Dr. Reddy's Laboratories, Ud., et al.
`V.
`Galderma Laboratories, lnc.
`IPR2015-__
`Exhibit 1018
`
`Exh. 1018
`
`

`
`THE J OIJHNAL OF lNVESTIGATIVE DERMATOLOGY, 65:532-536, 1975
`Copyright© 1975 by The Williams & Wilkins Co.
`
`Vol. 65, No. 6 ·
`Printed in U.S.A,
`
`ANTI~INFLAMMATORY EFFE(JTS OF ANTIMICROBIAL AGENTS:
`AN .IN VIVO STUDY
`
`GERD PLEWIG, M.D., AND ERWIN SCHÖPF, M.D.
`Department of Dermatology, University of Munich, Munich, West Germany (GP), and Department o{Dermatology,
`University of Heidelberg, Heidelberg, West Germany (ES)
`
`Antimicrobials are used emperically in a variety of inflammatory dermatoses such as
`rosacea and dermatitis herpetiformis, although these diseases are not believed to be of
`bacterial etiology. Wehave used potassium iodide ointment to induce follicular pustules and
`have found in this in vivo model properties of antimicrobials not related to their antibacterial
`actions.
`Topical' demethylchlortetracycline and erythromycin (5%) Iead to suppression of KI-
`induced inflammation (erythema, pustules) and systemic use of these drugs as well as
`diaminodiphenylsulfone produces the same effect.
`
`While investigating the inflammatory response
`due to patch tests with potassium iodide (KI), an
`inhibitory effect of concomitantly administered
`antibiotics was noted [1 ]. It was decided to con-
`duct a larger series of in vivo experiments to clarify
`the role of antimicrobials in inhibiting intrafollicu-
`lar abscess formation.
`These experiments were designed to disclose
`properties of drugs not strictly related to their
`antimicrobial actions, a clinical observation known
`for many years in the treatment of inflammatory
`dermatoses such as dermatitis herpetiformis,
`acrodermatitis atrophieans Herxheimer, rosacea,
`acne, benign lymphocytic infiltration of the skin,
`pustular psoriasis [2 ], and follicular mucinosis [3 ].
`
`MATERIALSAND METHODS
`
`Exp.eriments
`Two types of experiments were performed:
`Topical therapy. The effects of topically applied drugs
`were investigated. All patch tests were standardized: on
`the upper back a 4 x 4 cm field was outlined by tape
`· (Leukosilk, 1 1/.1 cm, Beiersdorf, Germany). The area was
`covered with liberal amounts of 40% KI ointment (w/w in
`Unguentum Cordes, Ichthyol Gesellscpaft, Germany);
`KI = USP granular, Merck No. 4271, Rahway, N. J.,
`USA, or KI = Merck No. 5044, D~iJ.rmstadt, Germany). A
`
`Manuscript received May 13, 1975; in revised form
`July 23, 1975; ('lcct::pted for publication July 29, 1975.
`This work was supported in part by Grant No. P1
`58/3 from the Deutsche Forschungsgemeinschaft.
`This paper was presented in part at the 30th Meeting
`of the German Dermatological Society, Graz, Austria,
`September 10-14, 1974.
`Reprint requests to: Dr. G. Plewig, Department of
`Dermatology, University of Munich, Frauenlobstrasse 9,
`8 Munich 2, West Germany.
`,
`* Martin R, Warr G, Y eage.r H, Couch R, Knight V:
`Effects of tetracycline · on leukotaxis. Abstract, XII In-
`. terscience Conference Antimicrobial Agents Chemo-
`therapy, Chalfonte-Haddon Hall Hotel, Atlantic City,
`New Jersey, Septetuber 26-29, 1972.
`
`woven gauze with 40% KI on one side was placed on top to
`ensure superhydration [4 ]. · A 4 x 4 cm piece of plastic
`film covered the site and was occluded with impermeable
`tape (Dermiclear,
`transparent, Johnson & Johnson,
`USA). The opposite, symmetrical side of the back
`received a similar patch; however, the ointment consisted
`of a mixture of KI (40% w/w) and one of the drugs
`mentioned in Table I. If there were less than 5 pustules at
`24 hr the patch was reapplied. This patch was then
`removed at 48 hr.
`Systemic therapy. Orally administered drugs were
`investigated. In a screening test, 53 men were exposed to
`10% and 40% KI concentrations in hydrophilic ointment
`(USP Torch Laboratories, USA). Seventeen of these 53
`men reacted to both concentrations of KI and were given
`tetracyclines (tetracycline-HCl, 1500 mg; demethyl-
`chlortetracycline, 600 mg) for 10 days. The patch tests
`were then repeated with both concentrations of KI.
