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`Docket No.: 512-53P li
`
`METHODS OF TREAIING ACNE
`
`Inventor:
`
`Robert A. Ashley
`
`Hoffmann & Baron, L.L.P.
`6900 Jericho Turnpike
`Syosset, New York 11791
`(516) 822-3550
`
`Dr. Reddy's Laboratories, Ltd., et al.
`V.
`Galderma Laboratories, lnc.
`IPR2015-__
`Exhibit 1003
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`METHODS OF TREATING ACNE
`
`BACKGROUND OF TBE INVENTION
`
`Acoe is a common disease characterized by various types of lesions. The
`areas affected typically are areas ofthe skin where sebaceous glands are largest, most
`numerous, and most active. The lesions associated ·with acne are usually categorized
`as either non-inflammatory or inflammatory.
`
`5
`
`Non-inflammatory lesions include comedones. Comedones appear in two
`forms, open and closed. Comedones are thought to arise from abnormal follicular
`differentiation. Instead ofundergoing shedding aod discharge through the follicular
`orifice, abnormal desquamated cells (keratinocytes) become unusually cohesive,
`forming a microcomedo or a microscopic hyperkeratotic plug in the follicular canal.
`The progressive accumulation of these microcomedones Iead to visible comedones.
`
`In its mildest form, acoe is a more or less superficial disorder characterized by
`slight, spotty skin irritations. In such cases, ordioary skin hygiene is typically a
`satisfactory treatment. In the more inflammatory types of acne, however, pustules;
`infected cysts; and in extreme cases, canalizing, in:flamed and infected sacs appear.
`Without effective treatment, these lesions may become extensive and leave
`permanent, disfiguring scars.
`
`Micro-organisms, especially Propionibacterium acnes, are strongly implicated
`in the pathogenesis of acne. The micro-organisms are thought to release microbial
`mediators of inflammation into the dermis or trigger the release of cytokines from
`ductal keratinocytes.
`
`10
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`15
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`20
`
`Accordingly, the efficacy of antibiotics in treating acne is thougbt tobe due, in
`significant part, to the direct inhibitory effect ofthe antibiotics on the growth and
`25 metabolism oftbese micro-organisms. SystemicaJly-administered tetracyclioe
`
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`antibiotics, especially minocycline hydrochloride, are particularly effective in treating
`acne.
`
`The tetracyclines are a class of cornpounds of which tetracycline is the parent
`compound. Tetracycline has the following general structure:
`
`ID
`
`ll lfC~n
`-
`OH
`~
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`OH 0
`Structure A
`
`The numbering system of the multiple ring nucleus is as follows:
`
`Structure B
`
`Tetracycline, as we11 as the 5-0H (oxytetracycline, e.g. Terramycin) and 7-Cl
`(chlorotetracycline, e.g. Aureomycin) derivatives, exist in nature, and areallweil
`known antibiotics. Semisynthetic derivatives such as 7 -dimethylamino-tetracycline
`(minocycline) and 6a-deoxy-5-hydroxy-tetracycline (doxycycline) arealso known
`tetracycline antibiotics. Natural tetracyclines may be rnodified without losing their
`antibiotic properties, although certain elements of the structure must be retained to do
`so.
`
`In addition to the direct antimicrobial activity of tetracyclines, further
`activities of antibiotic tetracyclines have been investigated for possible therapeutic
`effects on acne.
`
`5
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`For example, a study by Elewski et al., J Amer. Acad Dermata/., 8:807-812
`(1983) suggests that acne therapy, consisting of orally-administered tetracycline at a
`total daily dose of 1 OOOmg, may have therapeutic anti-inflammatory effects in
`addition to antirnicrobial effects. In particular, it was found that tetracycline inhibited
`neutrophil chemotaxis induced by bacterial chemotactic factors.
