The parenteral toxicity of clindamycin 2-phosphate in laboratory animals

27,308-321(1974)

TOXICOLOGYANDAPPLIEDPHARMACOLOCY

The

Parenteral

Toxicity of Clindamycin in Laboratory Animals1

2-Phosphate

J. E. GRAY, R. N. WEAVER, J. MORAN AND E. S. FEENSTRA Research

Division, Received

The Upjohn April

Company,

10,1973;

Kalamazoo,

accepted

June

Michigan

49001

IL?,1973

The Parenteral Toxicity of Clindamycin 2-Phosphate in Laboratory Animals. GRAY, J. E., WEAVER, R. N., MORAN, J. AND FEENSTRA, E. S. (1974). Toxicol. Appl. Pharmacol. 27,308-321. The ip and iv LD50 values of 1145 and 855 mg/kg of clindamycin 2-phosphate in the Swisswhite mouse were approximately 3 times higher than those of clindamycin hydrochloride. The lesion produced by single injection of 50 or 100 mg/ml of clindamycin 2-phosphate in the loin muscles of the New Zealand White rabbit was graded as slight. The 24-hr serum creatine phosphokinase value was 1500 IU/liter which was lessthan one-half that of the parent antibiotic. Body weight gains and food conversion ratios in groups of 10 SpragueDawley rats injected SCwith 120 mg/kg for 6 days were comparable to those of the control group; 90 mg/kg was tolerated in these terms nearly as well for 30 days as no treatment or doses of 30 and 60 mg/kg. From 22 to 33 injections (each equivalent to 30, 60 or 90 mg/kg) were made bilaterally in the posterior thigh muscles of groups of 3 beagle dogs. The terminal elevations of serum glutamic-oxaloacetic transaminase varied from 54 to 400 Reitman-Frankel units. The characteristic pathologic change resulting from the superimposed injections was dose-related progressive scarring of the muscle bundles. Intravenous administration of 60 and 120 mg/kg daily in divided doses in 2 groups of 4 dogs each for 30 days produced no detectable irritation in the peripheral veins or drug-related hemolysis. Tests for drug-induced hemolysis and changes in erythrocyte fragility by in vitro methods were negative. In dogs treated iv with 120mg/kg for 1 week, as light increasein neutral lipid droplets was present in hepatocytes from 3 hr to 3 days when examined by electron microscopy. This transient change was not observed in dogs injected im with 90 mg/kg for the same period. A major research goal of a derivative program of the antibiotic clindamycin was the modification of the parent molecule so as to maximize parenteral tolerance. Results of oral toxicity studies with clindamycin hydrochloride (Gray et al., 1972) and its ultrastructural effects in the hepatocyte (Gray et al., 1971) have been published. Morozowich et al. (1970) postulated that a phosphate ester of clindamycin would improve local tolerance because the lower partition coefficient and higher water solubility of the ester would permit more rapid diffusion from the injection site. Phosphate esters also offered the advantages of stability in the dianionic form and of rapid in vivo hydrolysis to the 1 This paper was presented in part at the 12th Annual Meeting of the Society of Toxicology, New York City, March 18-22, 1973. Copyright 0 1974 by Academic Press, Inc. 308

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parent compound. Position 2, as shown in the structural formula, was chosen for the synthesis of the phosphate ester.2 CH I H-C-Cl

CH3

rlr CH3CH2CH2 (Y

(HzO)~

C-NH-CH a

0

HO

SCH3

OH 0

P O-P-OH OH

7(S)-chloro-7-deoxylincomycin

2-phosphate

Inherent similarities in tissue injury at injection sites and in accompanying changes in serum enzymes, such as glutamic-oxaloacetic transaminase and creatine phosphokinase, render data from parenteral studies in laboratory animals particularly predictive of tolerance of parenterally administered drugs in man. The back muscles of the rabbit were used as a site for single injections to study the extent and nature of injury. Multiple dose studies (30-day) were done subcutaneously in the rat and intramuscularly in the dog to ascertain any accumulative effects, either local or systemic. During a 30-day iv tolerance study in the dog, special efforts were made to determine the significance of hemolysis. Local effects on the injected limb veins were studied. These animals were also examined for systemic effects that might be associated with immediate high blood concentrations produced by iv administration. In a study of the ultrastructural effects in the hepatocyte caused by im and iv injections of the phosphate ester, minimum toxic effects were characterized. METHODS

