ANTIMICROBIAL Aoeurs AND CHEMOTHERAPY, Feb. 1983, p. 284-288
`0066-4804/83/020284—05302.Wo
`Copyright 0 1983, American Society for Microbiology
`
`Vol. 23, No. 2
`
`Norfloxacin Disposition After Sequentially Increasing Oral
`Doses
`
`BRIAN N. SWANSON,‘ VENKATA K. BOPPANA. PETER H. VLASSES, HESCHI H. ROTMENSCH,
`AND ROGER K. FERGUSON
`
`Division of Clinical Pharmacology, Jefi'erson Medical College of Thomas Jeferson University, Philadelphia,
`Pennsylvania 19107
`
`Received 11 August 1982/Accepted 9 December 1982
`
`Single doses of norfloxacin (200, 400, 800, 1,200, and 1,600 mg) or placebo were
`administered orally at weekly intervals to 14 healthy male volunteers in a double-
`blind study. Norfloxacin was measured in serum and urine by high-pressure liquid
`chromatography with UV detection. The concentrations of this drug in serum
`peaked 1 to 2 h after each dose; the mean peak values for increasing doses were
`0.75, 1.58, 2.41, 3.15, and 3.87 pig/ml. Mean area under the serum concentration-
`time curves for the first 12 h after each dose were 3.56, 6.26, 11.4, 16.1, and 19.7
`pg - h/ml, respectively. The elimination half-life of norfloxacin was about 7 h and
`was similar for all doses. The concentrations of the drug in urine also peaked 1 to 2
`h after dosage; mean peak values for increasing doses were 200, 478, 697, 992, and
`1,045 pig/ml. Renal clearances approximated 285 ml/min. About 30% of each dose
`was excreted into urine as unmetabolized norfloxacin. Crystals of the drug were
`occasionally observed during microscopic examination of freshly voided urine
`collected after the 1,200 and 1 ,600-mg doses. Crystalluria was not encountered at
`lower doses.
`
`Norfloxacin (MK-0366) is a new quinoline
`carboxylic acid which exhibits high antimicrobi-
`al activity in vitro against a wide variety of gram-
`negative and gram-positive bacteria,
`including
`gentamicin-resistant Pseudomonas aeruginosa
`and B-lactamase-positive Neisseria gonorrhoeae
`(2, 3, 5—7). In addition, this drug is severalfold
`more potent than nalidixic acid and pipemidic
`acid in the treatment of systemic and urinary
`tract infections in experimental animals (4). Ini-
`tial clinical
`trials indicate that norfloxacin is
`elfective in chemotherapy of urinary cystitis,
`pyelonephritis, and urethritis (Y. Nishimura, H.
`Kishi, O. Tsukada, T. Tominaga, and T. Nii-
`jima, Program Abstr. Intersci. Conf. Antimi-
`crob. Agents Chemother. 20th, New Orleans,
`La., abstr. no. 76, 1980). The responsiveness of
`infections outside the urinary tract is not well
`characterized to date.
`
`Although norfloxacin has been nontoxic in
`most laboratory animals, high doses of this drug
`(2150 mg/kg) caused severe nephrotoxicity and
`crystalluria in dogs (Merck Sharp & Dohme
`Research Laboratories, personal communica-
`tion). Rational selection of a dose regimen in
`humans will require detailed information on the
`oral dosage necessary for maintaining bacteri-
`cidal drug concentrations in urine while mini-
`mizing the risk of in vivo crystalluria. To this
`end, we have measured norfloxacin in both urine
`
`and serum over a wide range of acute oral doses
`and have assessed the potential risk of drug-
`induced crystalluria in humans.
`
`MATERIALS AND METHODS
`
`Volunteers. Fourteen male volunteers (aged 24 t 2
`years) participated in the study. All weighed within
`10% of their ideal body weight (76 t 8 kg) for their
`ages and heights and were judged to be in good health
`before the study on the basis of medical history,
`physical examination, and laboratory evaluation. They
`were instructed not to use other medications from 7
`days before initiation of the study until completion of
`all treatment periods.
`Study design. In a double-blind manner, volunteers
`were randomly assigned to receive each of seven oral
`treatments, including sequentially increasing doses of
`norfloxacin (200, 400, 800, 1,200, and 1,600 mg) and
`two randomly interspersed placebos. Volunteers re-
`ceived their medication (four capsules) with 250 ml of
`water in the morning after an overnight fast. They
`resumed a normal diet 3 h after drug administration.
`Blood from a forearm vein and urine were collected
`periodically for up to 48 h. During the first 6 h after
`each treatment, 10 ml of each freshly voided urine
`specimen was centrifuged at 2,500 x g for 10 min, and
`the pellet was examined microscopically for the pres-
`ence of crystals. A period of 6 days separated each
`treatment.
`
`Analytical methods. The concentration of norfloxa-
`cin in urine and serum was measured according to
`published methods (1). Briefly, specimens were
`
`284
`
`ALCON 2099
`Apotex Inc. v. Alcon Pharmaceuticals, Ltd.
`Case |PR2013—00012
`
`

