Identification of a Unique Subset of 2-Methylene-19-Nor Analogs of Vitamin D with Comedolytic Activityin the Rhino MouseNirca J. Nieves1, Jamie M. Ahrens2, Lori A. Plum2, Hector F. DeLuca2 and Margaret Clagett-Dame1,2

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and a series of 2-methylene-19-noranalogs of 1,25(OH)2D3 were evaluated for their ability to reduce the size of utricles (comedolytic activity) in arhino mouse model of acne. All analogs tested, as well as the native hormone, increased the skin epidermalthickness. In contrast, only a subset of analogs that lacked a full side chain and 25-hydroxyl group were found topossess comedolytic activity. A reduction in comedone area could be achieved without adversely affectingserum calcium levels. Although all compounds that contained a side chain ranging from 2 to 5 carbons in lengthhad similar potency as comedolytic agents, increasing the length of the side chain resulted in a progressiveincrease in calcemic liability. Dose–response studies of the comedolytic analogs showed that an increase inepidermal thickness was achieved at a lower dose than that needed to induce comedolysis. Thus, we haveidentified a unique subset of vitamin D analogs that produce comedolysis in the absence of hypercalcemia.Further, the activity of vitamin D analogs in causing epidermal hyperproliferation has been distinguished fromthat resulting in a reduction in utricle size.

Journal of Investigative Dermatology (2010) 130, 2359–2367; doi:10.1038/jid.2010.142; published online 10 June 2010

INTRODUCTIONAcne is a disease of the pilosebaceous unit of the skin.The pathogenesis of this disease involves hyperkeratinizationof the upper region of the follicle, increased sebumproduction by the sebaceous gland, colonization of thefollicle by Propionobacterium acnes, and induction of aninflammatory response. Acne therapy is directed towardsthese pathogenic causes with localized topical treatmentused most often, whereas systemic therapy is reserved for themost severe cases (Gollnick, 2003). Topical treatmentsprimarily include retinoid therapy, antibiotics, benzoylperoxide, and combinations of these agents (Degitz andOchsendorf, 2008; Kimball, 2008). These treatments areall effective at controlling acne but also induce some

unwanted side effects such as photosensitivity and skinirritation. Alternative topical therapeutic agents for acne thatdo not possess these side effects, or have a wider therapeuticwindow would be of great advantage for patients withthis condition.

Animal models that simulate a subset of diseasecharacteristics have been developed, but none of themrecapitulate all aspects of human acne, which is a multi-faceted condition. The rhino mouse has been useful inassessing the comedolytic activity of compounds, includingretinoids (Van Scott, 1972; Kligman and Kligman, 1979;Ashton et al., 1984; Mezick et al., 1984, 1985; Bouclier et al.,1990, 1991; Bernerd et al., 1991a, b; Zheng et al., 1993; Fort-Lacoste et al., 1999; Sakuta and Kanayama, 2005; Mirshah-panah and Maibach, 2007). The rhino (hrrh/hrrh) phenotype isdue to an autosomal recessive mutation in the hairless (hr)gene. In the rhino mouse, utriculi are derived from theinfundibular zone of the initial follicular units, and arehistologically similar to comedones (Mann, 1971).

The active hormonal form of vitamin D, 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), modulates the differentiation andproliferation of keratinocytes in the skin. In culturedkeratinocytes, 1,25(OH)2D3 induces differentiation and ex-erts antiproliferative activity (Hosomi et al., 1983; Holick,1994; Bikle, 2005). This is believed to account for thepositive effects of 1,25(OH)2D3 and analogs in the treatmentof psoriasis. However, topical application of 1,25(OH)2D3

and analogs to normal skin has been shown to inducekeratinocyte proliferation and epidermal hyperplasia, both inmice (Gniadecki and Serup, 1995; Gniadecki et al., 1995;

& 2010 The Society for Investigative Dermatology www.jidonline.org 2359

ORIGINAL ARTICLE

Received 27 October 2009; revised 13 April 2010; accepted 20 April 2010;published online 10 June 2010

1Pharmaceutical Sciences Division, School of Pharmacy, University ofWisconsin-Madison, Madison, Wisconsin, USA and 2Department ofBiochemistry, College of Agricultural and Life Sciences, University ofWisconsin-Madison, Madison, Wisconsin, USA

Correspondence: Margaret Clagett-Dame, Department of Biochemistry,College of Agricultural and Life Sciences, University of Wisconsin-Madison,433 Babcock Drive, Madison, Wisconsin 53706, USA.E-mail: dame@biochem.wisc.edu

