Patent Application
20050113348
The renin-angiotensin system is involved in blood pressure, electrolyte and volume homeostasis. Inappropriate activation of the renin-angiotensin system may lead to hypertension, which is a risk factor for stroke, myocardial infarction, congestive heart failure, progressive atherosclerosis and renal failure. The mechanisms of renin-angiotensin processes are not well understood.
Renin, a protease synthesized and secreted predominantly by the juxtaglomerular (JG) apparatus in the nephron is a rate-limiting component of the system. Renin cleaves angiotensin (Ang) I from liver-derived angiotensinogen, which is then converted to Ang II by the angiotensin-converting enzyme. Ang II, through binding to its receptors, exerts diverse actions that affect the electrolyte, volume and blood pressure homeostasis. Inappropriate stimulation of the renin-angiotensin system has been associated with hypertension, heart attack and stroke.
Renin-producing granulated cells are mainly located in the afferent glomerular arterioles in the kidney. Renin secretion is regulated by renal perfusion pressure, renal sympathetic nerve activity and tubular sodium load. Renin secretion is stimulated by factors such as prostaglandins, NO and adrenomedullin, and inhibited by other factors, including Ang II (feedback), endothelin, vasopressin and adenosine. Stimulation of renin secretion is often mediated by an increase in intracellular cAMP and is accompanied by increases in renin gene transcription.
Relationships have been suggested between the vitamin D pathways and blood pressure. Vitamin D is a primary regulator of calcium homeostasis. Genetic inactivation of either the vitamin D receptor (VDR), a member of the nuclear receptor superfamily that mediates the action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or 25-hydroxyvitamin D3 1α-hydroxylase, the rate-limiting enzyme for the biosynthesis of 1,25(OH)2D3, results in impaired calcium homeostasis, leading to hypocalcemia, secondary hyperparathyroidism and rickets. However, the mechanism underlying the relationship between vitamin D, blood pressure and plasma renin activity is unknown.
Vitamin D and vitamin D analogs or derivatives disclosed herein are new anti-hypertensive agents to control renin production and blood pressure. Vitamin D is a negative regulator of renin expression in vivo.
Disruption of the vitamin D signaling pathway leads to a deregulated elevation of renin expression, and an increase in serum vitamin D levels leads to a suppression of renin expression. Vitamin D is an endocrine suppressor for renin biosynthesis. Mutant mice that lack a vitamin D receptor have much higher production of renin and angiotensin II and develop hypertension and cardiac hypertrophy, whereas injection of 1,25-dihydroxyvitamin D3 into normal mice reduces renin synthesis. Vitamin D analogs with less calcemic effect and higher potency than vitamin D are used for suppressing rennin biosynthesis and regulating blood pressure.
A cell culture system for vitamin D analog screening was developed to identify a group of vitamin D analogs, including Gemini compounds that have more potent renin-suppressing activity than 1,25-dihyroxyvitamin D3 were identified. Gemini compounds suppress renin expression, and a few are 10 to 100 times more potent that 1,25 (OH)2D3 (
A method of suppressing renin expression in a mammal includes the steps of:
A method of reducing blood pressure in a mammal includes the steps of:
The pharmaceutical composition may have an acceptable carrier and other sustained/extended release formulations. More than type of one vitamin D analogue or derivative can also be formulated as a pharmaceutical composition and administered to mammals including humans.
In an aspect, a vitamin D analogue or derivative may include a Gemini compound. The Gemini compound may have two side chains at C20. Exemplary Gemini compounds include vitamin D analogues or derivatives such as (1,25-dihydroxy-21(3-methyl-3-hydroxy-butyl)-cholecalciferol) (#4); (1,25-dihydroxy-21(3-methyl-3-hydroxy-butyl)-19-nor-cholecalciferol) (#9); (1,25-dihydroxy-20R-21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluoro-cholecalciferol) (#10); (1,25-dihydroxy-20S-21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluoro-cholecalciferol) (#11); (1,25-dihydroxy-20R-21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol) (#12); (1,25-dihydroxy-20S-21 (3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol) (#13); (1,25-dihydroxy-21(2R,3-hydroxy-3-methylbutyl)-20R-cholecalciferol) (#17); and_(1,25-dihydroxy-21(2R,3-hydroxy-3-methylbutyl)-20S-cholecalciferol) (#18).
A method of reducing blood pressure in a mammal without inducing hypercalcemia includes the steps of:
An assay for screening vitamin D analogues or derivatives including Gemini compounds to suppress renin expression in a mammal comprising:
An assay for screening vitamin D analogues or derivatives including Gemini compounds to regulate blood pressure in a mammal includes the steps of:
In an aspect, the cell culture expresses vitamin D receptor (VDR) and renin.
