Osteoclast-mediated bone resorption

Here, researchers establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC50] of 20.3 ± 1.0 μM). They also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate[1].

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In this report researchers confirmed that risedronate, a nitrogen-containing bisphosphonate (N-BP), has a potent activity against the blood stages of P. falciparum in vitro. The IC50 established for parasite growth is in the range obtained for the same isolate (3D7) in previous studies. Researchers also showed that the inhibitory effect induced by risedronate can be partially reversed by the simultaneous addition of FPP or GGPP during P. falciparum culture treatment (Fig. 1). The restoration observed after the addition of GGPP is plausible, since Couto et al. previously demonstrated that P. falciparum is able to convert GGPP into FPP. In contrast, when we added IPP to the cultures, the parasites could not recover, suggesting that the inhibition of FPPS is a potential target for risedronate, which could also act by inhibiting GGPPS. Luckman et al. verified the same event of restoration after coincubating J774 macrophages with alendronate and FPP or GGPP, observing a partial prevention of apoptotic events.[1]
The results regarding the effect of risedronate on isoprenoid biosynthesis (Fig. 2) suggest the inhibition of FPPS. The RP-TLC profile for treated parasites shows that bands with Rf values equivalent to FOH and GGOH are decreased compared to the signal from untreated parasites, leading us to speculate that risedronate inhibits enzymes involved in FPP and/or GGPP synthesis. It is known that the major target of N-BPs, as risedronate, is FPPS (11); therefore, we assume the possible role of risedronate as a potent inhibitor of the isoprenoid pathway in P. falciparum by inhibiting FPPS and, consequently, blocking protein farnesylation and geranylgeranylation. In J774 macrophages, the drug inhibited protein prenylation, including Ras protein prenylation[1].

In vivo experiments demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.[1]
After determining the antiplasmodial effect of risedronate in vitro, we verified its efficacy in BALB/c mice infected with P. berghei strain ANKA. The administration of 20 and 25 mg/kg risedronate for 4 days led to decreases of parasitemia of 68.9% and 83.6%, respectively. On the seventh day of treatment the inhibitions were 63% and 88.9% with 20 and 25 mg/kg, respectively (Fig. 4A). After recovering the parasitemia, a dose-response curve was obtained for estimating the ID50 (dose causing 50% inhibition), equivalent to 17 ± 1.8 mg/kg after 7 days of treatment. Four days after the interruption of treatment (11 days postinfection), the parasitemias of the groups treated with 10, 15, 20, and 25 mg/kg/day were 15.3%, 15.9%, 15.2%, and 5.7%, respectively. Conversely, the group that received PBS presented parasitemia of 25.6%. Among the groups treated with risedronate, only the animals that received 25 mg/kg had a significant inhibition of 77.8% (see Table S1 in the supplemental material), demonstrating that even after treatment discontinuation, the parasitemia of the animals remained low in relation to that of the controls; however, parasite recrudescence was observed for all treated groups. By Kaplan-Meier survival analysis there was no difference between risedronate-treated mice and PBS-treated groups (Fig. 4B)[2].

Inhibition tests with risedronate and rescue assays.[1]
Risedronate was dissolved in sterile deionized water, resulting in a 25 mM stock solution. The inhibition tests were carried out with flat-bottomed microtiter plates using the following drug concentrations: 3,000, 300, 30, 3, 0.3, 0.03, and 0.003 μM. We employed a method described previously by Desjardins and coauthors, with some modifications, to determine risedronate 50% inhibitory concentrations (IC50s) for P. falciparum intraerythrocytic stages after 48 h of treatment. Briefly, synchronic ring-stage parasite cultures (5% hematocrit and 1% parasitemia) were exposed to increasing drug concentrations, and the parasitemia and parasite morphologies were determined with Giemsa-stained smears immediately before the start and at intervals of 24 to 96 h, instead of [3H]hypoxanthine incorporation. All tests were performed in triplicates for three independent experiments. The IC50, IC90 (± standard deviation), and 95% confidence interval (CI95%) values for growth inhibition were calculated by using Origin 8.1 software. For the rescue assays, FPP, GGPP, and IPP were solubilized in RPMI medium (5 mM stock solution), and different concentrations of each compound (100 nM to 1,000 nM) were then added simultaneously to synchronous P. falciparum cultures in the ring stage previously treated with 20 μM risedronate. Parasitemia was assessed every 24 h. Statistical analysis was performed by using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. A P value of <0.05 was considered statistically significant.