`

`VOL. 23, 1983
`
`NORFLOXACIN DISPOSITION
`
`285
`
`adjusted to pH 7.5 with buffer and extracted with
`methylene chloride. The drug was concentrated by
`back-extracting into a small volume of 0.3 N NaOl-l
`and then was quantified by high-pressure liquid chro-
`matography with UV detection at 273 nm. This meth-
`od separates norfloxacin from its metabolites and
`provides reliable quantitation (i.e., less than 5% varia-
`tion in replicates) down to 0.1 aglml in serum and 1
`pig/ml
`in urine. Urine specimens were mixed well
`before assay to assure uniform sampling.
`Area under the serum concentration-time curves
`(AUCs) were determined by the trapezoidal rule.
`Serum elimination half-lives were calculated from the
`slopes of the log concentration versus time plots from
`6 to 12 h after dosage, where the slope = —k. and W: =
`0.693/k.. Urinary elimination half-lives were calculat-
`ed from the slopes of the log excretion rate (milligrams
`per hour) versus time plots from 8 to 48 h after dosage;
`the midpoint of each collection interval was used for
`computations. Renal clearances were calculated by
`dividing the amount of urinary norfloxacin (in micro-
`grams) by the serum AUC (micrograms - hour per
`milliliter).
`To assess drug solubility in urine. norfloxacin was
`added in excess to urine samples that were previously
`adjusted to pH values between 4 and 10 with small
`quantities of 6 N HCl or 6 N NaOH. Urine was
`incubated at 37°C for 4 h with occasional interim
`adjustments in pH to correct for bufl'ering effects of the
`drug. Drug-saturated urine samples were centrifuged
`at 37°C in an Eppendorf model 5412 high-speed centri-
`fuge for 5 min, and the supernatant was assayed for
`
`norlloxacin by high-pressure liquid chromatography.
`Some samples were cooled to 25°C after the 37°C
`incubation and were centrifuged at room temperature
`to simulate the temperature conditions that existed
`during microscopic examinations of clinical speci-
`mens.
`
`Statistical comparisons between means were ac-
`complished with two-way analysis of variance and
`Duncan's multiple range test. Variation in data is
`expressed as : standard deviation unless otherwise
`stated.
`
`RESULTS
`
`Mean senrm concentrations of norfloxacin
`after various oral doses are shown in Fig. 1.
`Mean peak concentration (Cam) occurred be-
`tween 1 and 2 h after drug administration, with a
`progressive delay in the peak as the dose in-
`creased (Table 1). The mean AUG (0 to 12 h) and
`Cm did not increase linearly with increasing
`dose. For example, the AUG/dose and Cm/
`dose ratios were significantly greater (P < 0.01)
`for the 200- and 400-mg doses than for the 1,600-
`mg dose. The serum elimination half-life of
`norfloxacin, based on 6— to 12-h data, was ap-
`proximately 7 h and was similar for all doses.
`The elimination half-life as detemiined from
`renal excretion rates during the 8- to 48-h period
`was also about the same for all doses. Renal
`
`4.0
`
`3.0
`
`P o
`
`d O
`
`.5
`
`P O
`
`O (a
`
`.° to
`
`NortloxaclnInSerum(up/ml)
`
`O
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`Hours
`
`FIG. 1. Mean concentrations of norfloxacin in serum. Norfloxacin was administered orally at weekly
`intervals as single doses: 200 mg (Cl), 400 mg (A), 800 mg (A), 1,200 mg (0), and 1,600 mg (0). Bars indicate the
`standard error of the mean.
`
`