Vitamin D and its analogs and derivatives including Gemini compounds suppress renin expression and regulate hypertension in mammals. Vitamin D, and its analogs and derivatives including Gemini compounds are novel anti-hypertensive agents.
A physiological function of the renin-angiotensin system is to maintain vascular resistance and extracellular fluid volume homeostasis, accomplished by the regulatory actions of Ang II on the peripheral vasculature, heart, central nerve system, kidney and adrenal glands. The renin-angiotensin cascade, a rate-limiting component of renin secretion and production, is mostly stimulated by volume or salt depletion, reduction in renal vascular perfusion pressure and sympathetic nerve activity.
VDR null mice have a sustained elevation of renin expression while still maintaining a normal level of blood electrolytes. The augmentation of renin synthesis leads to increased plasma Ang II production from angiotensinogen, which drives VDR null mice to increase water intake and intestinal salt absorption, because Ang II is a very potent thirst-inducing agent that acts on the central nervous system, as well as a potent stimulator of intestinal sodium absorption. The mutant mice have to excrete more urine and salt to maintain volume and electrolyte homeostasis. As a potent vasoconstrictor, Ang II augmentation also leads to the development of hypertension and cardiac hypertrophy in VDR null mice. Thus, a new steady state of the renin-angiotensin system is established in VDR null mice, in which the basal renin expression is higher but still responds appropriately to the same tubular salt load and volume stimuli as in the normal state. It is believed that the up-regulation of renin expression is a primary defect in VDR null mice.
Renin expression in VDR null mice, which reacts to high salt load or dehydration indicates that the mechanism underlying the sustained renin elevation is independent of the pathways activated by tubular salt load or volume depletion. The involvement of COX-2 implicated in mediating macula densa-mediating renin release, in renin elevation in VDR null mice is unlikely, because the same low COX-2 protein level was observed in the kidney of both VDR null and wildtype mice. Because adult VDR null mice develop hypocalcemia and secondary hyperparathyroidism, the up-regulation of renin expression could be due to VDR inactivation per se, hypocalcemia and/or high PTH. However, vitamin D regulation of renin gene expression is direct and independent of the calcium status because: (1) Pre-weaned VDR null mice that have not yet developed hypocalcemia already show an elevated renin expression; (2) When the blood ionized calcium of adult VDR null mice is normalized by the HCa-Lac diet, their renin expression and Ang II level are still elevated; (3) Conversely, Gcm2 null mice, which are as hypocalcemic as VDR null mice, do not manifest elevated renin expression; (4) In wildtype mice, reduction of 1,25(OH)2D3 biosynthesis also results in elevated renin expression, whereas injection of 1,25(OH)2D3 leads to reduced renin expression; and (5) 1,25(OH)2D3 directly suppresses renin gene transcription in As4.1 cells by a VDR-mediated mechanism. Vitamin D is a potent negative endocrine regulator of renin expression in vivo.
Secondary hyperparathyroidism may also contribute to the renin up-regulation in VDR null mice, because the serum PTH level in the normocalcemic pre-weaned or HCa-Lac diet-treated VDR null mice is still significantly higher than that of the wildtype mice (even though it is much lower than that of the untreated adult VDR null mice). PTH may indirectly regulate renin expression in vivo.
In most cases where 1,25(OH)2D3 acts as a positive regulator, the liganded VDR heterodimerizes with the RXR and binds to specific DNA sequences (VDRE) in the promoter of target genes to regulate gene expression. 1,25(OH)2D3 also acts as a negative regulator. VDR-mediated transcriptional repression 1,25(OH)2D3 appears to suppress renin gene expression through a cis-DNA element(s) in the renin gene promoter.
The results of vitamin D analog screening using the cell culture system are summarized in TABLE 1. Of the 9 compounds, two compounds (as indicated by an *) are found as active as, or more active than, 1,25-dihydroxyvitamin D3. Both the active compounds are Gemini compounds. The results of more vitamin D analog screening are summarized in TABLE 2. At least 8 compounds either were as active or more potent than 1,25-dihydroxyvitamin D3 in suppressing renin gene expression. These compounds are suitable for animal testing. Some of these active compounds (e.g. 10, 11, 12, 13, 17, 18) were at least 10- to 100-fold more potent than 1-25-dihydroxyvitamin D3, whereas they are known to have less side effects than 1,25-dihydroxyvitamin D3, rendering them candidates for further testing.
These results demonstrated the feasibility of using the screening system of the present invention to screen potentially a large number of vitamin D analog compounds to identify the most promising ones for animal and human trials. The renin-angiotensin system (RAS) is a direct target for vitamin D to regulate blood pressure. RAS as a vitamin D target plays a role in regulating hypertension. RAS can also be targeted by vitamin D analogues or derivatives including Gemini compounds and their analogues and derivatives.