`
`However, a more recent study performed by Eady et al., J lnvest. Dermata/.,
`101:86-91 (1993) found somewhat different results with respect to the effect of
`tetracyclines on cytokines. The study was desigoed to determine whether oral acne
`therapy with minocycline or tetracycline altered the cytokine content of open
`comedones. The total daily dose of minocycline adrninistered was 1 OOmg. The total
`daily dose of tetracycline administered was 1 OOOmg. It was found that these therapies
`upregulated the production ofbioactive IL-1 C<-like material and immunochemical IL-
`1 ß. IL-1 is considered to be a pro-inflammatory cytokine. The authors speculate that
`increased Ievels of IL-1 in comedones destined to become inflamed may enhance
`resolution and promote repair of the damaged follicular epithelium.
`
`Another possible activity oftetracyclines in acne therapy was investigated by
`Bodokh, 1., et al., Acta. Derm. Venerol., 77:255-259 (1997). Tbeir study was
`designed to evaluate the action of minocycline on sebaceous excretion in acne
`patients. A 1 OOmg daily dose of minocycline was administered. A subclinical
`increase in seborrhoea was reported. The authors propose that minocycline induces
`an increase in seborrhoea via a reduction in ductal obstruction. The mechanism by
`wbich the ductal obstruction is reduced is proposed to be the reduction in ductal
`irritation. The authors suggest that the reduction of ductal irritation is due to
`minocycline's direct effect on P. acnes, or minocycline's effect on the lipase
`produced by P. acnes.
`
`Bodokh et al. also found that during treatment no correlation exists between
`seborrhoea intensity and clinical severity of acne. The authors state that the lack of
`correlation shows that seborrhoea is pathogenic because it is the "culture medium" of
`
`5
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`15
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`20
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`25
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`30
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`3
`
`Exh. 1003
`
`

`
`P. acnes. Thus, it can be concluded that the authors consider the antimicrobial
`activity of minocycline as a significant therapeutic activity with respect to acne.
`
`Sirnilarly, in a recent clinical study it was found that tetracycline doses lower
`than antimicrobial doses bad no clinical effect on acne. (Cunliffe et al., J. Am. Acad.
`Dermatol., 16:591-9 (1987).) In particular, a 100mg total daily dose ofminocycline;
`and a l.Og total daily dose of tetracycline were found to be necessary to successfully
`treat acne.
`
`The antimicrobial effects of antibiotics are generally directly proportional to
`the dose administered ofthe antibiotics. Accordingly, in moderate to severe (i.e.
`inflammatory) forms of acne, oral antibiotics are typically administered at high doses.
`For example, in conventional acne therapy, tetracycline is administered at an initial
`dose of 500 to 2,000 mg/day, followed by a maintenance dose of250-500 mglday.
`
`Clearly, the state-of-the-art teaching isthat the clinical efficacy of
`systemically-administered tetracyclines in the treatment of acne is due in significant
`part to the antimicrobial effects ofthe tetracyclines. Additionally, tetracyclines are
`believed to reduce the number of inflammatory lesions (papules, pustules and
`nodules) by a variety of non-antimicrobial mechanisms. Such mechanisms include
`interfering with the chemotaxis of polymorphonuclear leukocytes (PMN) into the
`inflammatory lesion, inhibition of PMN derived collagenase, and by scavenging
`reactive oxidative species produced by resident inflarnmatory cells.
`
`The use of tetracycline antibiotics, however, can lead to undesirable side
`effects. For example, the long term administration of antibiotic tetracyclines can
`reduce or eliminate healthy microbial flora, such as intestinal flora, and can Iead to the
`production of antibiotic resistant organisms or the overgrowth of yeast and fungi.
`Other side effects include gastraintestmal symptoms such as nausea, vomiting,
`abdominal pain, diarrhea, rashes, and phototoxic and allergic reactions. Tetracyclines
`also can cause fetal bann ifused during pregnancy.
`
`5
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`15
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`
`Accordingly, there is a need for an effective treatment of acne which does not
`cause the undesirable side effects produced by the systemically-administered
`antibiotics used in conventional acne therapy.
`
`SUMMARY OF INVENTION
`
`The present invention provides a method of treating acne in a human in need
`thereof. The method comprises administering systemically to the human a
`tetracycline compound in an amount that is effecti ve to treat acne but has substantially
`no antimicrobial activity, without administering a bisphosphonate compound.
