Updated ResultsUpdated Results::From October 2007 to September 2009, thirty nine subjects have been From October 2007 to September 2009, thirty nine subjects have been enrolled in the study: 26 Caucasian (CC), 12 African American (AA), enrolled in the study: 26 Caucasian (CC), 12 African American (AA), and 1 Asian. Four subjects were removed from the study due to and 1 Asian. Four subjects were removed from the study due to exclusionary criteria: a diagnosis of colorectal cancer, non-compliance, exclusionary criteria: a diagnosis of colorectal cancer, non-compliance, elevated serum PSA (>10ng/mL), and voluntary withdrawal. Twenty two elevated serum PSA (>10ng/mL), and voluntary withdrawal. Twenty two subjects have completed the study and most of them have indicated that subjects have completed the study and most of them have indicated that they will continue taking the vitamin Dthey will continue taking the vitamin D3 3 daily supplement. The average daily supplement. The average

VD3 pre-treatment levels by race were AA 22.48ng/ml (n=12), and CC VD3 pre-treatment levels by race were AA 22.48ng/ml (n=12), and CC 36.50ng/ml (n=23). Supplementation with 4,000 IU/day appears to 36.50ng/ml (n=23). Supplementation with 4,000 IU/day appears to correct all cases of vitamin D deficiency within 8-16 weeks, although the correct all cases of vitamin D deficiency within 8-16 weeks, although the magnitude of the response varies among subjects. No adverse events have magnitude of the response varies among subjects. No adverse events have been observed thus far. We observed that in most cases, PSA levels been observed thus far. We observed that in most cases, PSA levels remain relatively constant. Overall, there was no statistically significant remain relatively constant. Overall, there was no statistically significant change in PSA over the course of the study. There was no obvious change in PSA over the course of the study. There was no obvious correlation between serum PSA levels and the results of the repeat biopsy correlation between serum PSA levels and the results of the repeat biopsy in the 13 subjects whom completed the study and had a repeat biopsy. in the 13 subjects whom completed the study and had a repeat biopsy. Out of these 13 subjects, 7 are defined as responders (because they had a Out of these 13 subjects, 7 are defined as responders (because they had a decrease in the number of positive cores) and 6 are defined as non-decrease in the number of positive cores) and 6 are defined as non-responders (because they had no decrease in the number of positive responders (because they had no decrease in the number of positive cores). These preliminary values correspond to an estimated response rate cores). These preliminary values correspond to an estimated response rate of 54% with a 95% confidence interval for the true response rate of of 54% with a 95% confidence interval for the true response rate of (25%, 81%). While the confidence interval is wide, it is informative by (25%, 81%). While the confidence interval is wide, it is informative by suggesting that the true response rate is greater than 25%. No patient had suggesting that the true response rate is greater than 25%. No patient had an increase in Gleason score on repeat biopsy after VD3 an increase in Gleason score on repeat biopsy after VD3 supplementation. PSA levels, clinical outcomes, and results of repeat supplementation. PSA levels, clinical outcomes, and results of repeat biopsies continue to be followed. biopsies continue to be followed.

BibliographyBibliography::1. Miller GJ, Stapleton GE, Ferrara JA, Lucia MS, Pfister S, Hedlund TE, Upadhya P. 1. Miller GJ, Stapleton GE, Ferrara JA, Lucia MS, Pfister S, Hedlund TE, Upadhya P. The human The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1α,25-prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1α,25-dihydroxyvitamin Ddihydroxyvitamin D33. . Cancer Res Cancer Res 5252: 515-520, 1992.: 515-520, 1992.

2. Barreto AM, Schwartz GG, Woodruff R, Cramer SD. 25-hydroxyvitamin D2. Barreto AM, Schwartz GG, Woodruff R, Cramer SD. 25-hydroxyvitamin D33, the prohormone of , the prohormone of

1α,25-dihydroxyvitamin D1α,25-dihydroxyvitamin D33, inhibits the proliferation of primary prostatic epithelial cells. , inhibits the proliferation of primary prostatic epithelial cells. Cancer Cancer

res Epidemiol Biomarkers Prev res Epidemiol Biomarkers Prev 99: 265-270, 2000.: 265-270, 2000.3. Chen TC, Wang L, Whitlach LW, Flanagan JN, Holick MF. Prostatic 25-hydroxyvitamin D-1α-3. Chen TC, Wang L, Whitlach LW, Flanagan JN, Holick MF. Prostatic 25-hydroxyvitamin D-1α-hydroxylase and its implication in prostate cancer. hydroxylase and its implication in prostate cancer. J Cell Biochem J Cell Biochem 8888: 315-322, 2003.: 315-322, 2003.4. Schwartz GG, Whitlach LW, Chen TC, Lokeshwar BL, Holick MF. Human prostate cells 4. Schwartz GG, Whitlach LW, Chen TC, Lokeshwar BL, Holick MF. Human prostate cells synthesize 1,25(OH)synthesize 1,25(OH)2 2 DD3 3 from 25(OH)Dfrom 25(OH)D33. . Cancer Epidemiol Biomarkers Prev Cancer Epidemiol Biomarkers Prev 77: 391-395, 1998.: 391-395, 1998.