LD.50 determinations. Acute toxicity (LD50) determinations of clindamycin 2-phosphate in aqueous solution were made in groups of white Swiss mice (20 g) following ip and iv administrations. The oral LD50 determination was made in Upjohn SpragueDawley (TUC/SD) rats (175 g) after a 24-hr period of starvation. Observations were made on surviving animals for 7 days. The Spearman-Karber method (Finney, 1952) was employed to determine the LD50. Intramuscular irritation in the rabbit. Single injections of 50, 100, 200 and 300 mg base equivalence in 1 ml of isotonic saline were made in the lumbar muscles of groups of New Zealand White rabbits. After 3 and 7 days, the rabbits were sacrificed and the lumbar muscles were fixed in situ by perfusion with 10 % formaldehyde. The injection sites, which were identified by code, were graded according to the degree of hemorrhage, degeneration, and necrosis present at the respective postinjection intervals. Serum creatine phosphokinase (CPK) determinations were made in a group of 10 2 Cleocin@ is the registered trademark of The Upjohn Company for clindamycin. outside of the U.S.A. are Dalacin C and Sobelin. 11

Other trademarks

310

GRAY ET AL.

rabbits prior to injection and 24 hr after injection of 50 mg of the phosphate ester in sterile water. Comparable groups of animals were injected with 1 ml of the parent antibiotic (50 mg/ml), sterile water, and isotonic saline. A modification (Dade3) of the procedure of Nuttal and Wedin (1966) was used. Rabbits with CPK values higher than 100 IU/liter on 2 occasions were rejected prior to injection. Subcutaneous tolerance studies in the rat. In the first study, 10 male Upjohn SpragueDawley rats (125 g) were treated with 120 mg/kg for 6 days. In the second study, daily regimens of 30, 60 and 90 mg/kg were injected in groups of 5 male and 5 female rats (115 g) for 1 month. In both studies a sterile aqueous formulation of 300 mg base equivalence per ml was injected into the intrascapular subcutaneous tissue. Similar groups were injected with sterile isotonic saline or aqueous vehicle. Clinical observations were made daily, and individual body weight and food consumption were determined either terminally (6-day study) or weekly (30-day study). Hemograms which included the hematocrit, hemoglobin, total leucocyte count and differential leucocyte count were obtained on the day following the last treatment. Complete necropsy and histopathologic examinations were performed on 2 males and 2 females from each group. Weights of the liver, kidney, adrenals, testes and heart were recorded for all rats. Intramuscular tolerance studies in the dog. The same formulation was injected into the posterior thigh muscles of groups of 3 beagle dogs for comparable periods of time. In the 6-day study a daily dose of 60 mg/kg was obtained with two l-ml injections. Dose levels of 30,60 and 90 mg/kg for 32 days were also achieved by dividing the daily dose in bilateral injection sites in the thigh and forelimb muscles. Control groups of 3 dogs received 2 injections of 1.5 ml of aqueous vehicle daily. Hemograms and urinalyses were performed prior to treatment and at the termination of the study. Determinations of blood glucose, blood urea nitrogen, prothrombin time, total protein, bromosulfophthalein retention, serum transaminases (SGPT and SGOT) and alkaline phosphatase were also performed before and after treatment. A necropsy was performed in all dogs and histopathologic examinations of hematoxylin and eosin stained sections of a complete series of organs were performed. Intravenous tolerance study in the dog. Two groups of 4 dogs each were injected for 1 month (6 days a week) with an aqueous formulation of 15 mg/ml. The respective doses were 60 and 120 mg/kg; dogs of the 120-mg/kg group received about 75 ml each day in divided doses. Injections were made slowly (10 min) in the cephalic veins of dogs which were preconditioned to a suspended harness. A third group of 4 dogs received a volume of vehicle daily comparable to that given the higher dose group. Hemograms, chemical determinations of blood constituents, and urinalyses were obtained similarly as was done during the im tolerance study. Plasma hemoglobin was determined by a modification of the method of Chiamori et al. (1961). The plasma was scanned in a recording spectrophotometer, and the absorbance at 415 nm was compared with that of a known hemoglobin standard prepared by the method of Hawks et al. (1960). Blood samples were obtained at 3 hr after the am-dose. Collections were made into Peel-away syringes4 containing the anticoagulant Sequestersol through 20-gauge needles. The samples were centrifuged for 10 min at 2000 rpm. The plasma was removed with dispo-pipettes and transferred to glass tubes and centrifuged again 3 Dade Reagents, Inc., Miami, Florida 33152. 4 Peel-away Scientific, Box 4265, Long Beach, California.