`
`Additionally, the present invention provides methods for reducing the number
`of comedones, inhibiting oxidation of melanin, and/or inhibiting lipid-associated
`abnormal follicular differentiation in a human in need thereof. These methods
`comprise administering systemically to the human a tetracycline compound in an
`amount that is effective for its purpose, e.g., to reduce the nurober of comedones, to
`inhibit oxidation ofmelanin, andlor to inhibit lipid-associated abnormal follicular
`differentiation, but has substantially no antimicrobial activity.
`
`BRIEF DESCRIPTION OF THE DRA WINGS
`
`Figure 1 shows the photoirritancy factor (PIF) for some tetracycline
`compounds. For structure K, the compounds indicated are as follows:
`
`COL
`
`308
`311
`306
`
`R7
`
`hydrogen
`hydrogen
`hydrogen
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`amino
`palmitamide
`dimethylamino
`
`For structures L, M, N or 0 the compounds indicated are as follows:
`
`5
`
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`
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`20
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`
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`
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`802
`804
`805
`
`R7
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`acetamido
`dimethylaminoacetamido
`nitro
`amino
`
`For structure P, R7 is hydrogen, R8 is hydrogen and R9 is nitro.
`
`DETAILED DESCRIPTION
`
`The present invention provides methods of treating acne. As used herein, the
`term "acne" is a disorder of the skin characterized by papules, pustules, cysts, nodules,
`comedones, and other blemishes or skin lesions. These blemishes and lesions are
`often accompanied by inflammation ofthe skin glands and pilosebaceous follicles, as
`well as, microbial, especially bacterial, infection.
`
`For the purposes of this specification, acne includes all known types of acne.
`Some types of acne include, for example, acne vulgaris, cystic acne, acne atrophica,
`bromide acne, chlorine acne, acne conglobata, acne cosmetica, acne detergicans,
`epidemic acne, acne estivalis, acne fulminans, halogen acne, acne indurata, iodide
`acne, acne keloid, acne mechanica, acne papulosa, pomade acne, premenstral acne,
`acne pustulosa, acne scorbutica, acne scrofulosorurn, acne urticata, acne varioliformis,
`acne venenata, propionic acne, acne excoriee, gram negative acne, steroid acne,
`nodulocystic acne and acne rosacea. Acne rosacea is characterized by inflarnmatory
`lesions (erythema) and permanent diJation ofblood vessels (telangectasia).
`
`The present invention is particularly effective in treating comedones, e.g.,
`reducing the number of comedones. Both open and closed comedones can be treated
`in accordance with the methods of this invention.
`
`The present invention can also be used to treat certain other types of acneiform
`dermal disorders, e.g. perioral dermatitis, seborrheic dermatitis in the presence of
`6
`
`Exh. 1003
`
`

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`
`J
`
`\
`
`acne, gram negative folliculitis, sebaceous gland dysfunction, hiddradenitis
`suppurativa, pseudo-folliculitis barbae, or folliculitis.
`
`5
`
`The method comprises the administration of a tetracycline compound to a
`human in an amount which is effective for its purpose e.g., the treatment of acne,
`including reducing the nurober of comedones, but which has substantially no
`antimicrobial activity.
`
`The tetracycline compound can be an antimicrobial or non-antimicrobial
`compound. The tetracycline compound has the general structure indicated above.
`
`10
`
`Some examples of antimicrobial tetracycline compounds include doxycycline,
`minocycline, tetracycline, oxytetracycline, chlortetracycline, demeclocycline,
`lymecycline and their pharmaceutically acceptable salts. Doxyclycline is preferably
`administered as its hyclate salt or as a hydrate, preferably monohydrate.
`
`!.11
`
`15
`
`Non-antimicrobial tetracycline compounds are structurally related to the
`antibiotic tetracyclines, but have bad their antibiotic activity substantially or
`completely eliminated by chemical modification. For example, non-antimicrobial
`tetracycline compounds are capable of achieving antibiotic activity comparable to that
`of tetracycline at concentrations at least about ten tim es, preferably at least about
`twenty five tim es, greater than that of tetracycline.