Renin expression was increased in VDR (vitamin D receptor) null mutant mice because of the disruption of the vitamin D signaling pathway. Quantitative northern blot analysis, showed that the renin mRNA level in the kidney of adult VDR−/− mice was more than 3-fold higher than that of wildtype littermates (
Ang II is a potent vasoconstrictor. The blood pressure of VDR−/− and wildtype mice were compared. Both the systolic and diastolic pressures of VDR−/− mice were significantly higher (>20 mmHg) than those of wildtype littermates (
Ang II is known to be a very potent stimulus for thirst and salt craving as well as an inducer of intestinal water and sodium absorption. Water and food intake as well as blood and urinary electrolyte parameters were measured. As shown in TABLE 3, VDR−/− mice ingested about twice the amount of water as the wildtype littermates, and consequently, excreted approximately twice the amount of urine. The abnormal drinking behavior is not due to diabetes, because the blood glucose and insulin levels of VDR−/− mice were normal (TABLE 4). Food intake of VDR−/− mice was similar to wildtype mice, but they excreted 37% and 19% more Na+ and K+ in the urine, respectively (TABLE 3), while maintaining a normal concentration of blood Na+ and K+ (TABLE 4). Thus, VDR−/− mice appeared to have an increase in the intestinal salt absorption due to the Ang II elevation.
Renin production is very sensitive to changes in tubular salt load or extracellular fluid volume. The effect of high salt diet or dehydration on the expression of renin in VDR−/− and wildtype littermates was investigated. When placed on a normal diet supplemented with 8% NaCl, both VDR−/− and wildtype mice responded by reducing the expression of renin mRNA, but VDR−/− mice still maintained a significantly higher renin mRNA level even after 7 days on the high salt diet (
Dietary strontium has been shown to block the biosynthesis of 1,25(OH)2D3 and is widely used to render animals vitamin D-deficient. To confirm that the disruption of the vitamin D signaling can lead to renin up-regulation, wildtype mice were treated with strontium. The blood ionized calcium was monitored, instead of the blood 1,25(OH)2D3 level, during the treatment because of the extreme difficulty to measure the serum 1,25(OH)2D3 concentration in live mice. As shown in
1,25(OH)2D3 indeed suppressed renin expression in vivo. Wildtype mice were treated with 1,25(OH)2D3 or vehicle and then the renin mRNA level in the kidney was determined. After two doses of 1,25(OH)2D3 (30 pmole/dose) in two consecutive days, renal renin expression was decreased by 35%, and after five doses in three days, the expression was decreased by 50% (
Because vitamin D is a primary regulator of calcium homeostasis, changes in the vitamin D status altered the blood levels of calcium and PTH in animals. Adult VDR−/− mice developed hypocalcemia and secondary hyperparathyroidism. As shown in
To exclude the possibility that hypocalcemia may increase renin expression, renin expression was examined in Gcm2−/− mice that lack the parathyroid glands (Gcm2 is a master regulatory gene for parathyroid gland development), but have normal circulating PTH (derived from the thymus) and 1,25(OH)2D3 concentrations (
Vitamin D directly suppressed renin gene expression. The effect of 1,25(OH)2D3 treatment was examined on renin mRNA expression in As4.1 cells, a JG cell-like cell line that was derived from kidney tumors of SV40 T antigen (Simian Virus 40) transgenic mice and maintains a high level of renin synthesis. Treatment with 5×10−8 M of 1,25(OH)2D3 caused a moderate reduction in renin mRNA expression; however, when the cells were transiently transfected with the pcDNA-hVDR plasmid that contained the full-length coding sequence of human VDR cDNA, the same 1,25(OH)2D3 treatment reduced renin mRNA expression by about 90% (
Effects of high PTH levels in VDR−/− mice were determined. As4.1 cells were treated with different doses of PTH, or transfected with the plasmid pcDNA-PTH/PTHrPR that contained the full-length rat PTH/PTHrP receptor cDNA and then treated with PTH(1-34). No increase in renin expression was observed in either of the PTH-treated cells (
In the As4.1 cells, which have lost expression of some nuclear receptors such as LXR, the VDR mRNA transcript was undetectable by northern blot. As4.1 clones stably transfected with the pcDNA3.1 vector or pcDNA-hVDR were established (
Germini compounds (analogues and derivatives) that show potent activity to suppress renin expression in vitro, need to work in vivo in order for them to be used as therapeutic renin inhibitors or as pharmaceutical compositions. The Gemini compound #9 was tested as a model compound in normal mice. As shown in
Thus, renin expression was significantly reduced in vivo in normal mice treated with compound #9 without a significant increase blood calcium levels. Vitamin D analogues/derivatives including Gemini compounds or their derivatives such as compound #9 can be used as anti-hypertensive agents.