Abbreviations: CAGE-3, 2-methylene-19-nor-(20S)-26,27-dimethylene-1a,25-(OH)2D3; hr, hairless; 2MbisP, 2-methylene-19-nor-(20S)-1a-hydroxybishomopregnacalciferol; 2MP, 2-methylene-19-nor-1a-dihydroxyhomopregnacalciferol; 2Mpregna, 2-methylene-19-nor-1a-hydroxypregnacalciferol; 2MtrisP, 2-methylene-19-nor-(20S)-trishomo-1a-hydroxypregnacalciferol; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3;20R-2MbisP, 2-methylene-19-nor-(20R)-1a-hydroxy-bishomopregnacalciferol; VDR, vitamin D receptor

Fujimura et al., 2000) and in humans (Levy et al., 1994).1,25(OH)2D3 can either stimulate or inhibit DNA synthesisin cultured human keratinocytes, depending on the composi-tion of the culture media (Gniadecki, 1996). Thus, the abilityof 1,25(OH)2D3 to promote or inhibit proliferation ordifferentiation may depend on the physiological context,raising the possibility that vitamin D and its analogs couldbe useful as therapeutic agents for other disorders ofkeratinization. Recently, maxacalcitol, an analog of vitaminD3 used in the treatment of psoriasis, was evaluated in therhino mouse and was found to have comedolytic activity(Hayashi et al., 2006). This observation suggests that1,25(OH)2D3 and its analogs may also have utility in thetreatment of comedonal acne.

In addition to its effects on cellular differentiation, thebest-described physiological function of the vitamin Dhormone is to maintain circulating levels of calcium andphosphorus (Jones et al., 1998). However, the ability of1,25(OH)2D3 to increase blood calcium level constitutes themajor side effect in the pharmacological use of 1,25(OH)2D3

and its analogs. The recent generation of 19-nor-1,25(OH)2D3 analogs with modifications in the 2-carbonposition has yielded compounds with physiological selectiv-ity and in some cases, analogs with lower calcemic activity(DeLuca et al., 2007). In this study, we examine the activity ofa series of 2-methylene-19-nor analogs of 1a,25-dihydrox-yvitamin D3 in the skin of the rhino mouse. Herein, we showthat, although all analogs tested induce epidermal thickening,only a subset exert comedolytic activity.

RESULTSThe ability of 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) anda number of structurally modified 2-methylene-19-norderivatives of 1,25(OH)2 D3 and D2 to induce comedolysisand/or epidermal thickening in the rhino mouse wasevaluated as described in Figure 1. Two general groups ofanalogs were studied: those containing a full side chain(containing carbons 20–25; Table 1) and those in whichthe side chain was shortened and the 25-hydroxyl groupeliminated. Figure 2a shows the structure of 1,25(OH)2D3

and the structure common to 2-methylene-19-nor analogs.Previous studies of 19-nor analogs containing a full side chainrevealed a selective activity profile including a higherpotency in inducing cellular differentiation than the parenthormone. Shortened side chain analogs were also previouslyshown to be biologically active in vivo and nearly devoid ofcalcemic liability (Plum et al., 2004).

1,25(OH)2D3 does not induce comedolysis

Mice treated with 1,25(OH)2D3 did not show a reduction incomedone size at either of the two doses tested (6 or19 nmol kgBW

�1 ), compared with vehicle control (Figure 2b–d).In fact, comedone area was significantly increased in both thegroups receiving 1,25(OH)2D3 (Figure 2e). When the effect of1,25(OH)2D3 on epidermal thickness was assessed, only amodest increase (33% above vehicle) was noted at the19 nmol kgBW

�1 dose (Figure 2f) and there was no visual skininflammation or influx of inflammatory cells (neutrophils,

data not shown). Animals in both hormone-treated groupswere exposed to drug, as evidenced by a significant increasein total serum calcium levels (11.6 and 12.0 mg per 100 ml,in the 6 and 19 nmol kgBW

�1 groups, respectively, comparedwith 9.5 and 8.7 mg per 100 ml in the vehicle groups(Figure 2g). Thus, 1,25(OH)2D3 was ineffective in inducingcomedolysis and only the highest dose of the native hormonewas able to modestly increase the epidermal thickness.