5. Hsu J-Y, Feldman D, McNeal JE, Peehl DM. 5. Hsu J-Y, Feldman D, McNeal JE, Peehl DM. Reduced 1α-hydroxylase activity in human Reduced 1α-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25(OH)Dprostate cancer cells correlates with decreased susceptibility to 25(OH)D33-induced growth -induced growth

inhibition. inhibition. Cancer Res Cancer Res 6161: 2852-2856, 2001.: 2852-2856, 2001.6. Zao XY, Peehl DM, Navone NM, Feldman D. 1α,25-dihydroxyvitamin D6. Zao XY, Peehl DM, Navone NM, Feldman D. 1α,25-dihydroxyvitamin D3 3 inhibits prostate inhibits prostate

cancer cell growthcancer cell growth by androgen-dependent and androgen-independent mechanisms. by androgen-dependent and androgen-independent mechanisms. Endocrinology Endocrinology 141141: 2548-2556, 2000.: 2548-2556, 2000.7. Yang ES, Maiorino CA, Roos BA, Knight SR, Burnstein KL. Vitamin D-mediated growth 7. Yang ES, Maiorino CA, Roos BA, Knight SR, Burnstein KL. Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer. inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer. Molecular & Molecular & cellular Endocrinology cellular Endocrinology 186186: 69-79, 2007.: 69-79, 2007.8. Krishnan AV, Peehl DM, Feldman D. Inhibition of prostate cancer growth by vitamin D: 8. Krishnan AV, Peehl DM, Feldman D. Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression. Regulation of target gene expression. J Cellular Biochem J Cellular Biochem 8888: 363-371, 2003.: 363-371, 2003.9. Moreno J, Krishnan AV, Swami S, Nonn L, Peehl DM, Feldman D. Regulation of prostaglandin 9. Moreno J, Krishnan AV, Swami S, Nonn L, Peehl DM, Feldman D. Regulation of prostaglandin metabolism by calcitriol attenuates growth stimulation in prostate cancer cells. metabolism by calcitriol attenuates growth stimulation in prostate cancer cells. Cancer research Cancer research 6565: : 7917-7925, 2005.7917-7925, 2005.10. Bao BY, Yeh SD, Lee YF. 1α,25-dihydroxyvitamin D10. Bao BY, Yeh SD, Lee YF. 1α,25-dihydroxyvitamin D3 3 inhibits prostate cancer cell invasion via inhibits prostate cancer cell invasion via

modulation of selective proteases. modulation of selective proteases. Carcinogenesis Carcinogenesis 2727: 32-42, 2006.: 32-42, 2006.11. Nonn L, Peng L, Feldman D, Peehl DM. Inhibition of p38 by vitamin D reduces interleukin-6 11. Nonn L, Peng L, Feldman D, Peehl DM. Inhibition of p38 by vitamin D reduces interleukin-6 production in normal prostate cells via mitogen-activated protein kinase phosphatase 5: production in normal prostate cells via mitogen-activated protein kinase phosphatase 5: implications for prostate cancer prevention by vitamin D. implications for prostate cancer prevention by vitamin D. Cancer Research Cancer Research 6666: 4516-4524, 2006.: 4516-4524, 2006.12. Wang TT, Tavera-Mendoza LE, Laperriere D, Libby E, MacLeod NB, Nagai Y, Bourdeau V, 12. Wang TT, Tavera-Mendoza LE, Laperriere D, Libby E, MacLeod NB, Nagai Y, Bourdeau V, Konstorum A, Lallemant B, Zhang R, Mader S, White JH. Large-scale in silico and microarray-Konstorum A, Lallemant B, Zhang R, Mader S, White JH. Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes. based identification of direct 1,25-dihydroxyvitamin D3 target genes. Mol EndocrinolMol Endocrinol 1919: 2685-: 2685-2695, 2005.2695, 2005.13. Schwartz GG. Vitamin D and the Epidemiology of Prostate Cancer. 13. Schwartz GG. Vitamin D and the Epidemiology of Prostate Cancer. Semin DialSemin Dial, , 1818: 276–289, : 276–289, 2005.2005.  