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for 10 min at 2000 rpm. A necropsy was performed on all animals, and a complete histopathologic examination (Gray et al., 1972) was made of each dog. For determination of in vitro hemolysis, clindamycin 2-phosphate was added to blood samples containing sequestersol from 10 untreated dogs. The antibiotic concentration was 100 pg/ml which approximated the bioassay value of blood taken 15 min after an iv injection of 60 mg/kg. After I hr of exposure (37.5”C), plasma hemoglobin was determined by the method described above. Samples from each dog to which no antibiotic was added were used for manipulative control. Wet preparations of incubated samples were also examined for Heinz body formation. For determination of erythrocyte osmotic fragility, the method of Dacie (Davidson and Wells, 1965) was used. Statistical interpretation of the variability in plasma hemoglobin samples was made by analysis of variance on individual values and on the log transforms. Effect on the ultrastructure qf the hepatocyte. Groups of 3 purebred beagle dogs were injected im and iv respectively, with clindamycin 2-phosphate for 1 week. These dogs were mature animals of both sexes, l-l .5 years of age; the average weight was 11 kg. Intramuscular injections of 90 mg/kg were made with the same formulation used in human clinical studies. The concentration was 150 mg base equivalence per ml; each ml also contained 9.45 mg of benzyl alcohol NF. Volumes of I .3 to 1.9 ml, depending on the weight of the individual dog, were injected in 4 muscle sites (shoulders and thighs) once daily. The same formulation, diluted to 30 mg/ml with sterile aqueous vehicle was injected iv. Injections of 120 mg/kg were made on alternate days in the right and left cephalic veins. A volume of approximately 4 ml/kg was injected over a IO-min period. Samples of the liver were obtained with a Silverman biopsy needle. Prior to puncture, the abdominal wall over the left central lobe of the liver was anesthetized with 2% lidocaine hydrochloride. Control samples were obtained at 3 intervals from each of the 6 dogs before treatment was started. During the course of the studies, samples were collected at 3 and 6 hr after the first injection, on days I, 2,3 and 7 during the treatment period and on day 7 after treatment was completed. At the same intervals that biopsies were obtained, blood samples were collected for determination of serum activities of glutamic-oxaloacetic (SGOT) and glutamicpyruvic (SGPT) transaminases. SGOT determinations (milliunits/ml) were made on the SMA 12/60,5 SGPT activity was expressed in Reitman-Frankel (RF) units using Enza-tro13 as an enzyme control sample. Samples of the liver were fixed in 2 % glutaraldehyde at 0°C for I .5 hr. After holding overnight or longer in several changes of 0.1 M phosphate buffer, they were subjected to Millonig’s fixative for 1.5 hr. Graded alcohols were used for dehydration, and Epon 812 was used for embedding. Thin sections were stained by the method of Reynolds for lead citrate and also with uranium acetate (0.2 %). Selected areas were photographed in an RCA EMU-3H electron microscope. RESULTS LD.50 Determinations

In comparison with clindamycin hydrochloride (Gray et al., 1972), the LD50 values ranged more closely together. The ip and iv LD50 values in the mouse were approxi5 Sequential Multiple

Analyzer, Technicon Corp., Tarrytown,

New York.

312

GRAY

ET AL.

mately 3 times higher than those of the hydrochloride salt of clindamycin whereas the oral value was slightly lower (Table 1). The most consistent observations were depression and convuIsions preceding death. The time of death in mice after iv administration was from 30 to 90 min and after ip administration varied from a few minutes to 1 day. Rats treated with an oral lethal dose also became depressed and died within 24 hr. TABLE

1

LD50 DETERMINATIONS

OF CLINDAMYCIN IN SEVERAL SPECIES

Route of administration Intraperitoneal Intravenous Oral

Species Mouse Mouse Rat

~-PHOSPHATE

LD50 value bdkg) 1145 (10224282) 855 (806.-908) (1832 1500-2236)