2-Methylene-19-nor analogs of vitamin D with a full side chainare not comedolytic

The structure and activities of 19-nor analogs of 1,25(OH)2D3 and D2 containing a 25-hydroxylated side chain andmodified to contain a methylene group in the 2-carbonposition are shown in Table 1. All of them bind with highaffinity to the vitamin D receptor (VDR) in vitro, activatetranscription, and induce bone calcium mobilization, andintestinal calcium transport in vivo (Sicinski et al., 2002;Barycki et al., 2009; see the patent information described inthe section Chemicals under Materials and Methods). Thedose of drug used to test for activity in skin was based onin vivo potency, with the goal of using a dose that wouldproduce a 1.0–3.0 mg per 100 ml increase in serum calciumlevel to ensure sufficient drug exposure. For some analogs, anoncalcemic dose was also examined. After 3 weeks oftreatment, all 2-methylene-19-nor analogs shown in Table 1were active in increasing epidermal thickness, whereas nonewere effective in reducing the comedone area. Epidermal

a

b

Comedone

Figure 1. Comedone area and epidermal thickness determination. Five

images per animal sample were captured. For each image, the (a) area of each

comedone and (b) epidermal thickness were determined with the use of a

Wacom Intuos Tablet linked to the Metamorph Imaging System. Comedone

area measurements were obtained by delineating the perimeter of each utricle

with the tablet; using Metamorph Image Software, an area value for each

individual comedone in pixels was determined. Epidermal thickness

measurements were determined using the distance feature of Metamorph to

determine the thickness of the epidermis at each interfollicular region of the

image. On average, a total of 8–12 measurements of epidermal thickness per

section were collected. Bar¼ 50mm.

2360 Journal of Investigative Dermatology (2010), Volume 130

NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

thickness was increased the most (nearly 3 times that ofvehicle) by the highly potent analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1a,25(OH)2D3 (CAGE-3); thicken-ing was observed at a dose of CAGE-3 as low as0.025 nmol kgBW

�1 and was maximal at a 3.2-fold higher dose(0.079 nmol kgBW

�1 ; Supplementary Figure S1 online). Anincrease in cellularity in the dermis comprised largely ofmononuclear cells and neutrophils was also noted inhematoxylin and eosin sections, and a doubling of neutrophilnumber occurred at the same dose that produced a significantincrease in epidermal thickness (data not shown). However,there was no evidence for an increase in the number ofeosinophils when examined by histological staining (Luna) inthe highest CAGE-3-dose group. Interestingly, an increasein sebaceous remnant area but not number was observedwith increasing doses of the compound starting at0.079 nmol kgBW

�1 (Supplementary Figure S1f online), althoughit is not known whether the amount or composition ofsebaceous lipid was altered. The ability of compounds to

increase epidermal thickness was not due to elevated serumcalcium levels, as the analogs 2-methylene-(20R,25S)-19,26-dinor-1a,25-dihydroxy D3 and CAGE-3 when tested atnoncalcemic doses still produced significant skin thickening(Table 1; Supplementary Figure S1g online).

2-Methylene-19-nor-(20S)-1a-hydroxybishomopregnacalciferol(2MbisP) induces both comedolysis and epidermal thickening

As none of the 19-nor analogs with a full side chain showedcomedolytic activity, the ability of 2MbisP, a low-calcemic19-nor analog that lacks a 25-hydroxyl group and most of thevitamin D side chain, was tested. Remarkably, 2MbisP notonly increased epidermal thickness but also reduced the sizeof comedones in a dose-dependent manner. As shownin Figure 3, a dose of 69 nmol kgBW

�1 was ineffective incomedolysis, but produced a significant increase in epider-mal thickness and some neutrophil infiltration (approximatelya fourfold increase in cell number over vehicle; data notshown). The effect of 2MbisP on epidermal thickening was

Table 1. Biological activity of 19-nor 1,25(OH)2 –D2 and -D3 analogs containing a 25-hydroxylated side chain

Chemical nameStructure(R group)

Dose(nmolkgBW

�1 )

Comedonearea

(% of vehicle)

Epidermalthickness

(% ofvehicle)

Serum calcium(D fromvehicle;

mg per 100 ml)

2-Methylene-1a,25-dihydroxy-(17E)-17(20)-dehydro-19-

norvitamin D3 (Vit-III 17–20E)

25 116±15 216*±13 2.0*±0.1

2-Methylene-(20R, 25S)-19,26-dinor-1a,25-dihydroxyvitamin

D3 (NEL)

7.5

25

156±34

147±24

145*±3.7

189*±11

�0.1±0.4

1.2*±0.2

2-Methylene-19-nor-(20S)-26,27-dimethylene-1a,25-

dihydroxyvitamin D3 (CAGE-3)

0.025

0.079

158±23

198±63

158*±11

277*±14

0.1±0.3

1.4*±0.4

2-Methylene-18,19-dinor-(20S)-1a,25-dihydroxyvitamin D3

(VD-03)