Conclusion:Conclusion: These preliminary observations reveal no adverse These preliminary observations reveal no adverse effects from the use of 4000 IU of VD3 daily. effects from the use of 4000 IU of VD3 daily. African American men in this population had African American men in this population had lower base-line vitamin D levels than Caucasians.lower base-line vitamin D levels than Caucasians. PSA changes did not correlate with changes in PSA changes did not correlate with changes in repeat biopsy results. repeat biopsy results. A trend was seen for decreased number of A trend was seen for decreased number of positive cores after VD3 supplementation.positive cores after VD3 supplementation. No patient had an increase in Gleason score after No patient had an increase in Gleason score after VD3 supplementation.VD3 supplementation.

UVB (290-315 nm)

Major Source: Sun

Minor Source: DietaryVitamin D2 : Plants/supplementsVitamin D3: Fish (cod liver oil), meat, fortified milk, egg yolk, butter

Vitamin D3 (Cholecalciferol)

7-dehydrocholesterol in skin

25-hydroxyvitamin D3

25(OH) D3

25-hydroxylase

1-hydroxylase1,25-dihydroxyvitamin D3

Calcium absorption (small intestine) Urinary calcium reabsorption (kidney) Bone mineralization

Parathyroid hormone

()

( )(SPF > 8, clothes, glass)

5’ 3’

Nucleus

Vitamin D3 pathway in SKIN

Biological

Response

Anti-proliferation

Pro-differentiation

Immunomodulation

VDRE

RXR VDR

BloodUVB

CYP27B1CYP27A11,25-D3

OH

1,25-D3

OH

1,25D3

OH OH

7-DHC

DRIP

OH

25-D3

D3

Pre- and Post-Treatment PSA and No. of + Cores for Pre- and Post-Treatment PSA and No. of + Cores for Patients that have Completed the Trial and had Re-biopsyPatients that have Completed the Trial and had Re-biopsy

Pre-treatment with VD3 Post-treatment with VD3

25(OH)DLevelsng/ml

PSA Bx: + cores(Total 12

cores)

25(OH)DLevelsng/ml

PSA Bx: + cores(Total 12 cores)

57.6 3.46 1 (+2 PIN) 67.5 3.64 012.6 3.98 4 (+1 PIN) 51.1 4.55 0 (1 PIN)

32.3 3.33 6 63.3 3.76 517.1 5.65 2 (+1PIN) 84.8 4.94 524.3 3.57 1 50.6 4.53 017.3 6.28 2 65.9 7.11 330.4 1.5 1 50.7 0.98 035.5 0.75 1 69.3 0.67 235.4 4.13 2 57.9 4.83 427 4.21 5 56.0 6.45 2

28.5 5.48 3 51.7 10.1 475 4.56 1 84.1 4.55 2

29.2 6.38 2 48.2 10.8 1PIN: Prostatic Intraepithelial NeoplasiaPIN: Prostatic Intraepithelial Neoplasia

Support:Support:This study was supported by The Cancer Treatment Research Foundation/ This study was supported by The Cancer Treatment Research Foundation/ Gateway for Cancer Research Clinical Investigation Grant # G-06-068Gateway for Cancer Research Clinical Investigation Grant # G-06-068

Daily Vitamin D Supplementation in the Active Surveillance of Daily Vitamin D Supplementation in the Active Surveillance of Low-Risk Prostate CancerLow-Risk Prostate Cancer

Medical University of South Carolina, Charleston, SCMedical University of South Carolina, Charleston, SC

L. Cannick, MD; D.T. Marshall, MD, MS; S.J. Savage, MD; L.H. Ambrose, RN; Elizabeth Garrett-Mayer, PhD; L. Cannick, MD; D.T. Marshall, MD, MS; S.J. Savage, MD; L.H. Ambrose, RN; Elizabeth Garrett-Mayer, PhD; S. Gattoni-Celli, MDS. Gattoni-Celli, MD