Intramuscular Irritation in the Rabbit The lesion caused by an injection of 50 or 100 mg of the phosphate ester in 1 ml of sterile water was graded as slight in terms of its 3-dimensional size (mm) and gross appearance. Lesions at the site of injections of 200 and 300 mg/ml were somewhat larger and still present 7 days after injection. The degree of irritation revealed by these morphometric evaluations was shown to be lesser for the phosphate ester than that caused by comparable injections of the parent antibiotic. Averaged values of serum CPK activity, 24 hr after injection, from groups of 10 rabbits substantiated these differences and gave a more precise basis for evaluating the degree of musculoirritancy. The CPK value for the phosphate ester was less than one-half (avg. 1500 III/liter) that of the hydrochloride salt (4100 IU/liter). CPK values in the animals from groups injected with 1 ml of sterile water and isotonic saline 537 III/liter and 223 III/liter, respectively. Subcutaneous Tolerance Studies in the Rat In the 6-day study in which a group of 10 rats was injected with 120 mg/kg daily, the body weight gains and food conversion ratios were regarded as comparable to the control group. Evidence of multiple epidermal breakdown with scab formation over the injection site was present in most of the treated rats. The underlying subcutaneous tissue and brown fat were moderately inflamed. Tissue damage appeared to be rapidly repaired. No systemic evidence of drug effect was detected during necropsy. Weights of the liver, adrenals, gonads, and heart in the 2 groups of rats were similar. Likewise, no significant deviations from the control values were present among the hematologic data of the treated group. Repeated injections of 30 mg/kg for 30 days in the interscapular site produced low grade inflammatory changes. Injections of 60 and 90 mg/kg caused more extensive inflammatory changes and were more frequently accompanied by focal necrosis of the subcutaneous tissues and overlying epidermis. No drug-related systemic effects as evidenced by behavioral, functional or morphologic change were observed.

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Intramuscular Tolerance Studies in the Dog Upon first injection in the 6-day study, each dog showed signs of pain for several minutes. This response generally lessened upon subsequent injections. After 5 days, slight induration was palpable in the posterior thigh muscles. The dogs remained healthy and body weights were steady. Serum transaminase values were slightly elevated terminally. Concentrations of SGOT in the 3 dogs injected with 600 mg/kg increased before SGPTelevations occurred. No other treatment-related changes were seen in the terminal hemograms, blood chemistry values, and urinalyses. Gross pathological changes were confined to the injection sites. The subcutaneous connective tissue showed slight hemorrhage and edema. Scattered bundles of fibers, in the biceps femoris chiefly, were lighter in color and slightly indurated. Degenerative changes were partially obscured by mild hemorrhages, but no tracts of necrosis were detected. The popliteal lymph nodes showed cortical discoloration and slight swelling. Upon the administration of a large number (22-33) of im injections in the posterior thigh muscles of 3 groups of dogs in doses of 30,60 and 90 mg/kg, clindamycin 2-phosphate was found to be mildly to moderately irritating. For comparison, it should be noted that the formulated concentration (300 mg/ml) was 3 times greater, the total daily injected volume was as much as 50 % larger, and the dosing duration in the respective groups was more than 4 times longer than that used in earlier unpublished studies with the parent antibiotic. The characteristic pathologic change was a progressive (as long as injections were continued) scarring of the muscle bundles. The degree ofthisdamage was dose-related. Microscopic examination revealed irregular areas of loss of muscle fibers with fibroblastic replacement. The outlines of the muscle bundles were frequently preserved by the more resistant perimysial connective tissues. Injections made in the smaller mass of tricep muscles of the forelimb in dogs of the 60- and 90-mg/kg groups produced a more severe response than in the thigh muscle. This usually resulted in bands of scarring. Hemorrhage in the muscle was more frequently observed. A small hematoma was found in 1 dog of the 90-mg/kg group. The axillary lymph nodes were swollen and discolored, usually more so than the popliteal nodes. Thigh muscles of the control dogs which received 1.5 ml of the sterile aqueous vehicle in each thigh daily showed mild hemorrhage along the needle tracts, but no obvious scar formation was present. Microscopically, the sections were essentially normal. Terminally, elevations (54-400 RF units) of SCOT were found in all dogs of the treated groups. Slight increases (So-100 RF units) of SGPT values also occurred. Other blood constituents and liver function values were in the normal range. Four treated dogs may have had a slight (5 %) weight loss. Terminal hematologic data and urinalyses were regarded as normal. A slight dose-related increase in liver weight was indicated on the basis of percent of body weight, but no morphologic evidence of drug effect in the liver was obtained. There were relatively few spontaneous pathologic changes in this group of 12 dogs. Lymphocytic infiltrations were present in the thyroid glands of 3 dogs (control, 30- and 90-mg/kg groups). Both glands of a fourth dog (60 mg/kg) had the appearance of hyperplasia of embryonal type of thyroid epithelium.