`Therapy was continued during patch testing.
`Thereafter the effect of a variety of drugs given orally
`to patients undergoing KI patch tests was investigated
`(Tab. II). Patients were tested before systemic therapy
`and while receiving the drugs. Subjects were female and
`male inpatients and outpatients of the Department of
`Dermatology, aged 16 to 46 years. None had received
`systemic therapy for at least 4 weeks prior to this study.
`In all cases, control patch tests were clone either simulta-
`'1eously or a few days or weeks apart on the same side of
`the upper back. Test materials were kept at 4 oc and not
`stored for more than 6 weeks.
`
`Bacteriology
`More than 40 pustules induced with 40% KI, 40% KI +
`topical antimicrobials, and 40% KI pustules raised in
`patients receiving systemic therapy were studied. Smears
`from pustules were Gram stained. A droplet of pus was
`collected into Triton X-100. Standard streak plates were
`prepared from the. homogenate on trypticase soy agar
`(TSA) for aerobic incubation at 37°C for 48 hr and on
`casein yeast extract lactate glucose agar (CYLG) for
`incubation in N 2 + 10% CO 2 at 37 oc for 7 days [5 ]. These
`cultures were done to identify Staphylococcus or Micro-
`coccus or Propionibacterium acnes ( Corynebacterium
`acnes group I) and P. granulosum ( C. acnes group II)
`[5,6].
`
`532
`
`Exh. 1018
`
`

`
`Dec. 1975
`
`ANTI-INFLAMMATORY EFFECTS OF ANTIMICROBIAL AGENTS
`
`533
`
`Scoring
`The number of pustules in each 4 x 4 cm pat.ch test
`field was recorded at 24 and 48 hr. In the case of severe
`pustular reactions, patches were removed af 24 hr.
`Statistical anal.vsis
`As every patient served as his own control in both
`experimental designs, paired comparisons of the number
`of pustules were made. The Wilcoxon matched-pairs
`signed-rank test was used (7 ]. This is a nonparametric
`test which is roughly equivalent to the paired t-test but
`does not require the assumption of a normal probability
`distribution. In the experimental design I the nurober of
`pustules from the 40% KI patch vs the 40% KI +
`antimicrobials, andin experimental design II the nurober
`of pustules from the 40% KI patches before and during
`. systemic therapy were compared.
`Cantrots
`Controls with ointment base alone, plastic occlusion,
`and 5% antimicrobial ointment without KI were used.
`RESULTS
`Patch tests with KI produced follicular pustules,
`usually within 24 hr. Pustules were often on an
`erythematous and edematous base. Erythema and
`edema without pustule formation also occurred in a
`few subjects. The reaction was usually confined to
`the patch test areas, although spillage under the
`occlusive tapes sometimes caused erythema and
`papulopustules outside the patch test areas be-
`neath the strips of tape.
`·
`The pustules were not caus.ed by bacteria [1 ].
`
`TABLE I. Antibiotics and chemotherapeutics tested
`topically (all concentrations were 5% (w/w) in a KI
`(40%) ointment)
`
`Drugs
`
`No.of
`patients
`
`No.of
`tri als
`
`Tetracycline-H Cl
`Oxytetracycline
`Demethylchlortetracycline
`Erythromycin
`Gentamycin
`Penicillin G-Na
`Ampicillin
`Sulfamethoxypyridazine
`Diaminodiphenylsulfone (DDS)
`
`12
`10
`15
`10
`16
`8
`14
`10
`8
`
`25
`20
`15
`20
`30
`16
`28
`10
`16
`
`Smears did not reveal bacteria except when a
`microcomedo or seborrheic filament was inciden-
`tally removed, which contained rods, most likely P.
`acnes. The majority of pustules was sterile. Micro-
`cocci were found in less than 10% of the pustules
`(streak plates). Small numbers of P. granulosum
`were grown in a few cases. Similarly P. acnes was
`rarely recovered. C. acnes group I and II were
`identified according to morphologic criteria on
`streak plates [6 ].
`The most prominent finding was the suppression
`of erythema and pustules by e'ither topically or
`systemically administered antibiotics or chemo-
`therapeutics. Representative clinical pictures are
`presented in Figures 1 and 2 .
`Topical application. Diminution of pustules was
`seen with tetracyclines, erythromycin, and löng-
`acting sulfonamides. Erythema and edema were
`also decreased. Questionahle or no inhibition was
`achieved with gentamycin, penicillin G-Na, am-
`picillin, and DDS.