`

`286
`
`SWANSON ET AL.
`
`Annmcnoa. Aoems Cucuoruran.
`
`
`TABLE 1. Pharnracokinetic parameters (mean 1': SD) for norlloxacm‘ in 14 healthy men
`.
`Renal
`Urinary
`5%“ are W...
`as”
`9'"
`(ml/min),
`
`Oral
`Serum
`.... W Sm"
`(me)
`(u - hrml)‘
`
`200
`400
`
`800
`1,200
`1,600
`
`3.56 .+. 1.13
`6.26 z 2.05
`
`11.4 t 2.12,
`16.1 1 4.00
`19.7 t 6.07
`
`0.75 .+. 0.20
`1.58 a 0.60
`
`2.41 t 0.43
`3.15 t 0.76
`3.87 t 1.27
`
`1.1 t 0.4
`1.3 t 04
`
`1.5 t 0.4
`1.8 :t 0.8
`1.9 .t 0.6
`
`7.3 t 2.9
`7.4 1. 2.5
`
`6.2 I 1.8
`5.7 .+.- 1.2
`6.8 :t 1.4
`
`7.1 t 1.2
`8.0 t 2.4
`
`7.9 t 1.6
`7.1 :t: 1.1
`6.7 t 1.2
`
`272 t 96
`292 .+. 76
`
`273 x 37
`288 t 56
`298 I 40
`
`‘ AUC"'12 ", Area under the curve from 0 to 12 h after drug administration.
`" Cm, Peak concentration.
`‘tn“, Time of peak concentration.
`‘tm 6—12 h, Half-life with 6- to 12-h data.
`‘,tms—48h Half-lifewithS-to48-hdata.
`’ Renal clearance= drug excreted/serum AUC (0- to 12-h data).
`
`clearances were large (about 285 ml/min), and
`urinary drug concentrations were consequently
`100- to 300-fold greater than concurrent concen-
`trations in serum (Table 2). Approximately 30%
`of each dose was excreted into urine as unme-
`tabolizcd norfloxacin in the first 48 h after drug
`administration (Fig. 2).
`The urinary solubility of norfloxacin was
`found to be dependent on both pH and tempera-
`ture (Fig. 3). The drug was least soluble at pH
`7.5, exhibiting-a maximal solubility of about 450
`rig/ml at 25°C and 1,200 rig/ml at 37°C. At 37°C,
`the drug was freely soluble (concentration >40
`mg/ml) at any pH value less than or equal to 5.5.
`Drug crystals were observed in only 5 of over
`300 clinical specimens. All five specimens had
`been collected after 1,200- or 1,600-mg doses
`and exhibited pH values between 7.0 and 7.8 and
`drug concentrations between 1,200 and 2,300
`rig/ml. Drug crystals in urine were spherical with
`ragged edges and orange and green highlights.
`This was in marked contrast to crystals precip-
`itated from water, which were colorless needles.
`Routine laboratory tests for proteinuria, blood
`urea nitrogen, and serum creatinine indicated
`that acute doses of norfloxacin were not nephro-
`toxic. For example, the values for blood urea
`nitrogen and serum creatinine were 13.7 i 3.9
`and 1.10 I 0.12 mg per 100 ml, respectively,
`before the first dose and were 14.8 t 3.6 and
`1.06 i 0.09 mg per 100 ml 3 days after the last
`dose.
`
`DISCUSSION
`
`This study has defined several important fea-
`tures of norfloxacin disposition after oral admin-
`istration. Maximal concentrations in serum and
`
`urine were rapidly achieved, typically within 1
`to 2 h. Assuming'that norfloxacin is 14% bound
`to serum proteins (Merck Sharp & Dohme Re-
`
`search Laboratories, personal communication),
`the free renal clearance for this antibiotic was
`
`about 325 mein, a remarkable 2.5 times the
`normal glomerular filtration rate. Presumably,
`norfloxacin is actively secreted as an organic
`acid. However, this antibiotic is both an acid
`and a base and could also be secreted by other
`mechanisms. The amphoteric nature of the drug
`is reflected in its urine solubility curve (Fig. 3),
`which shows a trough in solubility at pH 7.5 and
`greatly enhanced solubility under both more
`acidic and more basic conditions.
`
`the serum CM, serum
`With rising doses,
`AUC from 0 to 12 h, and the 0- to 12—h urinary
`recovery of unmetabolizcd drug all became pro-
`gressively lower relative to the amount of drug
`administered. This suggested that the fraction of
`the dose absorbed over the first 12-h period was
`lower at the higher doses. However, by 48 h, the
`mean percent recovery in urine was not substan-
`tially difi'erent for the various doses (range, 27.4
`to 31.0%), indicating that absorption may have
`been delayed at the higher doses. These dose-
`related changes in norfloxacin bio-availability
`may be of no clinical importance, as they oc-
`curred at excessively high doses. Although the
`apparent serum half-er of norfloxacin in our
`study was found to be approximately 7 h, accu-
`rate calculation of an elimination half-life in
`serum is not possible if drug absorption is sub-
`stantially delayed. However, approximately the
`same half-life can be derived from urinary excre-
`tion rates observed after drug absorption pro-
`cesses are complete.
`In our study, drug-related crystals were ob-
`served only at the highest (1 ,200 and 1,600 mg)
`doses of norfloxacin and only when urine pH
`values exceeded 7.0. Since norfioxacin appears
`to be clinically elfective at a total daily dose of
`less than 600 mg (20th ICAAC, abstr. no. 76),
`crystalluria will probably not pose a significant
`problem during chemotherapy with this agent at
`
`