The relationship between RAS activation and the abnormalities in electrolyte and volume homeostasis was performed by analyzing the effects of ATI receptor antagonist losartan and angiotensin-converting enzyme inhibitor captopril on VDR-null mice. Treatment with losartan or captopril normalized the water intake and urine excretion of VDR-null mice. However, the increase in salt excretion in VDR-null mice was not affected by either drug, indicating that this abnormality is independent of the RAS. Northern blot and immunohistochemical analyses revealed that both drugs caused a drastic stimulation of renin expression in both wild-type and VDR-null mice, but renin expression in the treated VDR-null mice remained much higher than in the treated wild-type mice as shown in
These data support a causative relationship between RAS over-stimulation and the abnormal volume homeostasis in VDR-null mice, and demonstrated that the vitamin D repression of renin expression is independent of the Ang II feedback regulation in vivo. The increase in renin expression seen in the treated animals is due to the disruption of the Ang II feedback regulation caused by the drug treatment, although changes in other physiological parameters (such as perfusion pressure, sympathetic output and/or tubular sodium load) that may also affect renin expression. These results support that the regulatory mechanisms for renin production, including the Ang II feedback regulation, are functionally intact in VDR(−/−) mice. Thus, Ang II feedback repression and vitamin D repression of renin expression are independent negative regulatory pathways to maintain the homeostasis of the RAS.
Cardiac hypertrophy, usually characterized by enlarged left ventricular myocytes, is a common and often lethal complication of arterial hypertension. At the molecular level, cardiac hypertrophy is often accompanied by activation of the so-called fetal gene program in the left ventricle. This program includes the genes encoding atrial natriuretic peptide (ANP), α-skeletal actin, and β-myosin heavy chain. These genes are normally expressed in late fetal and early neonatal heart tissues and are extinguished in adult ventricular myocardium. The increase of ANP is regarded as a cardio-protective response because of the associated natriuretic, anti-hypertrophic, anti-fibrotic and anti-hypertensive activities.
As disclosed herein, 1,25-dihydroxyvitamin D3 is an endocrine suppressor of renin biosynthesis. Genetic disruption of the vitamin D receptor (VDR) resulted in over-stimulation of the renin-angiotensin system (RAS), leading to high blood pressure and cardiac hypertrophy. Consistent with the higher heart-to-body weight ratio, the size of left ventricular cardiomyocytes in VDR knockout (KO) mice was increased compared to wild-type mice. The levels of atrial natriuretic peptide (ANP) mRNA and circulating ANP were also increased in VDRKO mice. Treating VDRKO mice with captopril reduced cardiac hypertrophy and normalized ANP expression (
Rrenin, AGT and AT-1aR mRNA levels were measured within the hearts of VDR(−/−) mice by real-time RT-PCR. As shown in
These data support the notions that the cardiac hypertrophy seen in VDRKO mice is a consequence of activation of both the systemic and cardiac RAS, and that 1,25-dihydroxyvitamin D3 regulates cardiac functions, at least in part, through the RAS.
*Indicates Gemini analogues.
*The inhibitory activity was determined by measuring the renin mRNA level by Northern blot analyses after treating As4.1-hVDR cells with each Gemini compound for 24 hours at 10-8, 10-9 and 10-10 M. The relative activity is based on that of 1,25-dihydroxyvitamin D3, which is arbitrarily set at ++.
#refers to the compound number in
BW, body weight; Cr, creatinine; ns, not significant; n = number of animals.
Ns, not significant; n = number of animals.
Materials and Methods
Animals and treatment. The generation and characterization of VDR−/− and Gcm2−/− mice have been described by Li et al. (1997) and Gunther et al. (2000). VDR−/− and Gcm2−/− mice were generated through breeding of heterozygous mice and identified by PCR with tail genomic DNA as the template, and the wild-type littermates were used as controls. Mice were housed in a pathogen-free barrier facility in a 12 hr light/12 hr dark cycle, and fed an autoclaved standard rodent chow. To normalize the blood ionized calcium level of VDR−/− mice, two-month old animals were placed on the HCa-Lac diet (Teklad, Madison, Wis.) that contained 2% calcium, 1.25% phosphorus, 4 IU/g vitamin D, and 20% lactose for 5 weeks. To increase the sodium load, mice were fed the normal rodent diet supplemented with 8% NaCl for 1, 3, 5 and 7 days. In dehydration experiments, mice were restricted from water, but had free access to food, for 24 hr before sacrifice. To block 1,25(OH)2D3 synthesis, 1.5-month old wildtype mice were placed on the normal diet supplemented with 2.5% strontium chloride until hypocalcemia was detected. Wild-type mice were injected (i.p.) with vehicle or 30 pmole of 1,25(OH)2D3 dissolved in propylene glycol. Mice were sacrificed by exsanguination under anesthesia and the blood was collected into ice-cold tubes for serum isolation, or into ice-cold tubes containing 50 μl of EDTA (pH 8.0) and 100 U/ml aprotinin for plasma isolation. The determination of water and food intake, as well as urine collection, were carried out by using metabolic cages.
