to a saline control make this less likely. Results with this individual can probably be considered to be variable or anomalous since, on being tested four separate times with J H U material, he reacted negatively twice. JHU (5R)-penilloate led to a low response in two individuals and no response in the third subject, JB."

Discussion The present study demonstrates for the first time the direct chromatographic separation of the four diastereoisomers of Journal of Pharmaceutical Sciences 1 451 Vol. 74, No. 4, April 1985

benzvl-I,-penicilloic acid that differ in asymmetry a t carbons 5 and 6, i.e., (5R,6R,3S)-, (5S,FR,3S)-, (5R,6S,3S)-, and (5S,GS,~iS)-henzylpenicilloicacids. As a n aid in the identification of the diastereoisomers of (3s)-benzylpenicilloic acid, these chromatographic procedures are more direct and convenient than the methods previously used which required conversion to t,he dimethyl benzylpenicilloate esters, followed by chromatographic purification on silica gel, and then physicochemical identification.12 Moreover, a risk of epimerization is present in some of the reported methylation procedures that involve cat.alysis by base. Recently, other investigators have found HPLC useful in detecting the formation of the (5s)epimer of penicic acid':' and of ampicillin penicilloic acid.I4,l 5 In this study, HPLC procedures were used to advantage for ( a ) examining the composition of various MDM reagents of the penicillin skin tests; ( b ) examining the stability of MDM components (5R,tiR,S)-penicilloicacid and (5R,3S)-penilloic acid in solution; (c) assessing the tendency of three diastereoisomeric (3S)-benzylpenicilloicacids to epimerize in solution under various conditions. An aged solution of penicillin that has found use as a "simple skin test reagent" was judged by HPLC to be inadequate with respect to its content of both penicilloate and penilloate, thus suggesting that it be replaced by a more suitable MDM reagent. An aged part,ialalkaline hydrolysate of penicillin, also employed as a "simple skin test reagent," likewise lacked penilloate. Its content of (5R,612,3S)-penicilloate was one-half of that in the MDM reagent of Levine and co-workers currently used. When supplemented with penilloic acid, it may be sufficient for detecting allergic individuals, except for those marginally sensitive to penicilloate. (5R,6R,3S)-Penicilloic acid and (5R,3S)-penilloic acid in solution a t pH 7 underwent rapid change, both when each compound was present individually and when present as a mixture in the MDM reagent in current use. Generally, the presence of one component accelerated the decomposition of the other in the MDM reagent in solution. The resulting aqueous mixtures contained, in addition to the starting components, diastereoisomers of (3S)-penicilloic acid differing in chirality at C-5 and/ or C-6, and the diastereoisomer of (3S)-penilloic acid differing in chirality a t C-5. The proportion of the epimers in the mixture was influenced by pH, temperature, length of storage, and the presence of other components. After 24 h at room temp, an MDM solution retained only 22% of the starting (5R,6R,3S)penicilloate and 60% of (5R,3S)-penilloate, respectively. The products present, were (5S,GR,3S)-and (5S,GS,3S)-penicilloate (62 and 16%) and (5S,3S)-penilloate (40%). No (5R,6S,3S)penicilloate was detected. Even when the MDM solution was stored in the refrigerator for 24 h, only about, one-half of the penicilloate and 70% of the penilloate were present in their original diastereoisomeric form. When the stabilities of three diastereoisomers of (3s)-penicilloic acid in solution were compared under various mild conditions (Table 11), it became clear that, except for certain restrictions of pH, the diastereoisomers had in common a pronounced tendency to epimerize a t C-5, regardless of the chirality at C-6. In solution a t p H 3, within 1 d a t room temperature, (5R,6R,3S)-,(5S,6R,3S)-,and (5S,GS,3S)-henzylpenicilloic acids each underwent -50% epimerization at C-5. Under mildly alkaline conditions, (5R,GS,3S)-benzylpenicilloate, like (5R,6R,3S)-benzylpenicilloate,epimerized extensively at C-5 (-50%), but the (5S,6R,3S)-diastereoisomer remained stable. The unusual stability in solution of the (5S,6R,3S)-diastereoisomerunder the mildly alkaline condition is consistent with its formation in high yield from (5R,6R,3S)penicilloic acid under such conditions observed here and elsewhere." Also in general agreement are the observations of Carroll et aI."who found that basic hydrolysis of 6-aminopenicillanic acid yields first the expected (5R,GR)-penicic acid from which the epimer 5-epi(5S,GR)-penicic acid forms as the ther452

Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985

modynamically stahle product in basic solution. Moreover, the observed tendency of (5S,GR)-benzyl-D-penicilloate to convert to the (F~R,GR)-enantiomer under acidic but not slightly alkaline conditions (Table 11) is consistent with deductions made by NMR that (2S)-5,5-trimethylthiazolidine-4-(S)-carboxylic acid undergoes rapid equilibration a t C-2 (corresponding to C5 of the penicilloates) in 1 M HC1 but is stable for 24 h in dilute sodium carhonate.l6 Instances of epimerization of certain (5R,6R)-benzylpenicilloate and (5R)-benzylpenicilloate derivatives a t C-5 have been interpreted in terms of the behavior of the thiazolidine moiety."." 2-Substituted thiazolidine-4-carboxylicacids are known to epimerize a t C-2 in water or methanol solutions a t room temperature.I6 The reaction is ascribed to hydrolytic opening of the thiazolidine moiety at the C-2/S linkage followed by proton loss to form a Schiff base a t N=C-2, where both steps are reversible."~ 2" In the penicilloates and penilloates, the corresponding labile linkage would be C-5/S, and the Schiff base intermediate would involve N=C-5. In addition to this study and those already cited, observations of instability/epimerization of penicilloates and penilloates have been accumulating in the chemical literature since these compounds were first described some 35 years ago. These include epimerization of a-benzyl-D-penilloic acid and its diastereoisomer;"* 22 mutarotation of methyl esters of penicilloic acid when heated in alcohols;" mutarotation of a-penicilloate the presence of two materials a t pH 7.5 in aqueous solution;23,24 in an alkaline hydrolysate and a p-lactamase digest of penicillin;2s epimerization at C-5 of penicilloyl a-amide in excess NH:,;26and, a t elevated temperatures, epimerization a t C-5 and C-6 of the 0-lactate ester of benzylpenicilloate formed from penicillin in meat.2' In general, the various observations have not been evaluated sufficiently in relation to the immunological uses of these compounds as skin test reagents. The early descriptions of a preparation of a minor determinant reagent, for detecting penicillin hypersensitivity in humans reported the aqueous reagent to be stable for some months a t 4°C.' Subsequently, "some lability of benzylpenicilloate" was noted." Clitiical studies have employed MDM reagent solutions ranging from fresh reagent solutions and those frozen directly after preparation,2to those maintained at 4°C for 6 months,' to those prepared daily,5" or those prepared weekly and maintained a t 4'C.'' In some instances, the conditions of storage have been unspecified. In view of the extensive change (5R,GR,3S)-penicilloic acid and (5R,3S)-penilloic acid can undergo in solution even a t 4"C, the MDM reagents used in some of these studies may have lacked homogeneity. The antigenicity in humans of the epimerization products of (5R,6R,3S)-benzylpenicilloate and (5R,3S)-benzylpenilloate is not yet established. Preliminary skin-test studies with several MDM-positive individuals indicate that, when tested near its minimal effective concentration, the aqueous reagent inactivates. Until further information becomes available, it is clear from the kinetics shown in Figs. 1, 4, and 5 that, the aqueous MDM reagent is too labile a t room temperature to justify use with confidence except immediately after it has been prepared. An MDM solution maintained a t 4°C can probably he reused within several hours inasmuch as it retains a t least 85% of the starting compounds a t 3.5 h. An MDM solution frozen immediately after preparation and stored frozen appears to be satisfactory, although it was observed for only a limited period of 9 d. The lyophilized, single-dose MDM preparation was developed in the present study based upon our ohservations that, in contrast to the behavior of these components in aqueous solution, sodium (5R,6R,3S)-benzylpenicilloateand (5R,3S)-benzylpenilloic acid can be stored as crystalline solids at 4°C over silica gel for extended periods with little change, as judged by melting point and confirmed by HPLC. The lyophilized MDM material proved to have satisfactory storage properties and to

provide the expected composition upon being placed into solution. When the MDM reagent solution is freshly reconstituted from the lyophilized material just before use, the physician can be assured that the reagent solution has the desired composition. When its biological properties were examined independently in another laboratory a t ,Johns Hopkins University by using it to skin test a number of' MDM-positive individuals, the lyophilized MDM reagent elicited positive reactions in much the same way as the freshly prepared reagent." In a recent clinical study of 240 individuals with a history of allergy t o penicillin C, or one of its analogues, in which (relsensitization to penicillin by the skin test and challenge was examined, the MDM preparation developed herein proved to be a suitable, convenient. substitute for freshly prepared MDM. This study has been reported separately in detail."