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GRAY ET AL.

Intravenous Tolerance Study in the Dog No drug-related effects were observed in any of the 8 dogs during or following the twice daily iv administrations of 60 or 120 mg/kg. The safe conduct of the study was further substantiated by the lack of any significant deviations among the hemograms, blood chemistry values and urinalyses or among the pathologic findings.

FIG. 1. Intramuscular administration of 90 After 6 hr both smooth and rough endoplasmic distorted. The SER shows vesiculation, and These findings were not observed at subsequent

mg/kg of clindamycin 2-phosphate in the beagle dog. reticulum of this hepatocyte from dog A70-403 appear the RER shows a tendency to become sequestered. intervals during the l-week study. ~22,900.

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In spite of considerable variation in the degree of spontaneous hemolysis among 118 pretest and test samples, no difference or trend could be recognized upon statistical analyses of 39 plasma hemoglobin values from treated dogs compared to 79 values from

FIG. 2. Intravenous administration of 120 mg/kg of clindamycin 2-phosphate in the beagle dog. Hepatocytes of dog B70-306 contain widely dispersed droplets of neutral lipids 1 day after injection. All dogs of this group showed this mild change the first 3 days of the l-week study. x4900.

control dogs or from pretreatment values of the treated dogs. The injected cephalic veins remained essentially free of inflammation in all 12 dogs. A few minor needleinduced defects were found in histologic sections of the veins.

316

GRAY ET AL.

Results of an in vitro hemolysis study indicated no difference in treated samples cbver Lll ntreated samples. The in vitro values were higher (27.1 mgj100 ml for treated, 2IO.0 m [g/100 ml for untreated) than the 7.7 mg/lOO ml median value of pretest samp des.

FIG. 3. Intravenous administration of 120 mg/kg of clindamycin 2-phosphate in the beagle dog (‘4.70-402). Fully developed myeloid figures were seen occasionally in hepatocytes the first few (jays

of the study. Usually they were more irregular and smaller. x22.900.

0 ther in vitro studies revealed that no Heinz body formation Oi ?erythrocytes occurred in treated blood samples.

or increased fragi lity

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of the Dog Hepatocyte

No consistent or characterizable changes were observed in the hepatocytes of dogs treated im with 90 mg/kg of clindamycin 2-phosphate for 1 week. At the 6-hr interval after the first injection, the rough endoplasmic reticulum (RER) of some hepatocytes in samples from 2 dogs appeared to be dilated. There was also a tendency for fragments of RER to be sequestered as illustrated in Fig. 1. Slight vesiculation of the smooth endoplasmic reticulum (SER) was also shown in these samples. These findings were not observed at subsequent intervals in dogs of this group. mo450 400 350a 300 2G 250::

zoo150 IO0 50.

HOURS DURATION

OAYS

POST TEST

FIG. 4. Serum activities of transaminases in beagle dogs prior to, during l-week im administration and 7 days posttreatment of clindamycin 2-phosphate (90 mg/kg im). o----o Dog A70-403 ; o---o Dog A70-502; a-----n Dog A70-401.

Slight treatment-related changes were observed in a portion of the hepatocytes in samples from all 3 dogs after the first iv injection from 3 hr until day 3 of the study. A mild increase in neutral lipid droplets was most consistently found in samples taken 24 hr after the first injection (Fig. 2). The droplets were dispersed widely in the hepatocyte; in one instance, a few droplets were seen in a nucleus. The second treatment related change was the occasional formation of a myeloid figure which resembled that produced by clindamycin hydrochloride when administered orally at 600 mg/kg (Gray et al., 1971). Rarely, these were well developed, as shown in Fig. 3; more often, they were smaller and less regular. Both of these changes were shown to be transient. Samples taken after 7 days of treatment were essentially free of both changes. Deviations

318

GRAY

ET AL.

in the endoplasmic reticulum, such as dilated RER and vesiculated SER, were not consistent, but these changes were slightly more evident than those observed in the im group of dogs. Any apparent increase of SER did not suggest a role of enzyme inducer for clindamycin 2-phosphate. There was a suggestion that secondary lysosomes were more prominent in samples after treatment was initiated, but this impression could not be substantiated. Increases in serum activities of both SGOT and SGPT occurred within 3 hr after the first injection. In general, elevations of both enzymes remained slight to moderate as long as the injections were made (Figs. 4 and 5). SGOT activity was slightly higher in