Measurement of blood and urine parameters. The concentration of blood ionized calcium was determined using a Ciba/Coming 634 Ca++/pH analyzer (Chiron Diagnostics, East Walpole, Mass.) from 50 μl of whole blood obtained from tail snipping. Blood glucose concentrations were determined by using One Touch Sure Step test strips (Life Scan, Milpitas, Calif.). Serum intact parathyroid hormone (iPTH) was determined using a commercial ELISA kit (Immutopics, San Clemente, Calif.). The concentration of serum and urinary Na+, K+, and creatinine was determined by a Beckman Coulter CX5 Autoanalyzer as described by Li et al. (2001).
Measurement of Ang II. Mouse plasma Ang II concentrations were determined by radioimmunoassays (RIA), using a commercial RIA kit (Phoenix Pharmaceutical, Mountain View, Calif.) according to the manufacturer's instructions.
Measurement of blood pressure. Mouse blood pressure was determined as described by Liu et al. (1996). Briefly, mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The left carotid artery was isolated from surrounding tissues, and cannulated with a polyethylene catheter filled with sterile phosphate-buffered saline containing heparin (50 U/ml) under a dissecting microscope. Arterial blood pressure was measured using a Harvard Apparatus pressure transducer and recorded. To investigate whether the increase in blood pressure in VD−/− mice was directly due to the increase in the Ang II level, wildtype and VDR−/− mice were treated with captopril (100 mg/day/kg body weight, dissolved in drinking water) for 5 days before blood pressure was determined. Wildtype and VDR−/− mice fed normal drinking water were used as controls.
Immunohistochemistry. Kidneys freshly dissected from wildtype and VDR−/− mice were fixed overnight with 4% formaldehyde in PBS (pH 7.2), processed, embedded in paraffin and cut into 5-μm sections with a Leica microtone 2030. The slides were stained with a rabbit polyclonal anti-renin antiserum (1:1600 dilution) (provided by Dr. T. Inagami, Vanderbilt University). After incubation with a peroxidase-conjugated anti-rabbit IgG (KPL, Gaithersburg, Md.), the renin signal was visualized with a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, Calif.), followed by a light hematoxylin counterstaining.
RNA isolation and northern blot. The kidney and liver were dissected and immediately placed into the Trizol Reagent (Invitrogen, Grand Island, N.Y.) for total RNA isolation according to the manufacturer's instruction. To determine renin or angiotensinogen mRNA expression, total RNA (20 μg/lane) was separated on a 1.2% agarose gel containing 0.6 M formaldehyde, transferred onto a Nylon membrane (MSI, Westborough, Mass.) and crosslinked in a UV crosslinker (Bio-Rad, Hercules, Calif.). Hybridization was performed as described by Li et al. (2001). Mouse renin and angiotensinogen cDNA probes were labeled with 32P-dATP (ICN, Costa Mesa, Calif.) using the Prime-a-gene Labeling System (Promega, Madison, Wis.). After hybridization and washing, membranes were exposed to X-ray films at −80° C. for autoradiography. The relative amount of mRNA was quantitated using a Phospholmager (Molecular Dynamics, Sunnyvale, Calif.) and normalized with 36B4 mRNA.
As4.1 cell culture and transfection. As4.1 cells (ATCC, Manassas, Va.) were cultured in DMEM supplemented with 10% FBS at 37° C. and 5% CO2. For transient transfection, the cells were grown in 10-cm dishes to 50% confluence and transfected with pcDNA3.1, pcDNA-hVDR or pcDNA-PTH/PTHrPR plasmid (10 μg DNA/dish) by the standard calcium phosphate method. Twenty-four hr after transfection, the cells were treated for 24 hr with 5×10−8 M of 1,25(OH)2D3 or ethanol in serum-free media, or with different doses of bovine PTH(1-34) as indicated. Total RNA was isolated and analyzed for renin mRNA expression by northern blot. For stable transfection, As4.1 cells were transfected with pcDNA3.1 or pcDNA-hVDR plasmid by the use of Superfect reagent (Qiagen, Valencia, Calif.) and selected with 350 μg/ml of G418 for two weeks. Individual colonies were picked, expanded and selected for VDR expression. The As4.1-hVDR stable clones were treated with different doses of 1,25(OH)2D3 for 24 hr in serum-free media, and total RNA were analyzed by northern blot to examine renin expression.