Experimental Section Chromatography-The HPLC apparatus consisted of a solvent delivery system (model 6000A; Waters Associates, Milford, MA), injector (model U6K; Waters), reversed-phase column (p-Bondapak Clx; Waters), 8-pL flow cell (model 154-02; Altex Scientific, Berkeley, CA) with detector (model 252; Gilford, Oberlin, OH) later replaced by a detection system (Holo chrome; Gilson Electronics, Middleton, WI), and a recorder (model 6051; Gilford). Absorption was measured at 225 nm. Peak integrations were performed with a laboratory data system (model 3390A; Hewlett Packard, Palo Alto, CA). Sensitivity was 0.1 AUFS. The limit of detection was 0.4 pg for penicilloic acid sodium salt trihydrate and 0.7 pg for penilloic acid hydrate. Chromatographic conditions are given in the legend of Fig. 1. Epimerization of (5R,6R)-,( 5 S , 6 R ) - ,and (5R,6S)Benzyl-D-Penicilloic Acids-The synthetic purified diastereoisomers were allowed to stand in dilute aqueous solution at 4°C and room temperature, a t pH 3 and 8, generally for 24 h. The solutions were then analyzed directly by HPLC. Experimental conditions and results are given in Table 11. Solution of Simple Skin Test R e a g e n t s (SSTR)-Aged Penicillin-A solution of benzylpenicillin K (Eli Lilly, Indianapolis, IN) in 0.05 M potassium phosphate (J.T. Baker Chemical, Phillipsburg, N J ) buffer: 0.4% saline, pH 7.75 (1000 II/ mL, 1.69 mM) was sterilized by filt,ration and allowed to st.and at room temperature in a closed serum bottle.' After 6 d, the starting penicillin solution was present as a mixture of 1.14 mM penicillin, 0.08 mM (5R,6R)-benzylpenicilloate, 0.17 mM (5S,6R)-/(5K,6S)-benzylpenicilloate,and 0.05 niM (5S,6S)benzylpenicilloate. Recovery was 85%. No further attempt, was made to account quantitatively for the starting material by modifying the chromatographic system. Aged Solution of Alkali-Treated Penicillin--To 8.2 m1, of saline the following was added: 1 mL of a saline solution of benzylpenicillin K (1.04 x 1 0 17, 65.5 mg) and 0.8 mL of a solution of benzylpenicillin K (8 x lo4 U, 50.3 mg) that had been allowed to stand at lo5U/mL in 0.085 M NaOH (0.5 mol equivalent) for 45 min at room temperature followed by 1 week at 4°C.'" The resulting solution contained 22.6 mM penicillin, 4.8 mM (5R,6R)-benzylpenicilloate, and2 mM (55*,6R)-/( 5R,6S)benzylpenicilloate. Recovery was 94%. (5R,6R)-benzyl-~-penicilloate O t h e r Materials-Sodium trihydrate,", '"(5R)-benzyl-~-penilloicacid hydrate,"' '* ( 5 s ) benzyl-D-penilloic acid,21 and (5S,6R)-I2 (5S,GS)"-benzyI-11penicilloic acids, and benzyl-n-penillic acid'" were prepared as reported. (5R,GS)-BenzyI-~-penicilloicacid was prepared by treatment of (5R,6S)-benzylpenicillin3'with aqueous alkali in analogy with la. Diastereoisomeric compounds l a , l b , and I d have been characterized as their methyl esters,'.'' and 2a and 2b have been characterized as the acids.7,'"22They all had the expected elemental analyses and appropriate 'H NMR spectra.