`Quantitative data for DMCT anci sulfamethoxy-
`pyridazine are given in Tables Ill and IV. A
`marked reduction (number of reactors, degree of
`reaction) of about 50% was achieved with DMCT
`(Tab. III) after 24 hr, and over 60% after 48 hr in a
`group of 15 subjects. Thirteen of 15 subjects had
`less reaction in the KI + antibiotic-containing
`patch test as compared to the KI patch. Topical
`application of sulfamethoxypyridazine led to a
`similar, but less pronounced depression of pustules
`(Tab. IV), statistically, though of marginal signifi-
`cance. The mean number of pustules at 24 and 48
`hr was halved. Seven of 10 subjects had either little
`or no reaction in the sulfonamide-containing patch
`vs the KI patch.
`Systemic treatment. From the screening test
`with 17 men exposed to antibiotics, qualitative and
`quantitative anti-inflammatory properties of an-
`tibiotics could be expected (Tab. V, Fig. 2). The
`number of reactors with pustules due to KI de-
`clined from 75 to 26% and even more so with the
`higher KI concentration (Tab. V). In those still
`reacting to KI patch tests while receiving systemic
`antibiotics, the degree of reaction (0 to 4+) was
`suppressed (Fig. 3).
`Tetracyclines, erythromycin, and the sulfone
`definitely suppressed the inflammatory reaction.
`
`TABLE II. Antibiotics and chemotherapeutics given orally at time of 40% Kl patch testing (range of therapy 7 to 30 days
`at time of patch testing)
`Range of dosage
`at patch testing
`(mg or U/day)
`1000-1500
`100-200
`600-900
`1000-1500
`1.5 X 106
`2000-4000
`100-200
`
`No.of
`patients
`
`No. of
`tri als
`
`%Inhibition
`
`>80
`>80
`>80
`>80
`none
`none
`>90
`
`Drugs
`
`Tetracy clirie-H Cl
`Oxytetracycline
`Demethylchlortetracycline
`Erythromycin
`Penicillin G-Na
`Ampicillin
`Diaminodiphenylsulfone (D-qS)
`
`8'
`8
`13
`8
`6
`6
`20
`
`10
`8
`13
`8
`6
`10
`20
`
`Exh. 1018
`
`

`
`534
`
`PLEWIG AND SCHÖPF
`
`Val. 65, No. 6
`
`The most pronounced anti-inflammatory response
`was seen with DDS. The penicillins showed little
`Suppression. Representative data are given in Ta.
`bles VI and VII. In the subjects receiving tetracy-
`clines, the mean nurober of pustules was reduced
`from almost 15 to 3 at 24 hr, and from 20 to 1 at 48
`hr (Tab. VI).
`FollQwing systemic therapy with DDS, the pus-
`tular response was reduced at 24 and 48 hr (Tab.
`VII). Thirteen subjects showed either a marked
`suppression or no reaction at all when receiving.
`DDS. Erythema and edema were suppressed
`within the confines of the patches, even if there
`were no pustules.
`
`DISCUSSION
`V arious antibiotics, sulfonamides, and sulfones
`have been used in the past to successfully treat a
`
`Fra. 1. Typical KI-induced follicular pustules 48 hr
`after occlusive application of 40% KI. Theseare two 4 x 4
`cm sites paravertebrally on the upper back.
`
`Fra. 2. Anti-inflammatory effects of antibiotics. Topi-
`cal application of 5% DMCT + 40% KI (B) reduces
`inflammation as compared to control patch (A) with 40%
`KI alone. Systemic treatment with DMCT abolishes
`erythema, edema, and pustules completely (D) as com-
`pared to control patch ( C). Both patches were done with
`40% KI for 48 hr.
`
`Exh. 1018
`
`

`
`Dec. 1975
`
`ANTI-INFLAMMATORY EFFECTS OF ANTIMICROBIAL AGENTS
`
`535
`
`TABLE III. Topical5% DMCT in 40% KI ointment
`Nurober ofpustules
`
`Sub-
`jects
`
`24hr
`40%KI + 5%
`DMCT
`
`40%KI
`
`40%KI
`
`28
`4
`11
`30
`19
`28
`33
`7
`0
`21
`0
`2
`3
`1
`0
`
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`
`35
`6
`4
`0
`6
`16
`9
`2
`0
`3
`0
`0
`0
`0
`0
`
`7
`50
`*
`50
`*
`
`50
`18
`32
`13
`12
`15
`9
`4
`
`48 hr
`40% KI + 5%
`DMCT
`*
`16
`7
`*
`30
`
`*
`11
`2
`18
`0
`0
`1
`3
`1.