Real-time RT-PCR. The mRNA levels of renin, AGT, and type Ia Ang II receptor (AT-1aR) in the heart were quantified by real-time RT-PCR. Briefly, first strand cDNAs were synthesized from 5 μg of total heart RNAs in a 50 μl reaction using M-MLV reverse transcriptase (Invitrogen Life Technologies) and oligo-dT12-18 as the primer. The cDNAs were then used as the template (5 μl per reaction) for real-time PCR amplification. Real-time PCR was carried out using a Cepheid Smart Cycler (Cepheid, Sunnyvale, Calif.) and a SYBR Green PCR Reagents kit (Applied Biosystems, Foster City, Calif.). The PCR primers for mouse renin, AGT, AT-1aR, and GAPDH genes were designed based on cDNA sequences deposited in GenBank database. GAPDH was used as the internal control for each reaction. All primers were tested for their specificity by conventional PCR before being used for the real-time PCR quantitative studies. The Ct value for each gene was obtained from the real-time PCR reactions, and the starting amount of each target mRNA was calculated based on a calibration curve and the Ct value. The relative amount of mRNA was normalized to GAPDH mRNA.
Therapeutic compositions: Pharmaceutical compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include materials that when combined with the therapeutic composition retain the anti-tumor function of the therapeutic composition. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, water, and the like. Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing sterile sodium chloride for injection. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection. Dosages and administration protocols will generally depend on a number of other factors appreciated in the art.
Renin gene promoter analysis. Plasmid pR1C-4.1 CAT that contains 4.1 kb 5′-flanking sequence of mouse Ren-1c gene (Petrovic et al. 1996) was provided by Dr. K. W. Gross (Roswell Park Cancer Institute, Buffalo, N.Y.). To generate pGL-117 bp reporter plasmid, the 123 bp renin minimal promoter fragment (+6 to −117) was released from pR1C-4.1 CAT with XbaI and BamHI and inserted into the HindIII site of pGL3-basic vector (Promega, Madison, Wis.). To generate pGL-4.1 kb reporter plasmid, the BamHI fragment (−4.1 kb to −118 bp) from pRI C-4.1 CAT was inserted into the BglII site of pGL-117 bp. To analyze the activity of renin gene promoter, As4.1-hVDR cells were transfected with the reporter plasmids by electroporation according to Shi et al. (2001) using a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.). pCMV-β-gal plasmid was co-transfected as an internal control. pGL3-control plasmid (Promega) was used as the positive control. The transfected cells were treated with ethanol or 10−8 M of 1,25(OH)2D3 in Opti-MEM medium (Invitrogen) containing 2% charcoal-treated FBS four hr after electroporation, and luciferase activity was determined at 48 hr after initial transfection, using the Luciferase Assay System (Promega). Luciferase activity was normalized to β-gal activity obtained from the same electroporation, and presented as fold induction based on the basal activity of pGL3-basic empty vector determined in the same experiment.
Statistical analysis. Data were presented as mean±SD and analyzed with student's t-test to assess significance. P values of 0.05 or lower were considered statistically significant.
Structural comDounds. Structural formulations for Vitamin D analogues or derivatives, Gemini compounds and Gemini analogues are as described in U.S. Pat. Nos. 6,030,962; 6,559,138; 6,329,538; 6,331,642; 6,452,028; and 4,225,525, each of which is herein incorporated by reference. As disclosed herein, analogues or derivatives of Vitamin D include Gemini compounds and Gemini analogues and any other molecule that structurally resembles Vitamin D.
An exemplary Vitamin D analog or derivative has a structure that resembles:
wherein: X is H2 or CH2;
Y is hydrogen, hydroxy or fluorine;
Z is hydroxy;
R1 and R2 are a (C1-C4) alkyl or fluoroalkyl, or R1 and R2 together with C25 form a (C3-C6) cycloalkyl or cyclofluoroalkyl;
R3 and R4 are a (C1-C4) alkyl or fluoroalkyl, or R3 and R4 together with C25, form a (C3-C6)cycloalkyl or cyclofluoroalkyl;
A is a single bond or a double bond;
B1 is a single bond, an E-double bond, a Z-double bond or a triple bond; and
B2 is a single bond, an E-double bond, a Z-double bond or a triple bond.
Data.
Diocuments
The following publications are incorporated by reference to the extent they relate to the protocols used in this disclosure.
Alroy, I., Towers, T. L., and Freedman, L. P. 1995. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol Cell Biol. 15:5789-5799.
Antonipillai, I., and Horton, R. 1985. Role of extra- and intracellular calcium and calmodulin in renin release from rat kidney. Endocrinology. 117:601-606.
Ballermann, B. J., Zeidel, M. L., Gunning, M. E., and Brenner, B. M. 1991. Vasoactive peptides and the kidney. In The kidney. B. M. Brenner, and F. C. Rector, editors. W. B. Saunders Company, Philadelphia. 510-583.
Broulik, P. D., Horky, K., and Pacovsky, V. 1986. Effect of parathyroid hormone on plasma renin activity in humans. Horm Metab Res. 18:490-492.