Methyl est,ers were prepared by treating a suspension of the benzy~-D-penicil~oic acid in CH2C12 with a slight excess of ethereal diazomethane at 5°C. After 10 min, the mixture was taken to dryness and the residue was examined by HPLC. Benzyl-wpenicillamine and benzyl-wpenicillenic acid were commercial products (Sigma Chemical Co., St. Louis, MO). All other materials were of the highest grade available. P r e p a r a t i o n , Analysis, and Stability Studies-Solutions of MDM and Components-The MDM solution consisted trihydrate and of 10 mM sodium (5R,6R)-benzyl-~-penicilloate 10 mM (5R)-benzyl-~-penilloicacid hydrate in 0.07 M sodium phosphate buffer, p H 7.5.2R The solution was sterilized by filtration through a 13-mm membrane (Millipore), 0.45-pm pore size, int,o sterile 2-mL glass ampules. Separate solutions of' 10 mM sodium (5R,GR)-benzyI-~-penicilloate and of 10 mM (E&)-benzyl-D-penilloate a t p H 7.5 were prepared similarly. Ampules were sealed, and some were maintained a t 23°C and 4°C. Others were frozen in dry ice:EtOH and stored a t -17°C. At, intervals, the ampules were opened and samples of 0.05 pmol were chromatographed on the Waters reversed-phase column. Lyophilized MDM-MDM solution was prepared as just described, cooled in ice-water, dist,ributed in 0.2-mL portions into ampules or vials, and then frozen immediately in dry ice:EtOH. From the time the solutions were prepared to this point required 4 5 min. The ampules were freeze-dried a t 0.1 Torr. Rigorous anhydrous precautions were taken from the time the ampules were removed from the lyophilizer until they were sealed. Sealed ampules were stored a t room temperature, 3"C, and a t -25°C for specified periods. Just prior to use they were opened, and the MDM reagent was reconstituted to 10 mM by the addition of 0.2 mL of sterile water for analysis or normal saline for clinical use. Resulting solutions were immediately analyzed or otherwise used. The identity and concentration of the components in the lyophilized MDM were determined by quantitative HPLC using as standards freshly prepared solutions of each compound at increasing concentrations. Lyophilized M D M Biological Studies-Skin tests were carried out,by Dr. N. F. Adkinson, Jr. and co-workers a t Johns Hopkins University (JHU) on three individuals with a history of positive reaction to MDM and giving a current positive reaction to PPI,. UConn and J H U preparations were administered simultaneously on separate arms. Response was measured as millimeters induration a t the site of intradermal injection after 1520 min. Lyophilized MDM (UConn) was compared in the skin tests with MDM solutions freshly prepared a t JHU. Both reagents contained 10 mM (5R,6R)-benzylpenicilloateand (5R)-benzylpenilloate, but JHU contained benzylpenicillin as well. UConn lyophilized (5R,6R)-benzylpenicilloate and J H U (5R,6R)-benzylpenicilloate solutions were also compared. Separate J H U benzylpenicillin and (5R)-benzylpenilloate solutions were included in the study. The composition of all UConn and JH1J materials was confirmed by HPLC analysis a t UConn.

References and Notes 1. Voss, H. E.; Redmond, A. P.; Levine, B. B. J. A m . Med. Assoc. 1966, 196,679. 2 . Levine, B. B.; Zolov, D. M. J . Allergy 1 9 6 9 , 43, 231. 3. Budd, M. A,; Parker, C. W.; Norden, C. W. J . A m . Med. Assoc. 1964,190,203. 4. Erffmeyer, J. E. Ann. Allergy 1 9 8 1 , 47, 288; Parts I and 11. 5. (a) Sullivan, T. J.; Wedner, J.; Shatz, G. S.; Yecies, L. D.; Parker, C. W. J . Allerm Clin. Immunol. 1 9 8 1 . 68. 171. (b) Van Dellen. R. G. J . Allergy Clin. Immunol. 1981,'68,' 169; (cj Solley, G. '0.; Gleich, G. .J.; Van Dellen, R. G. J . Allergy Clin. Immunol. 1982, 69, 238. (dj Adkinson, N.F., Jr.; Thompson, W. L.; Maddrey, W. C.; Lichtenstein, L. M. N. Eng. J . Med. 1 9 7 1 , 285, 22. 6. Mendelson, L. M.; Ressler, C.; Rosen, J. P.; Selcow, J. E. J. Allergy C'lm Inimunol 1 9 8 4 , 73, 76 Nomenclature: tu-benzvl-D-Denicilloicacid has the (5R,6R,3S)configuration of natural 6-penicillin and corresponds to CY of refs. 21 I,"

,.

Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985

453

and 12; @ has (5S,6R,3S)-configurationand corresponds to @ of ref. 21 and d of ref. 12; y has (5S,6S,3S)-configuration and corresponds to d of ref. 21 and y of ref. 12; 6-epi has (5R,6S,3S)configuration and corresponds to y of ref. 12 and @ of ref. 12. aBenzyl-D-penilloic acid has the (5R,3S)-configuration of natural D-penicillin; @ has (5S,3S)-configuration. 8. Van Arsdel, P. P., Jr., in “Allergy, Principles and Practice”; Middleton, E., Jr.; Reed, C. F.; Ellis, E. F., Eds.; C. V. Mosby Co.: St. Louis, 1978; pp 1153-1154. 9. Bierman, C. W.; Van Arsdel, P. P., Jr. J. Allergy, 1969, 43, 267. 10. Edwards, R. B.; Wagle, S. S, personal communication. 11. Adkinson, N. F., Jr., personal communication. 12. Busson, R.; Claes, P. J.; Vanderhaege, H. J. Org. Chem. 1976, 41, 2556. 13. Carroll, R. D.; Jung, S.; Sklavounos, C. G. J. Heterocycl. Chem. 1977, 14, 503. 14. Bird, A. E.; Cutmore, E. A.; Jennings, K. R.; Marshall, A. C. J. Pharm. Pharmacol. 1983,35, 138. 15. After this manuscript had been submitted for publication, an HLPC procedure was used to examine the formation of (5S,6R,3S)from (5R,GR,SS)-benzyE Ghebre-Sellassie, I.; Hem, S. L.; Knevel, A. M. J . Pharm. Sci. 1984, 73, 125. 16. Nagasawa, H. T.; Goon, D. J. W.; Shirota, F. N. J. Heterocycl. Chem. 1981, 18,1047. 17. Herak, J. J.; Kovacevic, M.; Gaspert, B. Croat. Chem. Acta. 1977, 49, 141; Chem. Abstr. 1977, 87, 102216~. 18. Herak, J . J.; Kovacevic, M.; Gaspert, B. Croat. Chem. Acta. 1978, 51, 265; Chem. Abstr. 1970, 90, 186246~. 19. Pesek, J. J.; Frost, J. H. Tetrahedron Lett. 1975, 31,907. 20. Luhowy, R.; Meneghini, F. J . Am. Chem. Soc. 1979, 101,420. 21. Mozingo, R.; Folkers, K. in “The Chemistry of Penicillin”; Clarke,

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22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

H. T.; Johnson, J. R.; Robinson, R., Eds.; Princeton University Press: Princeton, 1949; pp 535-577. Kiener, P. A,; Waley, S. G. Biochern. J . 1978,169, 197. Levine, B. B. Nature (London) 1960, 187, 939. Schneider, G. H.; de Weck, A. L. Helu. Chim. Actu, 1967,50,2011. Sabath, L. D.; Jago, M.; Abraham, E. P. Biochern. J. 1965, 96, 739. Hamilton-Miller, J. M. T.; Richards, E.; Abraham, E. P. Biochern. J . 1970. l l f i . 385. DePaolii, A.-M.; Denney, D. Z.; Rosen, J. D. J . Agric. Food Chem. 1979, 27, 199. Levine, B. B.; Redmond, A. P. Znt. Arch. Allergy 1969,35,445. Adkinson, N. F., Jr.; Ressler, C.; Mendelson, L. M., unpublished results. Cook, A. H. in “The Chemistry of Penicillin”; Clarke, H. T., Johnson, J. R., Robinson, R., Eds.; Princeton University Press. Princeton, 1949; p 106. Vlietinck, A.; Roets, E.; Claes, P.; Janssen, G.; Vanderhaeghe, H. J . Chem. Soc. Perkin I1973 937.

Acknowledgments This study was aided by University of Connecticut Research Foundation Grant 35-158. We thank Dr. M. Tsutsumi for preliminary HPLC experiments. We are indebted to Dr. N. F. Adkinson, Jr., and colleagues for testing the lyophilized MDM in several MDM-positive patients. The procedure for preparing a “simple skin test reagent” by alkali treatment of benzypenicillin was kindly provided by Drs. R. S. Edwards and S. S. Wagle of Kremers-Urban Co. This study was presented in part a t the American Academy of Allergy and Immunology Meetings, Chicago, IL, March 7, 1984.