0.62 90±14 225*±5.9 2.5*±0.2

2-Methylene-19-nor-24-epi-1a,25-dihydroxyvitamin

D2 (20R-2MD-24-epi)

2.3 176±55 189*±13 2.6*±0.2

The activities of compounds in binding to the receptor (kd; nM), and in inducing HL60 cell differentiation (IC50; nM) are as follows: 2-methylene-1a,25-dihydroxy-(17E)-17(20)-dehydro-19-norvitamin D3 (Vitamin-III 17–20E) (0.4, 20; US 7,241,752 B2); 2-methylene-(20R,25S)-19,26-dinor-1a,25-dihydroxy-vitamin D3 (NEL) (0.04, 1; US 7,528,122 B2); 2-methylene-19-nor-(20S)-26,27-dimethylene-1a,25-dihydroxyvitamin D3 (CAGE-3) (0.03, 0.04; Sicinskiet al., 2002); 2-methylene-18,19-dinor-(20S)-1a,25-dihydroxyvitamin D3 (VD-03) (0.02, 0.4; Barycki et al., 2009); 2-methylene-19-nor-24-epi-1a,25-dihydroxyvitamin D2 ((20R)2MD-24-epi) (0.04, 0.1; US 7,232,810 B2). Values for 1,25(OH)2D3 are given for comparison (0.07, 4). Significant differencesfrom vehicle are indicated by an asterisk, *Po0.05.

www.jidonline.org 2361

NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

maximal at a dose 0.5 log units higher (218 nmol kgBW�1 ), and

an increase in sebaceous gland area was also seen, but therewas no further increase in neutrophil number. At this dose, asignificant reduction in comedone area was observed (44%),with a maximal effect observed at 690 nmol kgBW

�1 . Higherdoses of compound (up to 2,180 nmol kgBW

�1 ) did not improvethe extent of comedolysis, and produced a significantincrease in serum calcium level (2.1±0.2 mg per 100 mlabove vehicle; data not shown). No redness or swelling of the

skin was noted after 21 days of treatment, although in somecases exfoliation was evident (data not shown).

The activity of 2MbisP in decreasing comedone areaand increasing epidermal thickness did not change whencompared at 3 and 6 weeks, indicating that the effectswere maximal at the earlier time point (SupplementaryFigure S2 online). Groups treated topically with 2MbisP(218 nmol kgBW

�1 ) for 3 or 6 weeks showed a 43% reduction incomedone area, as well as a 230% increase in epidermalthickness compared with the vehicle groups. The ability of2MbisP to increase epidermal thickness was the result of anincrease in cell proliferation as assessed by an increase inbromodeoxyuridine incorporation in the basal cell layer as aresult of this treatment (Supplementary Figure S3 online).

The effect of side chain length on activity

In the next experiment, the importance of side chain length tocomedolytic activity was explored in more detail after3 weeks of topical drug application. 2-methylene-19-nor-1a-hydroxy-pregnacalciferol (2MPregna) and 2-methylene-19-nor-1a-hydroxy-homopregnacalciferol (2MP), each with afurther reduction in side chain length compared with 2MbisP(two and one carbon(s), respectively), as well as a compoundwith an additional carbon compared with 2MbisP,

0

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2-Methylene-19-nor analogs1α,25(OH)2D3

HO

1,25 (OH)2D3

(nmol kg–1BW)

1,25

(O

H) 2

D3

(nm

ol k

g–1 BW

)

Figure 2. Activity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in rhino

mouse skin. (a) Chemical structure of 1,25(OH)2D3 and the structure of

the A ring of the 2-methylene-19-nor analogs. The effect of topical application

of (b) vehicle, (c) 6 nmol kgBW�1 , or (d) 19 nmol kgBW

�1 of 1,25(OH)2D3

on skin morphology. Bar¼ 100mm. (e) Comedone area and (f) epidermal

thickness were measured by image analysis as indicated in the methods

section and Figure 1. (g) Change in total serum calcium level from vehicle

was measured after 21 days of dosing. Dosing solutions were prepared so

that the average mouse (21.3 g) would receive a total of 0.13 or 0.41 nmol

1,25(OH)2D3 for the low and high doses, respectively. Significant differences

from vehicle are indicated by an asterisk, *Po0.05.

69 218 690

69 218 690

69 218 690

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2Mbi

sP (

nmol

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W)

2MbisP (nmol kg–1BW)

*

**

**

*

690

218

Figure 3. Effect of 2MbisP on comedone area, epidermal thickness, and

serum calcium levels. The effect of topical application of (a) vehicle and (b–d)

2MbisP at 69, 218, and 690 nmol kgBW�1 on rhino mouse skin morphology.