AbstractAbstract:Purpose: Purpose: Vitamin D inhibits the stress-activated protein kinase p38, an Vitamin D inhibits the stress-activated protein kinase p38, an activator of the pro-inflammatory cytokine interleukin 6, implicated in activator of the pro-inflammatory cytokine interleukin 6, implicated in the initiation and progression of prostate cancer.the initiation and progression of prostate cancer. The objective of this The objective of this study is to determine if daily doses of vitamin D3 (VD3) at 4,000 IU by study is to determine if daily doses of vitamin D3 (VD3) at 4,000 IU by mouth, taken for 12 months will result in decreased PSA levels in mouth, taken for 12 months will result in decreased PSA levels in patients with low-risk prostate cancer on active surveillance, and to patients with low-risk prostate cancer on active surveillance, and to compare percent positive prostate cores pre and post treatment. compare percent positive prostate cores pre and post treatment. Methods: Methods: 80 male patients with early stage low risk prostate cancer, 80 male patients with early stage low risk prostate cancer, serum PSA a serum PSA value of ≤10.0 ng/ml, and a Gleason score of 6 serum PSA a serum PSA value of ≤10.0 ng/ml, and a Gleason score of 6 or less will be enrolled. All subjects will be monitored via active or less will be enrolled. All subjects will be monitored via active surveillance for at least one year, which will include routine labs every 8 surveillance for at least one year, which will include routine labs every 8 weeks and a prostate biopsy recommended at the end of 12 months. weeks and a prostate biopsy recommended at the end of 12 months. Subjects will be given 4,000 IU of (VD3) each day. Upon completion of Subjects will be given 4,000 IU of (VD3) each day. Upon completion of the protocol the patients may elect to continue with active surveillance or the protocol the patients may elect to continue with active surveillance or proceed to definitive treatment.proceed to definitive treatment.  Results: Results: From October 2007 to April 2009, 33 subjects have enrolled From October 2007 to April 2009, 33 subjects have enrolled onto the study, 12 African American (AA), 20 Caucasian (CC), and 1 onto the study, 12 African American (AA), 20 Caucasian (CC), and 1 Asian. One subject was terminated because he was diagnosed with Asian. One subject was terminated because he was diagnosed with colorectal cancer shortly after enrollment; a second was removed due to colorectal cancer shortly after enrollment; a second was removed due to noncompliance with VD3 supplementation. 10 subjects have completed noncompliance with VD3 supplementation. 10 subjects have completed treatment thus far. For subjects with VD3 levels <20ng/ml (severely treatment thus far. For subjects with VD3 levels <20ng/ml (severely deficient) the average pre-treatment PSA was 5.183 (n=9), and for deficient) the average pre-treatment PSA was 5.183 (n=9), and for subjects with VD3 levels >20ng/ml their average PSA was 4.02 (n=20). subjects with VD3 levels >20ng/ml their average PSA was 4.02 (n=20). The average VD3 levels by race were AA 22.48ng/ml (n=12), and The average VD3 levels by race were AA 22.48ng/ml (n=12), and Caucasian 36.18ng/ml (n=19). All patients’ VD3 levels rose above 20 Caucasian 36.18ng/ml (n=19). All patients’ VD3 levels rose above 20 ng/ml after only 8 weeks of supplementation. There have been no ng/ml after only 8 weeks of supplementation. There have been no toxicities associated with the trial. For the 21 patients with at least 5 toxicities associated with the trial. For the 21 patients with at least 5 months of VD3 supplementation, 4 were VD3 deficient prior to initiation months of VD3 supplementation, 4 were VD3 deficient prior to initiation of supplementation and 17 were not. The average increase in PSA during of supplementation and 17 were not. The average increase in PSA during this time frame for these 21 patients was 0.028 ng/ml for the 4 VD3 this time frame for these 21 patients was 0.028 ng/ml for the 4 VD3 deficient patients and 1.075 ng/ml for the 17 patients who were not VD3 deficient patients and 1.075 ng/ml for the 17 patients who were not VD3 deficient at the initiation of VD3 supplementation. Of the 10 subjects that deficient at the initiation of VD3 supplementation. Of the 10 subjects that have completed the study the average pre-treatment PSA was 3.88 and have completed the study the average pre-treatment PSA was 3.88 and post-treatment PSA was 4.03. Four of five patients who have had post-post-treatment PSA was 4.03. Four of five patients who have had post-study prostate biopsies have had a decrease in the number of positive study prostate biopsies have had a decrease in the number of positive cores.cores.  Conclusions: Conclusions: These preliminary observations reveal no adverse effects These preliminary observations reveal no adverse effects from the use of 4,000 IU of VD3 daily. These data also suggest that from the use of 4,000 IU of VD3 daily. These data also suggest that vitamin D levels are lower in AA prostate cancer patients than in CC vitamin D levels are lower in AA prostate cancer patients than in CC patients. The use of VD3 supplementation may decrease PSA velocity in patients. The use of VD3 supplementation may decrease PSA velocity in men with early-stage, low-risk prostate cancer, especially those who are men with early-stage, low-risk prostate cancer, especially those who are severely vitamin D-deficient (<20 ng 25-OH- Dseverely vitamin D-deficient (<20 ng 25-OH- D3 3 per mL of serum). per mL of serum).