`
`Examples of chemically modified non-antimicrobial tetracyclines (CMTs)
`include, 4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile (CMT-2), 6-
`demethyl-6-deoxy-4-de( dimethylamino )tetracycline (CMT -3), 7 -chloro-4-
`de(dinlethylamino)tetracycline (CMT-4), tetracycline pyrazole (CMT-5), 4-hydroxy-
`4-de( dimethy lamino )tetracycline ( CMT -6), 4-de( dimethylamino-12et-
`deoxytetracycline ( CMT-7), 6-deoxy-5et-hydroxy-4-de( dimethylamino )tetracycline
`(CMT-8), 4-de(dimethylamino)-12a-deoxyanhydrotetracycline (CMT-9), 4-
`de( dimethylamino )minocycline (CMT -1 0).
`
`20
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`
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`
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`
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`
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`
`Further examples of chemically modified non-antimicrobial tetracyclines
`include Structures C-Z. (See Index of Structures.)
`
`The minimal amount ofthe tetracycline compound administered to a human is
`the lowest amount capable of providing effe.ctive treatment of acne. Effective
`treatment is a reduction or inhibition of the blemishes and lesions associated with
`acne.
`
`The amount of a tetracycline compound that has substantially no antimicrobial
`activity is an amount that does not significantly prevent the growth of microbes, e.g.
`bacteria.
`
`For example, tetracycline compounds that have significant antimicrobial
`activity may be administered in an amount which is 10-80% of the antimicrobial
`amount. More preferably, the antimicrobial tetracycline compound is administered in
`an amount which is 40-70% ofthe antimicrobial amount.
`
`Some examples of antimicrobial an1ounts of members of the tetracycline
`family include 1 OOmg/day of doxycycline, 200mg/day of minocycline, 250mg of
`tetracycline four times a day, lOOOmg/day of oxytetracycline, 600mg/day of
`demeclocycline and 600mg/day of lymecycline.
`
`An example of an antimicrobial tetracycline administered in a non-
`antimicrobial amount is doxycycline hyclate administered at a 20 milligram dose
`twice daily. Such a formulation is sold for the treatment ofperiodontal disease by
`CollaGenex Pharmaceuticals, Inc. ofNewtown, Pennsylvania under the trademark
`Periostat ®.
`
`30
`
`Any of the tetracycline compounds mentioned above, especially doxycycline
`and minocycline, is unexpectedly effective in reducing the nurober of comedones
`
`8
`
`Exh. 1003
`
`

`
`when administered at a dose which has substantial!y no antimicrobial effect.
`Preferably the reduction is at least about 20% greater than for a placebo control, more
`preferably at least about 30% greater than for a placebo control, most preferably at
`least about 40% greater than for a placebo control, and optimally at least about 50%
`greater than for a placebo control.
`
`5
`
`Example 38 below summarizes a clinical study using 20 mg doxycycline
`hyclate tablets administered twice a day. A significant reduction in the number of
`comedones was observed. Tbis reduction in the number of comedones is unexpected.
`10 This reduction is particularly unexpected since, as can be seen from tbe microbiology
`results in Example 38, the treatrnent with doxycycline resulted in no reduction of skin
`microflora vis-a-vis a placebo control.
`
`15
`
`The inventors are not certain of, and do not wish to be limited by, any
`particular mecbanism. Nevertbeless, it is believed that the ability of doxycycline to
`inhibit oxidation of melanin and lipid-associated abnormal follicular differentiation
`prevents keratinocytes from becoming cohesive, thereby inhibiting the formation of
`comedones.
`
`20
`
`The tetracycline compound may be administered by sustained release.
`Sustained release administration is a method of drug delivery to achieve a certain
`Ievel of the drug over a particular period of time. The Ievel typically is measured by
`serum concentration. Further description of methods of delivering tetracycline
`compounds by sustained release can be found in the patent application, "Controlled
`25 Delivery ofTetracycline and Tetracycline Derivatives," filed on AprilS, 2001 and
`assigned to CollaGenex Pharrnaceuticals, lnc. ofNewtown, Pennsylvania. Tbe
`aforementioned application is incorporated herein by reference in its entirety.