Bar¼100 mm. (e) Change in comedone area and (f) epidermal thickness with

increasing doses of 2MbisP. (g) Change in total serum calcium level from

vehicle was measured after 21 days of dosing. Dosing solutions were

prepared so that the average mouse would receive a total of 1.47, 4.64, or

14.9 nmoles at the 69, 218, or 690 nmol kgBW�1 doses, respectively. Significant

differences from vehicle are indicated by an asterisk, *Po0.05.

2362 Journal of Investigative Dermatology (2010), Volume 130

NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

2-methylene-19-nor-(20S)-trishomo-1a-hydroxypregnacalciferol(2MtrisP), were studied. All of the abbreviated sidechain 2-methylene-19-nor analogs, when administered at690 nmol kgBW

�1 , showed significant comedolytic activitycompared with the vehicle control (Figure 4a). At the lowerdose tested (218 nmol kgBW

�1 ), all of the analogs, with theexception of 2MP, produced a slight reduction in comedonearea. All analogs tested also caused a significant increasein epidermal thickness at both doses (Figure 4b). Althoughsmall changes in the side chain length did not alter

comedolytic activity, increasing the length had a pronouncedeffect on the total serum calcium level (Figure 4c). Serumcalcium levels at day 21 remained unchanged for both2-methylene-19-nor-1a-hydroxy-pregnacalciferol, and 2MPat each dose, and for 2MbisP at the lower dose. However,at the highest dose of 2MbisP and both doses of 2MtrisP,serum calcium levels were significantly increased to abovethat of the vehicle group. When the side chain of 2MtrisPwas further extended by two carbons, the comedolyticpotential of the analog could not be tested because ofthe severe hypercalcemia that was apparent by day 4 of theexperiment (data not shown). Therefore, 19-nor-1a,25-(OH)2D3 analogs that significantly reduce the comedonearea at doses that do not cause hypercalcemia wereidentified; however, increasing the length of the sidechain increased calcemic liability. Furthermore, this workshows that a terminal hydroxyl group is not necessary for2-methylene-19-nor analogs of 1,25(OH)2D3 to showcomedolytic activity.

0

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nmol kg–1BW

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2MPregna

2MbisP 2MtrisP2MP2MPregnaR group:

218 690

2MbisP

218 690

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218 690

2MP

218 690

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218 690

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218 690

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Figure 4. 2-Methylene-19-nor analogs of 1,25-dihydroxyvitamin D3

lacking a full side chain possess comedolytic activity. The effect of topical

application of 2-methylene-19-nor-1a-hydroxy-pregnacalciferol (2Mpregna),

2-methylene-19-nor-1a-dihydroxyhomopregnacalciferol (2MP), 2-methylene-

19-nor-(20S)-1a-hydroxybishomopregnacalciferol (2MbisP), and 2-

methylene-19-nor-(20S)-trishomo-1a-hydroxypregnacalciferol (2MtrisP) at

218 and 690 nmol kgBW�1 on (a) comedone area, (b) epidermal thickness,

and (c) serum calcium levels after 3 weeks. The low and high doses of

drug were analyzed in two independent experiments. Animals treated with

2MtrisP at 690 nmol kgBW�1 were evaluated after 2 weeks. The response for

each drug at a given dose was compared with the vehicle control group in

that experiment; the data are shown together on the graph. Significant

differences from vehicle are indicated by an asterisk, *P-value o0.05.

Analog structure in Figure 1 with modification to the R group is shown

above the graphs.

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20R-2MbisP (nmol kg–1BW)

20R-2MbisP (nmol kg–1BW)

*

*

*

Figure 5. 2-Methylene-19-nor-(20R)-1a-hydroxy-bishomopregna-

calciferol (20R-2MbisP) induces comedolysis. (a) Comedone area and (b)

change in total serum calcium level from vehicle measured after 21 days of

dosing. Significant differences from vehicle are indicated by an asterisk,

*Po0.05.

www.jidonline.org 2363

NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

2-Methylene-19-nor-(20R)-1a-hydroxy-bishomopregnacalciferol(20R-2MbisP) is an effective comedolytic agent but is slightly lesspotent than the 20S isomer

The importance of the configuration of the methyl group atcarbon 20 within the 2MbisP structure in comedolyticactivity was further explored. At 218 nmol kgBW

�1 , 20R-2MbisPwas ineffective in reducing the comedone area (Figure 5),whereas a higher dose of 690 nmol kgBW

�1 was similar inefficacy to the 218 nmol kgBW

�1 dose of 20S-2MbisP (seeFigure 3). Furthermore, 20R-2MbisP was able to produce a55% reduction in comedone size without an accompanyingincrease in serum calcium levels. Thus, the 20R isomer of2MbisP seemed to be approximately 0.5 log units less activein comedolytic activity than the 20S configuration. However,both compounds were efficacious at doses that did notincrease blood calcium levels.