SCPT

0

1” PRETEST

” 3

6

- Serum glutamic mnsaminare 1

HOURS

2

pyruvic

ji 3 OAYS

OUAATION

+-77 POST TEST

FIG. 5. Serumactivitiesof transaminasesin beagledogsprior to, during 1-weekin administration and 7 days post-treatment of clindamycin 2-phosphate (120 mg/kg iv). o----o Dog A70-402; 0-0 Dog

B70-306;

n----n

Dog

B70-102.

dogs of the im group (Fig. 4), and SGPT activity tended to be slightly higher, except at the 3-hr interval, in dogs of the iv group (Fig. 5). The data suggested that SGOT activity was highest in both groups in a few hours after injection and then decreased u.ntil the next injection was made on the following day. The SGOT values on successive days, i.e., on days 1,2,3 and 7, tended to be similar and probably these values represent the “lows” on the daily curve of activity for the individual dogs. (Samples were obtained just prior to next injection.) A daily peak in SGPT activity was not obvious from the data. It should also be noted that SGOT activity returned more uniformly to the normal range (by 7 days after the last injections by both routes of administration) than SGPT activity.

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DISCUSSION

Parenteral toxicity studies in laboratory animals require an exaggeration, both in dose and duration, of im and iv treatment regimens. A frequent limiting factor in the performance of such studies, especially when the duration is from 2 to 4 weeks in length, is local site intolerance rather than systemic injury. The fact that the muscle masses of the rabbit, dog, and monkey, for example, are smaller than in man imposes a compromise on the number of injections of even a mildly irritating drug that a muscle site will tolerate well. L.ocal tolerance during a series of iv injections also has a similar inherent limitation. As a general rule, the greater the number of injections in a periphera1 vein the more likely the vessel will sustain an impairment. When injections are superimposed in time, the injury to a muscle site or peripheral vein may, in effect, be compounded, and an evaluation in terms of potential tolerance of a drug in man is rendered more difficult. Standardized injections in the back muscles of a rabbit offers the best opportunity to study the inherent musculoirritant property of a new drug. In addition to morphologic delineation of the site of a single injection by gross and microscopic observation, the effect of the injection can be translated numerically in terms of creatine phosphokinase units in the blood on a rather precise basis. The results in groups of 10 rabbits have been quite uniform. The 24-hr postinjection interval was selected for determination of the serum CPK activity because, in our experience, it was the closest practical interval to the maximum blood activity. It is important to handle the rabbits assigned for CPK determination carefully, and it is generally best not to put them on test too soon after they have been transported or housed in new quarters. Otherwise, some difficulty may be experienced in obtaining values uniformly within the normal range, which is 62 f 19 IU/liter in our experience. Parallel studies have shown that excellent correlations may be obtained between the extent of muscle injury and elevation of serum CPK content. Clindamycin Zphosphate, given in a single dose of 50 mg in 1 ml, produced a slight lesion in the back muscles of the rabbit whereas clindamycin hydrochloride produced a moderate to marked lesion. The respective serum CPK activities were 1500 and 4100 II-j/liter. It is interesting in this respect that control injections of sterile water and isotonic saline raised the serum CPK values to about 500 and 200 IU/liter, respectively. From the data we have obtained with a number of parenterally administered antibiotics in rabbit im tolerance studies, the zone of questionable im human tolerance would appear to be between 2000 and 3000 IU/liter ofCPKin the serum. Values higher than 3000 IU/liter would likely be too irritating, and values lower than 2000 probably would be clinically tolerable. Spontaneous hemolysis in samples collected from dogs occurs rather commonly. At times, when the degree of hemolysis is detectable visually, it may interfere with the determination of certain biochemical values such as that of the bromosulfophthalein retention test for hepatic function. Hemolysis in samples on which drug-induced hemolysis will be determined by the plasma hemoglobin content is obviously undesirable. Yet quantitation of hemolysis cannot be absolute as a small amount of free hemoglobin is present in the blood at all times as a result of the normal turnover of erythrocytes. Hemolysis becomes grossly detectable at about 10 mg/lOO ml. Of 118 plasma hemoglobin values, two-thirds were obtained prior to the start of the test and

320

GRAY

ET AL.