`

`NORFLOXACIN DISPOSITION
`
`287
`
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`
`VOL. 23, 1983
`
`5 oo
`
`
`
`A
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`
`EV
`
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`lized norfloxacin after various single oral doses: 200
`m8(A).400ms(E|),800ms(A),1,200ms(0).and
`1,600 mg (0). Points and bars indicate means 1-
`standard errors of the mean.
`
`these lower doses. The urinary concentration
`data indicate that the 1,200- and 1,600-mg doses
`should not be required in the treatment of llI'i-
`nary tract infections, as the minimal inhibitory
`concentration of norfloxacin for most pathogens
`is <4 ug/ml (2—7); this concentration was ex-
`ceeded in all urine specimens collected between
`0 and 12 h after even the ZOO-mg dose.
`This study in normal volunteers has shown
`that norfloxacin rapidly reaches therapeutic con-
`centrations in urine after oral administration and
`
`that drug-induced crystalluria is unlikely to oc-
`
`our atusual therapeutic doses. Future studies on
`
`norfloxacin disposition should be undertaken to
`assess the extent of drug cumulation and the
`potential risk of crystalluria after chronic admin-
`istration.
`
`

`

`288
`
`SWANSON ET AL.
`
`Aunmcaos. AoaN'rs Cnauonraa.
`
`Funding for this study was provided by Merck Sharp a
`Dohme Research Laboratories.
`
`LITERATURECH'D
`
`1. In". V. K., ad I. N. Sw-aa. 19s2. Determination
`ofnorfloxacinmnewnalidixicacidanalogfiihumanserum
`and urine by high-performance liquid chromatography.
`Antimicrob. Agents Chemother. mans-810.
`2. m1" V.'l'. Ant-Isle, sultan-.1982. Invitro
`activity of Mil-0366 against clinical urinary pathogens
`including gentamicin-resistant Pseudomonar aeruglnosa.
`Antimicrob. AgentsChemother. 21:610-672.
`3. W,B.B.,D.L.ME.WM.IS.K.
`(“.1982.Comparisonoftheanlibacterialactivityof
`nortloxacinOlK-OJ“, AM715).aneworganicacid. with
`that of other orally absorbed chemotherapeutic agents.
`Infection 10:41-44.
`4. MK..A.lsa,Y.Ahe.S.s-Ie,1'. MMJH.
`.I s.m 1981. Comparative activities “AM-715
`andpipemidicandnalidixicacidsagainstexperimentally
`induced systemic and urinary tract infections. Antimicrob.
`Agents Chemother. 19:188-189.
`5. lb,A.,K.llm‘al,M.llans,ll.lqa,S.SlIe,T.lrI-a,
`ad 3. Mia-had. 19!). In vitroaritibacter'nl activity of
`AM-715. a new nalidixic acid analog. Antimicrob. Agents
`Chemother. 17:103-1m.
`6. “an. M.Y.,l.l'. G”, 8.“. N“ .dLE.
`Klein. 1982. Comparative in vitro activity of norfloxaein
`(MK-0366) and ten other oral ntimierobial agents against
`urinary bacterial isolates. Antimicrob. Agents Chemother.
`21:848-851.
`7. In, M.Y., Y. M, II LP. Or“. 1981.
`Comparativeinvitmaetivityot‘hfltMandotherselect—
`ed oral antimicrobial agents againstNeiueria gonorrhea.
`Antimicrob. Agents Chemother. ”dos-266.
`
`10
`
`9.0
`
`8.0
`
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`
`u 5
`
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`
`2.0
`
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`
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`
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`
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`
`4
`
`5
`
`8
`7
`8
`Urinary pH
`
`9
`
`10
`
`FIG. 3. Eifect of pH and temperature on urinary
`solubility of nodoxacin.
`
`ACKNOWLEDGMENTS
`
`WethankAnthanasioChremosandWilliamAbramsof
`Merck Sharpabohlnellesearch LaboratoriesMest Point,
`Pa.) for assisting in the successful execution of this study.
`
`