`
`For example, 40 milligrams of doxycycline may be administered by sustained
`release over a 24 hour period.
`
`30
`
`9
`
`I I I
`~
`
`Exh. 1003
`
`

`
`The antimicrobial tetracycline compound can be administered in an amount
`which results in a serwn tetracycline concentration which is 1 0-80% of the minimum
`antimicrobial serum concentration. The minimum antimicrobial serum concentration
`is the lowest concentration known to exert a significant antimicrobial effect.
`
`Some examples of the approximate minimum antimicrobial serum
`concentrations ofmembers ofthe tetracycline family follow.
`
`A single dose of two 1 OOmg minocycline HCl tablets administered to adult
`humans results in minocycline serum levels ranging from 0.74 to 4.45 J,tg/ml over a
`period of an hour. The average level is 2.24 J,tglml.
`
`Two hundred and fifty milligrams of tetracycline HCl administered every six
`hours over a twenty-four hour period produces a peak. plasma concentration of
`approximately 3 J.Lg/ml. Five hundred milligrams of tetracycline HCI administered
`every six hours over a twenty-four hour period produces aserum concentration Ievel
`of 4 to 5 J.lg/ml.
`
`5
`
`10
`
`15
`
`The minimum antimicrobial serum concentration of doxycycline is 1 J.lg/ml.
`20 Accordingly, aserum concentration of doxycycline in the range of about 0.1 to about
`0.8 J.Lg/ml is 10-80% ofthe minimum antimicrobial serum concentration of
`doxycycline. The more preferred range is about 0.4 to about 0.7 J.Lg/ml.
`
`To achieve this serum concentration, doxycycline can be administered by
`sustained release, or by interrnittent administration.
`
`25
`
`In another embodirnent, the tetracycline compound can be administered in an
`amount which results in aserum concentration between about 0.1 and 10.0 J.lg/ml,
`more preferably between 0.3 and 5.0 J.l.g/ml. For example, doxycycline is
`administered in an amount which results in a serum concentration between about 0.1
`and 0.8 J..Lg/ml, more preferably between 0.4 and 0.7 ~-tg/ml.
`
`30
`
`10
`
`Exh. 1003
`
`

`
`In a preferred embodiment, to reduce the number of comedones, doxycycline
`is administered in a daily amount of from about 30 to about 60 milligrams, but
`maintains a concentration in human plasrna below the threshold for a significant
`antimicrobial effect.
`
`Non-antimicrobial tetracycline compounds can be used in higher amounts than
`antirnicrobial tetracyclines, while avoiding the indiscriminate killing of microbes, and
`the emergence ofresistant rnicrobes. For example, 6-demethyl-6-deoxy-
`4-de(dimethylamino)tetracycline (CMT-3) may be administered in doses ofabout 40
`to about 200 mg/day, or in amounts that result in serum Ievels of about 1.55ug/ml to
`about 10 ul/ml.
`
`The actual preferred amounts of tetracycline compound in a specified case will
`vary according to the particular compositions formulated, the mode of application,
`and the particular sites and subject being treated.
`
`5
`
`10
`
`15
`
`20
`
`The tetracycline compounds can be in the form of pharmaceutically acceptable
`salts of the compounds. The term "pharmaceutically acceptable salt" refers to a salt
`prepared from tetracycline compounds and pharmaceutically acceptable non-toxic
`acids or bases. The acids may be inorganic or organic acids of tetracycline
`compounds. Examples of inorganic acids include hydrochloric, hydrobromic,
`hydroiodic, sulfuric, and phosphoric acids. Examples of organic acids include
`carboxylic and sulfonic acids. The radical of the organic acids may be aliphatic or
`aromatic. Some examples of organic acids include formic, acetic, phenylacetic,
`propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic,
`anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic ), methanesulfonic,
`ethanesulfonic, panthenoic, benzenesulfonic, stearic, sulfanilic, alginic, tartaric, citric,
`gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriate organic bases
`30 may be selected, for example, from N,N-dibenzylethylenediamine, chloroprocaine,
`
`25
`
`11
`
`Exh. 1003
`
`

`
`' l
`
`choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and
`procaine.