`
`TABLE V. Percent ·ot subjects who react to KI
`Systemic therapy with antibiotics suppresses pustules
`induced by KI patch tests. Fifty-three supjects (controls)
`were exposed to two concentrations of KI öintment, 17 of
`these were subsequently given tetracycline-HC11500 mg,
`or DMCT 600 mg for 10 days.
`Controls
`(N =53)
`
`Systemic antibiotics
`(N = 17)
`
`10%KI
`40%KI
`
`74.9
`80.7
`
`25.6
`23.6
`
`Number of
`Subjects
`1~
`13
`12
`11
`10
`9
`
`Mean
`T
`p
`
`12.5
`
`5.4
`
`23.6
`
`8.1
`
`3.0
`9.0
`.005
`.02
`* Patch not repeated at 48 hr because of severe reac-
`tion at 24 hr.
`
`TABLE IV. Topical5% sulfamethoxypyridazine (SMP)
`in 40% KI ointment
`· ·
`Nurober of pustules
`
`Sub-
`jects
`
`24hr
`40% KI + 5%
`SMP
`
`48hr
`40%KI + 5%
`SMP
`
`40%Kl
`
`40%KI
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`6
`0
`50
`25
`0
`8
`0
`3
`0
`0
`
`0
`1
`40
`15
`0
`8
`0
`0
`0
`0
`
`12
`4
`
`*
`10
`4a
`5
`8
`2
`0
`
`0
`8
`*
`*
`2
`4a
`3
`4
`1
`0
`
`Mean
`T
`p
`
`9.2
`
`6.4
`
`5.6
`
`2.8
`
`3.5
`1
`.14
`.08
`* Patch not repeated at 48 hr because of severe
`reaction·at 24 hr.
`a Several pustules broke from 24 to 48 hr interval,
`therefore the smaller numbers of lesions at day 2.
`
`~ Reoction.
`Non-
`Grade
`Reoctors
`FIG. 3. Systemic antibiotics suppress KI-induced follic-
`ular pustules. Screening experiment with good reactors to
`KI (N = 17). Darker, striped bars indicate pronQunced
`reactions prior
`to exposure to antibiotics (tetracy-
`cline-HCl and DMCT·). Stippted bars indicate the sup-
`pressed reaction in these 17 subjects following systemic
`tetracyclirie treatment. Twelve subjects were made non-
`reactive to KI patch tests.
`
`TABLE VI. Systemic antibiotic administration; effects of
`DMCT given orally
`Nurober of pustules in the 40% KI patch test
`24hr
`48 hr
`before Rx
`duringRx
`beforeRx
`duringRx
`
`Sub-
`jects
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`
`0
`9
`9
`8
`0
`0
`21
`8
`5
`10
`50
`50
`9
`
`0
`0
`5
`5
`3
`2
`0
`0
`0
`0
`2
`3
`3
`
`3
`29
`32
`33
`4
`4
`32
`4
`
`*
`*
`*
`30
`
`0
`0
`8
`6
`8
`4
`0
`0
`*
`*
`*
`*
`10
`
`variety of inflam~atory diseases showing either
`erythema, vesicles, pustules, or granulomas. These
`diseases are believed of nonbacterial origin. Exam-
`ples
`include erythema chronicum migrans,
`acrodermatitis chronica atrophicans, dermatitis
`
`Mean
`T
`p
`
`13.8
`
`1.8
`
`19.0
`
`4.0
`
`3'.5
`2.5
`.02
`.005
`* Patch not repeated at 48 hr because of severe
`reaction at 24 hr.
`
`Exh. 1018
`
`

`
`536
`
`PLEWIG AND SCHÖPF
`
`TABLE VII. Systemic §Ulfone administration (effects of
`DDS given orally)
`Nuniber of pustules in the 40% KI patc.h test
`~· ..
`24hr
`48 hr
`duringRx
`beforeRx
`duringRx
`before Rx
`
`Sub-
`jects
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`
`1
`0
`5
`3
`0
`1
`10
`9
`0
`0
`4
`9
`4
`3
`28
`33
`0
`0
`2
`6
`
`1
`0
`3
`3
`0
`0
`0
`0
`0
`0
`0
`0
`0
`1
`2
`0
`0
`0
`0
`0
`
`42
`43
`21
`13
`8
`7
`27
`23
`14
`8
`37
`32
`32
`24
`*
`*
`13
`5
`+
`+
`
`Mean
`T
`p
`
`5.9
`
`0.0
`.001
`
`0.5
`
`21.8
`
`0.0
`.0001
`
`1
`0
`5
`6
`3
`2
`4
`8
`3
`2
`0
`1
`2
`2
`
`0
`3
`+
`+
`2.6
`
`*Patch not repeated at 48 hr because of severe
`reaction at 24 hr. + = not clone.