DISCUSSIONFrom these studies, a specific class of vitamin D analogs thatalters the size and shape of utricles and thickness of theepidermis in the skin of the rhino mouse in a dose-dependentmanner was identified. Whereas 1,25(OH)2D3 and all 2-methylene-19-nor analogs tested cause an increase inepidermal thickness, only those that lack the 25-hydoxylgroup and have a side chain less than five carbons in lengthexhibit comedolytic activity.

1,25(OH)2D3 functions by binding to the nuclear VDR,and this ligand–receptor complex regulates the transcriptionof target genes (DeLuca, 2008; Haussler et al., 2008). TheVDR is expressed in a number of cell types in the skin,including the keratinocyte (Stumpf et al., 1979; Hosomi et al.,1983; Pillai et al., 1988; Reichrath et al., 1997). All of the2-methylene-19-nor-1,25(OH)2 D3 and D2 analogs testedbind with high affinity to the VDR, and have been shown tobe transcriptionally active in reporter cell systems (Sicinskiet al., 2002; Plum et al., 2004; Barycki et al., 2009; seeTable 1 footnote). However, the compounds that are mosteffective in inducing comedolysis do not have the highestaffinity to the VDR. Therefore, there is a specificity of actionin comedolysis that cannot be explained merely on the basisof the binding affinity to the VDR. It is possible that tissueand/or cell-specific factors present in the skin are responsiblefor the ability of some compounds to produce both ahyperplastic epidermis and a reduction in utricle size.It has been shown that hormone binding alters theconformation of the C-terminus of the VDR, creating abinding surface for the productive interaction with coacti-vator proteins involved in modulating chromatin structureand in interacting with the basal transcriptional machinery(Rochel et al., 2000; Tocchini-Valentini et al., 2001; Vanhookeet al., 2004, 2007). Multiple coactivator proteins have beendescribed, and thus it is possible that the shortened side chainanalogs influence the nature of this interaction in a uniquemanner.

The VDR has been shown to interact with the hairless gene(Hsieh et al., 2003). There are some similarities with respectto defects in hair follicle cycling in VDR knockout andhairless mutant mice (Li et al., 1997; Yoshizawa et al., 1997;

Zarach et al., 2004); however, it should be noted that thefunction of the VDR in this process is independent of the VDRligand (Skorija et al., 2005). Cell transfection studies suggestthat Hairless (Hr) may alter the responsiveness of the VDR toits ligand, but evidence for this in vivo is currently lacking.Whether the rhino mouse serves as an accurate predictor ofall vitamin D analog-related effects in humans remains to beestablished.

The shortened side chain 2-methylene-19-nor analogsshow many similarities to the effects of retinoids in the rhinomodel, including hyperproliferation of the epidermis andreversion of utricles to follicles, or in some cases, theircomplete involution (Kligman and Kligman, 1979). It hasbeen proposed that the ability of retinoids to induceepidermal proliferation is key to their ability to reduce thesize of the utricle diameter (Ashton et al., 1984). However,our studies clearly show that vitamin D analogs that increasethe thickness of the epidermis do not always have comedo-lytic activity. Furthermore, analogs that possess comedolyticactivity induce hyperplasia at doses at which they are not yetactive as comedolytic agents. Only one vitamin D analog haspreviously been reported to have comedolytic activity in therhino mouse and it was suggested that the resultantcomedolysis was due to an induction of hyperplasia (Hayashiet al., 2006). Our work supports the conclusion that, at leastfor vitamin D analogs, activity in increasing epidermalproliferation or thickness, per se, is not sufficient to inducecomedolysis.