`
`5
`
`Preferably, the tetracycline compounds have low phototoxicity, or is
`administered in an amount that results in a serum Ievel at which the phototoxicity is
`acceptable. Phototaxicity is a chemically-induced photosensitivity. Such
`photosensitivity renders skin susceptible to damage, e.g. sunburn, blisters, accelerated
`aging, erythemas and eczematoid lesions, upon exposure to light, in particular
`ultraviolet light. The preferred amount of the tetracycUne compound produces no
`10 more phototoxicity than is produced by the administration of a 40mg total daily dose
`of doxycycline.
`
`Phototaxicity can be evaluated in terrns of a photoirritancy factor (PIF), as
`described in the examples. A PIF value of about 1.0 indicates that a compound is
`considered to have no measurable phototoxicity.
`
`Tbe low phototoxic derivatives preferably have PIF values no greater than
`about 5, preferably no greater than about 2, more preferably no greater tban about 1.5,
`most preferably no greater than about 1.2, and optimalJy about 1.
`
`15
`
`20
`
`Some antimicrobial tetracyclines having low phototoxicity include, for
`example, minocycline and tetracyline.
`
`Some non-antirnicrobial tetracyclines having low phototoxicity include, but
`are not limited to, tetracycline compounds having the general formulae:
`
`25
`
`STRUCTUREK
`
`wherein: R7, R8, and R9 taken together in each case, have the following meanings:
`
`30
`
`R7
`
`R8
`
`12
`
`R9
`
`Exh. 1003
`
`

`
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`hydrogen
`hydrogen
`
`hydrogen
`hydrogen
`hydrogen
`
`amino
`palmitamide
`dimethylamino
`
`and
`
`5
`
`STRUCTUREL
`STRUCTUREN
`
`STRUCTUREM
`STRUCTUREO
`
`wherein: R7, R8, and R9 tak:en together in each case, have the following meanings:
`
`R7
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`and
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`acetamido
`dimethylaminoacetamido
`nitro
`amino
`
`STRUCTUREP
`
`wherein: R7, R8, and R9 taken together are, respectively, hydrogen, hydrogen and
`nitro.
`
`The tetracycline compounds are administered without administering a
`bisphosphonate compound. Bisphosphonates compounds are related to inorganic
`pyrophosphonic acid. The bisphosphonates include, as non-limiting examples,
`alendronate ((4-amino-1- hydroxybutylidene) bisphosphonic acid), clodronate
`(dichloromethane diphosphonic acid), etidronate ((1-hyd.roxyethylidene) diphosphanic
`acid) and pamidronate ((3-amino-1- hydroxypropylidene) bisphosphonic acid); also
`risedronate ([ -hydroxy-2-(3-pyridinyl)ethylidene] bisphosphonic acid), tiludronate,
`i.e., tiludronic acid ([(4-chlorophenyl) thio] methylene] bisphosphonic acid) and
`zolendronate.
`
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`13
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`Exh. 1003
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`\
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`I
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`The tetracycline compounds are administered systemically. For the purposes
`ofthis specification, "systemic administration" means admin.istration to a human by a
`method that causes the compounds to be absorbed into the bloodstream.
`
`For example, the tetracyclines compounds can be administered orally by any
`method known in the art. For example, oral administration can be by tablets,
`capsules, pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the
`like.
`
`Additionally, the tetracycline compounds can be administered enterally or
`parenterally, e.g., intravenously, intramuscularly, or subcutaneously, as injectable
`solutions or suspensions. Administration can also be intranasally, in the form of, for
`example, an intranasal spray; or transdermally, in the form of, for example, a patch.