`
`herpetiformis, rosacea, and follicular mucinosis
`[2,3 ].
`Why do antibiotics, sulfonamides, or sulfones
`work in these conditions? They possibly have
`anti-inflammatory properties not identical with
`their bactericidal or bacteriostatic effects. One
`possible property could be, for instance, the inhibi-
`tion of perivascula:r leukocyte migration. To test
`this anti-inflammatory hypothesis, an experimen-
`tal design was chosen to induce a modest degree of
`inflammation. The KI patchtest seemed suitable.
`The pustules in this test are not caused by bacte-
`ria. Two procedures were undertaken. One was to
`deliver the drugs topically. Occlusive conditions
`and superhydration were useful pr<'lrequisites. In
`the second experimental design drugs were given
`orally. After 7 to 30 days of systemic therapy
`reexposures with KI were made.
`The hypothesis that some antimicrobials would
`show anti-inflammatory effects, proved correct.
`When tested topically, the tetracyclines and eryth-
`romycin were most active, especially DMCT. Sul-
`fam:ethoxypyridazine was also effective. Topical
`penicillin G-Na, ampicillin, or DDS were without
`effect. When tested systemically, the tetracyclines
`
`Vol. 65, No.6
`
`and DDS were potent anti-inflammatory drugs,
`Anions, such as iodides, have leukotactic proper-
`ties (unpublished data). The pustules are the
`result of an irritant dermatitis due to KI [1 ]. The
`formation of pustules, however, is not specific for
`KI, as other salts related chemically (e.g., Nai,
`KCl, N aCl) have similar effects, although to a
`lesser extent (unpublished data). Likewise, some
`antimicrobials have effects opposite to KI, for
`instance by antagonizing the leukotactic effects of
`KI, namely by inhibiting leukotaxis. It may be
`speculated that leukocytes with their potent tis-
`sue-destroying enzymes may perpetuate the chro-
`nicity of various dermatoses. The prevention of
`leukotaxis, that is inhibition of the formation of
`cellular infiltrates, can be considered a helpful
`adjunct of these drugs. This, in fact, may be the
`explanation for why certain dermatoses obviously
`not of bacterial origin are benefitted by antimi-
`crobials. The possibility that inhibitiory effects of
`topically applied antibiotics could result from a
`chemical binding between the antibiotic and KI
`has been ruled out (unpublished data).
`Recently, inhibitory effects of tetracycline on
`leukotaxis in vitro were reported. * In this report
`leukocyte migration was studied using a modified
`Boyden chamber. Random migration of white blood
`cells and chemotaxis toward Mycoplasrri·a pneu-
`moniae antigen were reduced by tetracycline by
`30 to 60%. * Others have studied the effects of
`tetracyclines on the phagocytic function of human
`leukocytes [8 ]. Human leukocytes incubated with
`tetracyclines in vitro showed a decreased capacity
`to phagocytize yeast and bacteria, and leukocytes
`harvested from healthy volunteers after ingestion
`of
`tetracycline also demonstrated decreased
`phagocytic capacity for yeast [8 ].
`
`The assistance of Dr. Cornelia Hofman is kindly
`acknowledged.
`
`REFERENCES
`1. Plewig· G, Kligman AM: Follikuläre Pusteln im Kali-
`umjodid-Epicutantest. Arch Dermatal Forsch
`242:137-152, 1972
`2. Macmillan AL, Champion RH: Generalized pustular
`psoria:sis treated with dapsone. Br J Dermatal
`88:183-185, 1973
`3. Kubba RK, Stewart TW: Follicular muci.nosis re-
`sponding to dapsone. Br J Dermatal 91:217-220,
`1974
`4. Hojyo-Tomoka MT, Marples RR, Kligman AM: Pseu-
`domonas infection in superhydrated skin. Arch
`Dermatal 107:723-727, 1973
`.
`5. Marples RR, Izumi AK: Bacteriology ofpustular acne.
`J Invest Dermatal 54:252-255, 1970
`6. Marples RR, McGinley KJ: Corynebacterium acnes
`and other anaerobic diphtheroicl~> from human skin.
`J Med Microbiol 7:349-357. 1974
`7. SiegelS: Nonparametrl.c Statistfes for the Behavioral
`Sciences. New York, McGraw-Hill, 1956, pp 75-83
`8. Forsgren A, Schmeling D, Quie PG: Effect of tetracy-
`cline on the phagocytic function of human leuko-
`cytes. J Infect Dis 130:412-415, 1974
`
`'
`
`,
`
`Exh. 1018