Vitamin D analogs are used to treat psoriasis and theycause remission of psoriatic plaques by inhibiting keratinocytehyperproliferation and inducing differentiation (Reichrathet al., 1997). In this study, however, an increase in epidermalthickness was observed in response to topical application ofall 2-methylene-19-nor analogs. In normal human skin, avitamin D analog used in the treatment of psoriasis,calcipotriol, has been shown to produce a significantincrease in skin thickness after 3 weeks of topical applicationin vivo (Levy et al., 1994). In addition to epidermalthickening, the size of sebaceous gland remnants increasedwith the comedolytic compound 2MbisP, as well as withthe noncomedolytic analog CAGE-3. The increase in thesebaceous area and thickening of the epidermis both seem toresult from increased cell proliferation (SupplementaryFigure S3 online). Interestingly, enlargement of sebaceousglands was also reported after topical treatment of therhino mouse with retinoid (Kligman and Kligman, 1979;Zheng et al., 1993). In vitro studies on sebaceous gland cellshave shown that 1,25(OH)2D3 exerts a biphasic effecton sebocyte proliferation that is dependent on dose andcell culture conditions (Kramer et al., 2009). Thus, theeffect of vitamin D compounds on skin seems to differdepending on the physiological or pathological state ofthe organism at the time of application. This highlightsthe potential of vitamin D analogs as treatment forkeratinization disorders such as psoriasis and acne, as wellas in conditions in which an induction of epidermalthickening is desired, such as adjunct therapy to corticoster-oid drug use.

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NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

The major limitation in the use of 1,25(OH)2D3 and itsanalogs in clinical medicine is the potent effect that thesecompounds have on calcium metabolism. Pharmacologicaladministration of the native hormone by both oral and topicalroutes can lead to hypercalcemia (Langner et al., 1993;Osborn et al., 1995). A recent major advance has been thedevelopment of vitamin D analogs with lesser calcemicliability. The analogs 2MbisP, 2MP, and 2-methylene-19-nor-1a-hydroxy-pregnacalciferol given intraperitoneally havebeen shown to be very active in inducing the 24-hydroxylaseenzyme in skin and suppressing the secretion of parathyroidhormone into the bloodstream in vivo, while having verylittle effect, if any, on intestinal calcium absorption orcalcium mobilization from bone (Plum et al., 2004; DeLucaet al., 2007). These compounds, as well as the isomer20R-2MbisP, also function as comedolytic agents whenapplied topically to the rhino mouse at doses that cause noincrease in serum calcium levels. Increasing the vitamin Dside chain up to five carbons in length does not alter thecomedolytic potential of the molecule, but it does cause anincrease in calcemic liability. Thus, efficacy can be obtainedwith specific analogs in the absence of perturbing bloodcalcium.

In conclusion, we have shown that a subset of2-methylene-19-nor-1,25(OH)2D3 analogs exert potent co-medolytic activity in rhino mouse skin, whereas all analogstested, as well as the native hormone, increase the epidermalthickness. Thus, the low-calcemic shortened side chain2-methylene-19-nor-1,25(OH)2D3 analogs described hereinrepresent a class of vitamin D analogs with potential for usein skin disorders including acne.

MATERIALS AND METHODSChemicals

1,25(OH)2D3, 2MbisP, 2MP, and 2-methylene-19-nor-1a-hydroxy-

pregnacalciferol were synthesized and purified by SAFC (Madison,

WI, USA) using previously described methods and as modified by

SAFC (Sicinski et al., 1998; Plum et al., 2004). 20R-2MD-24-epi

(DeLuca HF, Sicinski RR, Gowlugari S, inventors; Wisconsin Alumni

Research Foundation Madison, WI, USA assignee. 2-Methylene-19-

nor-Vitamin D Compounds. United States patent US 7,232, 810 B2,

19 June 2007), Vit-III 17-20E (DeLuca HF, Tadi BP, Plum LA, Clagett-

Dame M, inventors; Wisconsin Alumni Research Foundation

Madison, WI, USA assignee. 17,20(E)-dehydro vitamin D analogs

and their uses. United States patent US 7,241,752 B2, 10 July 2007),

2-methylene-(20R,25S)-19,26-dinor-1a,25-dihydroxy D3 (DeLuca

HF, Chiellini G, Gryzwacz P, Plum LA, Clagett-Dame M, inventors;

Wisconsin Alumni Research Foundation Madison, WI, USA assign-

ee. Vitamin D analog-2-methylene-(20R,25S)-19,26-dinor-1a,25-

dihydroxy D3, Methods and uses thereof. United States patent US

7,528,122 B2, 5 May 2009), CAGE-3 (Sicinski et al., 2002), VD-03

(Barycki et al., 2009), 20R-2MbisP (DeLuca HF, Plum LA, Clagett-

Dame M, inventors; Wisconsin Alumni Research Foundation,

Madison, WI, USA assignee. 2-Methylene-19-nor-(20R)-1alpha-

hydroxy-bishomopregnacalciferol. United States patent US 2006/

0135799 A1, 22 June 2006), and 2MtrisP (DeLuca HF, Plum LA,

Clagett-Dame M, inventors; Wisconsin Alumni Research Foundation

Madison, WI, USA assignee. 2-Methylene-19-nor-(20S)-1alpha-

hydroxy-trishomopregnacalciferol. United States patent US 2006/

0135798 A1, 22 June 2006) were prepared as described. The

compounds were determined by HPLC and nuclear magnetic

resonance to be 498% pure.