Introduction:Introduction:It has been known for some time that human prostate cells express the It has been known for some time that human prostate cells express the vitamin D receptor (1). Subsequently it was reported that normal prostate vitamin D receptor (1). Subsequently it was reported that normal prostate cells also synthesize 1-calcidiol (2,3), as prostate cells express CYP27A1, cells also synthesize 1-calcidiol (2,3), as prostate cells express CYP27A1, a vitamin Da vitamin D33 25-hydroxylase, and the 25(OH)D 25-hydroxylase, and the 25(OH)D33 1α-hydroxylase (4). 1α-hydroxylase (4).

Furthermore, neoplastic progression in prostate tissue appears to be Furthermore, neoplastic progression in prostate tissue appears to be associated with loss of 25(OH)Dassociated with loss of 25(OH)D33 1α-hydroxylase activity (5). 1α-hydroxylase activity (5).

Additional studies have shown that 1,25(OH)Additional studies have shown that 1,25(OH)2 2 DD33 can: a) inhibit the can: a) inhibit the

proliferation of PCa cells by androgen-dependent and androgen-proliferation of PCa cells by androgen-dependent and androgen-independent mechanisms (6,7); b) independent mechanisms (6,7); b) induce the expression of insulin growth induce the expression of insulin growth

factor binding protein-3 (IGFBP-3), which increases the levels of the cell-factor binding protein-3 (IGFBP-3), which increases the levels of the cell-cycle inhibitor p21 (8); c) suppress the expression of COX-2, the key cycle inhibitor p21 (8); c) suppress the expression of COX-2, the key enzyme for the synthesis of prostaglandins, mediators of inflammation and enzyme for the synthesis of prostaglandins, mediators of inflammation and thought to be important for cancer progression (9); d) decrease matrix thought to be important for cancer progression (9); d) decrease matrix metalloproteinases and cathepsin activities, while increasing the activities metalloproteinases and cathepsin activities, while increasing the activities of their counterparts, tissue inhibitors of metalloproteinase-1 and cathepsin of their counterparts, tissue inhibitors of metalloproteinase-1 and cathepsin inhibitors, suggesting that vitamin D-based therapies may benefit patients inhibitors, suggesting that vitamin D-based therapies may benefit patients with advanced PCa (10); e) inhibit the stress-activated protein kinase p38, with advanced PCa (10); e) inhibit the stress-activated protein kinase p38, an activator of the pro-inflammatory cytokine interleukin 6, implicated in an activator of the pro-inflammatory cytokine interleukin 6, implicated in the initiation and progression of PCa (11). the initiation and progression of PCa (11).

Furthermore, recent studies have demonstrated that the vitamin D receptor Furthermore, recent studies have demonstrated that the vitamin D receptor may recognize cognate vitamin D response elements present within the may recognize cognate vitamin D response elements present within the regulatory sequences of hundreds of human genes, implicating vitamin D regulatory sequences of hundreds of human genes, implicating vitamin D in a vast network of gene regulation, and underlying its broad in a vast network of gene regulation, and underlying its broad physiological actions (12). Therefore, it appears that vitamin Dphysiological actions (12). Therefore, it appears that vitamin D33

supplementation helps to promote the differentiation of prostate cancer supplementation helps to promote the differentiation of prostate cancer cells, maintains the differentiated phenotype of prostate epithelial cells, cells, maintains the differentiated phenotype of prostate epithelial cells, and can induce prostate cancer cell death, raising the possibility that and can induce prostate cancer cell death, raising the possibility that vitamin D deficiency over time promotes the progression of subclinical vitamin D deficiency over time promotes the progression of subclinical prostate cancer to clinical disease (13).prostate cancer to clinical disease (13).

0 50 100 150 200 250 300 350

Time in Days

PSA

0.2

0.5

12

510

Serum PSA levels (in mg/mL) Over Time Serum PSA levels (in mg/mL) Over Time Time=0 represent the serum PSA level at enrollment. Time=0 represent the serum PSA level at enrollment. Red Red lines lines represent serum PSA levels of responders (subjects represent serum PSA levels of responders (subjects who experienced a decrease in positive cores at repeat who experienced a decrease in positive cores at repeat biopsy); biopsy); green lines green lines represent serum PSA levels of non-represent serum PSA levels of non-responders (subjects who experienced an increase of responders (subjects who experienced an increase of positive cores at repeat biopsy). positive cores at repeat biopsy). Black lines Black lines represent represent serum PSA levels of subjects who have yet to undergo a serum PSA levels of subjects who have yet to undergo a repeat biopsy.repeat biopsy.