from the 4 control dogs during the study. In nearly one-third (25/79) of these samples the value was >I0 mg/lOO ml. Excessive hemolysis apparently is induced more readily by the collection technique in the samples of some dogs than of others. The values obtained at different times from the same dogs also vary widely. A comparison of median pretest and test values (5 of each) of the 8 treated dogs reveal that the values fall in 4 categories as follows :

Group Treated (8) Control (4)

Remain

Remain

Decrease to

Increase to

~10 mg/lOO ml

>lO mg/lOO ml


>lO mg/lOO ml

4 3

1

2 1

1

It is apparent from the data that no evidence of drug-induced hemolysis is reflected in values of the treated dogs when compared to the pretest and control dog values. There is some indication that with repetitive sampling the chance of obtaining a grossly detectable hemolyzed sample was slightly lessened, but certainly spontaneous hemolysis was not eliminated by the collection method employed. Analysis of variance on the individual plasma hemoglobin values and also on the log transforms confirmed the high variability occurring from dog to dog on the same day-treatment and the high variability from day to day for the same dog-treatment, and did not reveal any statistically significant effects associated with treatment. The experimental design of the ultrastructure study of the hepatocyte of the dog was arranged to imitate clinical usages of clindamycin 2-phosphate. However, both the im and iv regimens were several times greater than those employed therapeutically in man. The results provide guidance as to the limits of tolerance. The observations also have prognostic value in terms of the onset, nature and outcome of drug-related effects in the hepatocyte should such occur under extenuating circumstances during therapy in man. What has been observed morphologically can be regarded as a “threshold of effect,” i.e., an expression of minimal change. The results further suggest that the effect on the ultrastructure of the liver cell is dependent on the concentration of clindamycin exceeding a critical level in the blood (or hepatocyte), even for a very brief period. Under the conditions of these studies, the concentration of clindamycin when clindamycin 2-phosphate was given iv was for a brief period higher than when it was injected im. The first iv injection appeared to produce most of the mild ultrastructural effects in the hepatocyte. Subsequent daily injections appeared to add little to the morphologic changes. Indeed, the evidence indicates that the later injections were metabolized without provoking any morphologic effect. Obviously the hepatocyte improved its capacity to handle clindamycin within 1 or 2 days after treatment was started.

ACKNOWLEDGMENT The technical assistanceof Mildred Prestrud, Andrejs Purmalis, John Mathews and Robert Simonds is much appreciated. We are also grateful to Jack Northam for statistical analysesof the plasma hemoglobin data of the intravenous tolerance study in the dog.

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REFERENCES H., HENRY, R. J. AND GOLUB, 0. J. (1961). Studies on the determination of bile pigments. II. Spectrophotometric determination of bilirubin and hemoglobin in serum. Clin. Chem. Acta 6, 1-15. DAVIDSON, I. AND WELLS, B. B. (1965). Todd-Sanford Clinical Diagnosis by Laboratory Methods, 13th ed., pp. 97-98, Saunders, Philadelphia, Pennsylvania. FINNEY, D. J. (1952). Statistical Methodof Biological Assay, p. 524. Hafner, New York. GRAY, J. E., PURMALIS, A., PURMALIS, B. AND MATHEWS, J. (1971). Ultrastructural studies of the hepatic changes brought about by clindamycin and erythromycin in animals. Toxicol. Appl. Pharmacol. 19, 217-233. GRAY, J. E., WEAVER, R. N., BOLLERT, J. A. AND FEENSTRA, E. S. (1972). The oral toxicity of clindamycin in laboratory animals. Toxicol. Appl. Pharmacol. 21, 516-531. HAWKS, G. E., CASSELL, M., RAY, R. N. AND CHAPLIN, H. (1960). A further modification of the benzidine method for measurement of hemoglobin in plasma. J. Lab. Clin. Med. 56,486-498. MOROZOWICH, W., LAMB, D. J., DEHAAN, R. M. AND GRAY, J. E. (1970) Clindamycin-2phosphate, an injectable derivative of clindamycin with improved muscle tolerance. J. Pharm. Sci., p. 63, Abstr. APHA Acad. Pharm. Sci., 117th Meeting of Amer. Pharm. Ass., April 12-17, Washington, D.C. NUTTAL, F. Q. AND WEDIN, D. S. (1966). A simple rapid calorimetric method for determination of creatine kinase activity. J. Lab. Clin. Med. 68,324332. CHIAMORI,