Brunner, H. R., Laragh, J. H., Baer, L., Newton, M. A., Goodwin, F. T., Krakoff, L. R., Bard, R. H., and Buhler, F. R. 1972. Essential hypertension: renin and aldosterone, heart attack and stroke. N Engl J Med. 286:441-449.
Burgess, E. D., Hawkins, R. G., and Watanabe, M. 1990. Interaction of 1,25-dihydroxyvitamin D and plasma renin activity in high renin essential hypertension. Am J Hypertens. 3:903-905.
Cheng, H. F., Wang, J. L., Zhang, M. Z., Wang, S. W., McKanna, J. A., and Harris, R.C. 2001. Genetic deletion of COX-2 prevents increased renin expression in response to ACE inhibition. Am J Physiol Renal Physiol. 280:F449-456.
Dardenne, O., Prud'homme, J., Arabian, A., Glorieux, F. H., and St-Arnaud, R. 2001. Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)- hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D- deficiency rickets. Endocrinology. 142:3135-3141.
Fitzsimons, J. T. 1980. Angiotensin stimulation of the central nervous system. Rev Physiol Biochem Pharmacol. 87:117-167.
Geisterfer, A. A., Peach, M. J., and Owens, G. K. 1988. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 62:749-756.
Gunther, T., Chen, Z. F., Kim, J., Priemel, M., Rueger, J. M., Amling, M., Moseley, J. M., Martin, T. J., Anderson, D. J., and Karsenty, G. 2000. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature. 406:199-203.
Hackenthal, E., Paul, M., Ganten, D., and Taugner, R. 1990. Morphology, physiology, and molecular biology of renin secretion. Physiol Rev. 70:1067-1116.
Imaoka, M., Morimoto, S., Kitano, S., Fukuo, F., and Ogihara, T. 1991. Calcium metabolism in elderly hypertensive patients: possible participation of exaggerated sodium, calcium and phosphate excretion. Clin Exp Pharmacol Physiol. 18:631-641.
Kimura, Y., Kawamura, M., Owada, M., Oshima, T., Murooka, M., Fujiwara, T., and Hiramori, K. 1999. Effectiveness of 1,25-dihydroxyvitamin D supplementation on blood pressure reduction in a pseudohypoparathyroidism patient with high renin activity. Intern Med. 38:31-35.
Kjaer, A., Knigge, U., Jorgensen, H., and Warberg, J. 1998. Dehydration-induced renin secretion: involvement of histaminergic neurons. Neuroendocrinology. 67:325-329.
Kong, J., Li, X. J., Gavin, D., Jiang, Y., and Li, Y. C. 2002. Target expression of human vitamin D receptor in the skin promotes the initiation of postnatal hair follicular cycle and rescue the alopecia in vitamin D receptor null mice. J. Invest. Dermatol. 118:631-638.
Krause, R., Buhring, M., Hopfenmuller, W., Holick, M. F., and Sharma, A. M. 1998. Ultraviolet B and blood pressure. Lancet. 352:709-710.
Kristal-Boneh, E., Froom, P., Harari, G., and Ribak, J. 1997. Association of calcitriol and blood pressure in normotensive men. Hypertension. 30:1289-1294.
Li, Y. C., Amling, M., Pirro, A. E., Priemel, M., Meuse, J., Baron, R., Delling, G., and Demay, M. B. 1998. Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice. Endocrinology. 139:4391-4396.
Li, Y. C., Bolt, M. J. G., Cao, L.-P., and Sitrin, M. D. 2001. Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism. Am J Physiol Endocrinol Metab. 281:E558-E564.
Li, Y. C., Pirro, A. E., Amling, M., Delling, G., Baron, R., Bronson, R., and Demay, M. B. 1997. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA. 94:9831-9835.
Lind, L., Hanni, A., Lithell, H., Hvarfner, A., Sorensen, O. H., and Ljunghall, S. 1995. Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men. Am J Hypertens. 8:894-901.
Lind, L., Wengle, B., Wide, L., and Ljunghall, S. 1989. Reduction of blood pressure during long-term treatment with active vitamin D (alphacalcidol) is dependent on plasma renin activity and calcium status. A double-blind, placebo-controlled study. Am J Hypertens. 2:20-25.
Liu, S. Q., and Fung, Y. C. 1996. Indicial functions of arterial remodeling- in response to locally altered blood pressure. Am J Physiol. 270:H1323-1333.
Nishishita, T., Okazaki, T., Ishikawa, T., Igarashi, T., Hata, K., Ogata, E., and Fujita, T. 1998. A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D. J Biol Chem. 273:10901-10907.
Omdahl, J. L., and DeLuca, H. F. 1971. Strontium induced rickets: metabolic basis. Science. 174:949-951.
Pan, L., Black, T. A., Shi, Q., Jones, C. A., Petrovic, N., Loudon, J., Kane, C., Sigmund, C. D., and Gross, K. W. 2001. Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression. J Biol Chem. 276:45530-45538.