`
`For the pharmaceutical purposes described above, the tetracycline compounds
`of the invention can be formulated per se in pharmaceutical preparations optionally
`with a suitable pharmaceutical carrier (vehicle) or excipient as understood by
`practitioners in the art. These preparations can be made according to conventional
`chemical methods.
`
`Examples of carriers and excipients include starch, milk, sugar, certain types
`of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc,
`vegetable fats or oils, gums and glycols.
`
`In the embodiment in which the tetracycline compound is a non-antimicrobial
`tetracycline compound, administration can include topical application. Particular non-
`antimicrobial tetracycline compounds have only limited biodistribution, e.g. CMT-5.
`In such cases, topical application is the preferred method of administration of the
`compound.
`
`5
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`10
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`14
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`Exh. 1003
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`' •,
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`Carrier compositions deemed to be suited for topical use include gels, salves,
`lotions, creams, ointments and the like. The non-antimicrobial tetracycline compound
`can also be incorporated with a support base or matrix or the like which can be
`directly applied to skin.
`
`Topical application of non-antimicrobial tetracycline compounds in amounts
`of up to about 25% (w/w) in a vehicle are effective in treating acne while not inducing
`significant toxicity in the human. Amounts of from about 0.1 % to about 10% are
`preferred.
`
`Combined or coordinated topical and systemic administration ofthe
`tetracycline compounds is also contemplated under the invention. For example, a
`non-absorbable non-antimicrobial tetracycline compound can be administered
`topically, while a tetracycline compound capable of substantial absorption and
`effective systemic distribution in a human can be administered systemically.
`
`5
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`10
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`15
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`The tetracycline compounds are prepared by methods known in the art. For
`exarnple, natural tetracyclines may be modified without losing their antibiotic
`properties, although certain elements of the structure must be retained. The
`20 modifications that may and may not be made to the basic tetracycline structure have
`been reviewed by Mitscher in The Chemistry o[Tetracyclines, Chapter 6, Marcel
`Dekker, Publishers, New York (1978). According to Mitscher, the substituents at
`positions 5-9 of the tetracycline ring system may be modified without the complete
`loss of antibiotic properties. Changes to the basic ring system or replacement of the
`substituents at positions 1-4 and 10-12, however, generally lead to synthetic
`tetracyclines with substantially less or effectively no antimicrobial activity.
`
`25
`
`Further methods of preparing the tetracycline compounds are described in the
`exarnples.
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`30
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`Exh. 1003
`
`

`
`EXAMPLES
`
`5
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`
`The following examples serve to provide further appreciation of the invention
`but are not meant in any way to restriet the effective scope of the invention.
`
`Preparation of Compounds
`
`EXAMPLEl
`4-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-nitrotetracycline sulfate
`
`To a solution of one millimole of 4-dedimethylamino-7-dimethylamino-6-
`demethyl-6-deoxytetracycline in 25 ml of concentrated sulfuric aeid at 0°C was added
`1.05 mmole of potassiurn nitrate. The resulting solutionwas stirred at ice bath
`temperature for 15 minutes and poured in one liter of cold ether with stirring. The
`precipitated solid was allowed to settle and the majority of solvent decanted. The
`remaining material was filtered through a sintered glass funnel and the collected solid
`was washed weil with cold ether. The product was dried in a vacuum desiccator
`ovemight.
`
`EXAMPLE 2
`9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline
`sulfate
`
`To a solution of300 mg ofthe 9-nitro compound from example 1, in 30 ml of
`ethanolwas added 50 mg ofPt02. The mixturewas hydrogenated at atmospheric
`pressure until the theoretical amount of hydrogen was absorbed. The system is
`flushed with nitrogen, the catalyst Pt02 is fittered and the filtrate added dropwise to
`300 ml of ether. The product that separates is filtered and dried in a vacuum
`desiccator.