Compounds were dissolved in ethanol and their concentration

was determined by a UV spectrophotometer using a molar extinction

coefficient of 42,000 at a wavelength of 252 nm for the 2-methylene-

19-nor analogs and 18,200 at 265 nm for 1,25(OH)2D3. Dosing

solutions were prepared in an ethanol:propylene glycol vehicle

(70:30, v/v) so that the dose administered to a 30 g mouse was

delivered in a maximum volume of 100ml.

Animals

All procedures involving animals were reviewed and approved by

the University Committee on Use and Care of Animals at the

University of Wisconsin Madison, WI, USA, which is a facility

accredited by the Association for the Assessment and Accreditation

of Laboratory Animal Care International. Rhino mice (RHJ/LeJ),

heterozygote female (hrrh-J/þ ) and homozygote male (hrrh-J/hrrh-J)

pairs, were obtained from Jackson Laboratory (Bar Harbor, ME).

Homozygote rhino mice (hrrh-J/hrrh-J) were obtained from an in-house

breeding program. Homozygote rhino mice were assigned to study

groups at 6–8 weeks of age, with three males and three females per

group (n¼ 6 per group) with an average weight of 21.3±0.1 g.

Compounds were applied on the entire dorsal (back) area of the

mouse once a day for 21 consecutive days. Animals were weighed

three times per week, and the dose of drug administered was

adjusted weekly on the basis of body weight. Blood was taken

24 hours after the seventh and twenty-first dose by maxillary bleed

for determination of serum calcium levels (see Supplementary

Materials and Methods for details). Skin biopsy samples were

collected 3 days after the last application of compounds.

Skin histologySkin biopsy samples for histology (approximately 6 mm in length)

were laid flat on wooden sticks and fixed overnight in 4%

paraformaldehyde, dehydrated with increasing percentages of

methanol, and embedded in paraffin. Five 10 mm sections were

prepared from each biopsy sample at 150mm intervals. Slices

were stained with Gill’s hematoxylin and eosin, imaged, and

analyzed using MetaMorph Software (Molecular Devices, Down-

ingtown, PA). The area of every comedone in each of five slices was

calculated and the average comedone area was determined for each

mouse. Similarly, epidermal thickness was determined by measuring

the thickness of the epidermis between the comedones

using MetaMorph Software. The area of sebaceous glands was

measured in a similar manner in a subset of experiments. From

these data, mean was determined for the vehicle group and for each

group of animals, the percent of vehicle was determined and

expressed as mean (percent of vehicle)±SEM. The average

comedone area in vehicle-treated mice in these studies was

6615±345mm2 and the thickness of the epidermis was

21.4±0.3mm. Neutrophils were counted in random fields (five per

animal; 400-fold magnification) for the 1,25(OH)2D3

(19 nmol kgBW�1 ), 2MbisP (69, 218, 690 nmol kgBW

�1 ) and CAGE-3

(0.0079, 0.025, 0.079, 0.25 nmol kgBW�1 ) dosing groups. Statistical

analysis of variance was performed as described in Supplementary

Materials and Methods.

www.jidonline.org 2365

NJ Nieves et al.Vitamin D Analogs with Comedolytic Activity

CONFLICT OF INTERESTThe authors are listed as inventors on patents assigned to the WisconsinAlumni Research Foundation that relate to a number of compoundsdescribed in this report. LAP, HFD, and MCD are officers in DeltanoidPharmaceuticals.

ACKNOWLEDGMENTSWe thank Shinobu Miyasaki, Mary Kaiser, and Wendy Hellwig of theDepartment of Biochemistry for their technical assistance. We also thankLaura Vanderploeg of the Biochemistry Media Lab for the artwork andNicholas S Keuler of the College of Agriculture and Life Sciences statisticsgroup for help in statistical analysis. We also thank Ruth Sullivan, DVM, PhDfor assistance in evaluation of hematoxylin and eosin sections. Nirca Nievesreceived support from an Advanced Opportunity Fellowship from theUniversity of Wisconsin-Madison Graduate School. This work was alsosupported in part by a research grant to the University of Wisconsin-Madisonfrom Deltanoid Pharmaceuticals.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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