Pan, L., Xie, Y., Black, T. A., Jones, C. A., Pruitt, S. C., and Gross, K. W. 2001. An Abd-B class HOX.PBX recognition sequence is required for expression from the mouse Ren-1c gene. J Biol Chem. 276:32489-32494.
Panda, D. K., Miao, D., Tremblay, M. L., Sirois, J., Farookhi, R., Hendy, G. N., and Goltzman, D. 2001. Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA. 98:7498-7503.
Park, C. W., Oh, Y. S., Shin, Y. S., Kim, C. M., Kim, Y. S., Kim, S. Y., Choi, E. J., Chang, Y. S., and Bang, B. K. 1999. Intravenous calcitriol regresses myocardial hypertrophy in hemodialysis patients with secondary hyperparathyroidism. Am J Kidney Dis. 33:73-81.
Petrovic, N., Black, T. A., Fabian, J. R., Kane, C., Jones, C. A., Loudon, J. A., Abonia, J. P., Sigmund, C. D., and Gross, K. W. 1996. Role of proximal promoter elements in regulation of renin gene transcription. J Biol Chem. 271:22499-22505.
Pfeifer, M., Begerow, B., Minne, H. W., Nachtigall, D., and Hansen, C. 2001. Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women. J Clin Endocrinol Metab. 86:1633-1637.
Polly, P., Herdick, M., Moehren, U., Baniahmad, A., Heinzel, T., and Carlberg, C. 2000. VDR-Alien: a novel, DNA-selective vitamin D(3) receptor-corepressor partnership. Faseb J 14:1455-1463.
Resnick, L. M., Muller, F. B., and Laragh, J. H. 1986. Calcium-regulating hormones in essential hypertension. Relation to plasma renin activity and sodium metabolism. Ann Intern Med. 105:649-654.
Ritthaler, T., Scholz, H., Ackermann, M., Riegger, G., Kurtz, A., and Kramer, B. K. 1995. Effects of endothelins on renin secretion from isolated mouse renal juxtaglomerular cells. Am J Physiol. 268:F39-45.
Rostand, S. G. 1997. Ultraviolet light may contribute to geographic and racial blood pressure differences. Hypertension. 30:150-156.
Shi, Q., Gross, K. W., and Sigmund, C. D. 2001. Retinoic acid-mediated activation of the mouse renin enhancer. J Biol Chem. 276:3597-3603.
Sigmund, C. D., Okuyama, K., Ingelfinger, J., Jones, C. A., Mullins, J. J., Kane, C., Kim, U., Wu, C. Z., Kenny, L., Rustum, Y., et al. 1990. Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct. J Biol Chem. 265:19916-19922.
Silver, J., Naveh-many, T., Mayer, H., Schmeizer, H. J., and Popovtzer, M. M. 1986. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin. Invest. 78:1296-1301.
Skott, O., and Briggs, J. P. 1987. Direct demonstration of macula densa-mediated renin secretion. Science. 237:1618-1620.
Smith, J. M., Mouw, D. R., and Vander, A. J. 1983. Effect of parathyroid hormone on renin secretion. Proc Soc Exp Biol Med. 172:482-487.
Tamura, K., Chen, Y. E., Horiuchi, M., Chen, Q., Daviet, L., Yang, Z., Lopez-Ilasaca, M., Mu, H., Pratt, R. E., and Dzau, V. J. 2000. LXRalpha functions as a cAMP-responsive transcriptional regulator of gene expression. Proc Natl Acad Sci USA. 97:8513-8518.
Towers, T. L., and Freedman, L. P. 1998. Granulocyte-macrophage colony-stimulating factor gene transcription is directly repressed by the vitamin D3 receptor. Implications for allosteric influences on nuclear receptor structure and function by a DNA element. J Biol Chem. 273:10338-10348.
Yoshizawa, T., Handa, Y., Uematsu, Y., Takeda, S., Sekine, K., Yoshihara, Y., Kawakami, T., Arioka, K., Sato, H., Uchiyama, Y., et al. 1997. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nature Genetics. 16:391-396.
U.S. Pat. No. 6,030,962
U.S. Pat. No. 6,559,138
U.S. Pat. No. 6,329,538
U.S. Pat. No. 6,331,642
U.S. Pat. No. 6,452,028
U.S. Pat. No. 4,225,525
This application is a continuation in part of U.S. Ser. No. 10/865,624 filed Jun. 10, 2004, now abandoned, which claims priority from U.S. Ser. No. 60/477,900 filed Jun. 12, 2003.
The U.S. Government has rights in the present invention due to partial support of the Digestive Disease Research Center Grant DK42086 and NIH grant DK 59327.
| Number | Date | Country | |
|---|---|---|---|
| 60477900 | Jun 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10865624 | Jun 2004 | US |
| Child | 10962215 | Oct 2004 | US |