`
`16
`
`Exh. 1003
`
`

`
`',
`
`)
`
`\
`
`EXAMPLE3
`9-Acetamido-4-dedimethy lamino-7 -dimethylamino-6-demethyl-6-deoxytetracycline
`sulfate
`
`To a well stirred cold solution ofSOO mg of9-amino-4-dedimethylamino-7-
`dimethylamino-6-demethyl-6-deoxytetracycline sulfate from example 2, in 2.0 ml of
`1.3-dimethyl-2-imidazolidinone, 500 mg of sodium bicarbonate was added followed
`by 0.21 ml of acetyl chloride. The mixture is stirred at room temperature for 30
`minutes, fittered and the filtrate was added dropwise to 500 ml of ether. The product
`that separated was fittered and dried in a vacuum desiccator.
`
`EXAMPLE4
`4-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-cliazonimntetracycline
`sulfate
`
`5
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`10
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`15
`
`To a solution of 0.5 g of 9-amino-4-dedimethylamino-7-dimethylarnino-6-
`demethyl-6-deoxytetracycline sulfate, from example 2, in 10 ml ofO.lN hydrochloric
`acid in methanol cooled in an ice bath, 0.5 m1 ofn-butyl nitritewas added. The
`solutionwas stirred at ice bath temperature for 30 minutes and then poured into 250
`20 ml of ether. The product that separated was filtered, washed \\ri.th ether and dried in a
`vacuum desiccator.
`
`EXAMPLE5
`9-Azido-4-dedimethylamino-7-dimethylarnino-6-demethyl-6-deoxytetracycline
`sulfate
`
`25
`
`To a solution of 0.3 mmole of 4-dedimethylamino-7-dimethylamino-6-
`demethyl-6-deoxy-9-diazoniumtetracycline sulfate, from example 4, 10 mJ of 0.1 N
`methanolic hydrogen chloride was added 0.33 mmole of sodium azide. The mixture
`30 was stirred at room temperature for 1.5 hours. The reaction mixturewas then poured
`
`17
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`Exh. 1003
`
`

`
`into 200 ml of ether. The product that separated was flltered and dried in a vacuum
`desiccator.
`
`EXAMPLE6
`9-Amino-8-chloro-4-dedimethylamino-7 -dimethylamino-6-demethyl-6-deoxy-
`tetracycline sulfate
`
`One gram of 9-azido-4-dedimethylamino-7 -dimethy lamino-6-demethyl-6-
`deoxytetracycline hydrochloride, from example 4, was dissolved in 10 ml of
`concentrated sulfuric acid saturated with HCL at 0°C. The mixture was stirred at ice
`bath temperature for 1.5 hours and then slowly added dropwise to 500 ml of cold
`ether. The product that separated was filtered, washed with ether and dried in a
`vacuum desiccator.
`
`EXAMPLE7
`4-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-ethoxythiocarbonylthio-
`tetracycline sulfate
`
`A Solution of 1.0 mmole of 4-dedimethylamino-7 -dimethylamino-6-demethyl-
`6-deoxy-9-diazoniumtetracycline sulfate, from example 4, in 15 ml of water was
`added to a solution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water. The
`mixturewas stirred at room temperature for one hour. The product separated and was
`fittered and dried in a vacuum desiccator.
`
`EXAMPLE8A
`General Procedure for Nitration
`
`To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml of
`concentrated sulfuric acid at 0°C was added 1 mmole of potassium nitrate with
`stirring. The reaction solutionwas stirred for 15 minutes and then poured into 100 g
`of chopped ice. The aqueous solution was extracted 5 tim es with 20 ml of butanol
`
`18
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`t:~l
`
`~F.l n.i
`b.fl
`0
`j..l.
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`Exh. 1003
`
`

`
`each time. The butanol extracts were washed three times with 10 ml of water each
`time, and concentrated in vacuo to a volume of25 ml. Tbe light yellow crystalline
`solid which precipitated was filtered, wasbed with 2 ml of butanol and dried in vacuo
`at 60°C for 2 hours. This solid was a mixture of the two mononitro isomers.
`
`EXAMPLE8B
`4-Dedimethylamino-6-deoxy-9-nitrotetracycline
`
`To 980 mg ofthe nitrationproduct from 4-dedimetbylamino-6-
`deoxytetracycline (a mixture ofthe 2 isomers) in 25 ml ofmethanol was added
`enough