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Treatment with risedronate and metabolic labeling.[1]
Asynchronous cultures of P. falciparum were treated with 15 μM risedronate for 36 h and labeled with [1-3H]GGPP (3.125 μCi/ml) or [1-3H]FPP (3.125 μCi/ml) in normal RPMI 1640 medium for the last 12 h in the presence of drug. After labeling, ring, trophozoite, and schizont forms were purified on a 40%-70%-80% discontinuous Percoll gradient, followed by cell lysis in a solution containing ice-cold 10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2% (vol/vol) Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM iodoacetamide, 1 mM N-(p-tosyl-lysine)chloromethyl ketone, and 1 μg/ml leupeptin. After incubation for 15 min at 4°C, lysates were centrifuged at 10,000 × g for 30 min. Supernatants of parasites were stored in liquid N2 for subsequent SDS-PAGE analysis. For the analysis of isoprenoids, synchronic cultures in the ring stage were treated with 15 μM risedronate for 36 h and metabolically labeled with [1-14C]IPP for the last 12 h. After labeling, schizont-stage parasites were purified on a 40%-70%-80% discontinuous Percoll gradient as described above and freeze-dried prior to lipid extraction as described elsewhere previously (30). Risedronate at 15 μM was considered the ideal drug concentration to be used in our metabolic labeling experiments, since approximately 90% of the parasite population remained viable after 36 h of treatment.

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Reverse-phase thin-layer chromatography (RP-TLC).[1]
Similar amounts of schizont-stage pellets of untreated or risedronate-treated parasites labeled with [1-14C]IPP as described above were extracted with hexane for the subsequent separation of alcohols on reverse-phase Silica Gel 60 plates with acetone-H2O (6:1, vol/vol). Plates were sprayed with En3Hance and subjected to autoradiography for 45 days at −70°C. The standards were visualized with iodine vapor, and Rf values were determined. Hexane extracts of uninfected erythrocytes were used as a control group.

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Immunoprecipitation assays.[1]
Synchronic cultures in the ring stage were treated with 15 μM risedronate for 24 h and metabolically labeled with [1-3H]FPP or [1-3H]GGPP for an additional 12 h in the presence of the drug. After labeling, schizont-stage parasites were purified on a 40%-70%-80% discontinuous Percoll gradient as described above. Pellets of untreated and treated schizont-stage parasites were resuspended in immunoprecipitation buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% [vol/vol] Triton X-100, 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] SDS, 5 μg/ml protease inhibitor cocktail [0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 mM β-mercaptoethanol, chymostatin {5 mg/ml}, and 1 μg/ml leupeptin, antipain, and pepstatin A]) and then precleared with protein A-Sepharose beads. Schizont forms were then incubated with anti-human Ras or anti-Rap1/Krev-1 monoclonal immunoglobulins (1:20 dilution) for 2 h at 4°C. The antigen-antibody complex was precipitated by using 100 μl of a 10% protein A-Sepharose slurry. After five washes with phosphate-buffered saline (PBS), the bound antigen was released in SDS sample buffer and analyzed by SDS-PAGE and autoradiography (20). Densitometric analyses were performed by using Image J software.

Each male BALB/c mouse (3 to 4 weeks old) (n = 10 to 15 per group) was injected intraperitoneally (i.p.) with 106 blood-stage Plasmodium berghei strain ANKA parasites. Our laboratory previously established this parasite burden as the 50% lethal dose 14 days after inoculation. Risedronate treatment with different concentrations was initiated in 2 h after infection on day 0 and continued for 7 days. The drug was diluted in PBS and administered i.p. at 10, 15, 20, and 25 mg/kg of body weight/day. Parasitemia levels were monitored microscopically by examining Giemsa-stained thin blood smears on days 4, 7, 11, 14, and 17 postinfection. Throughout this period, the spontaneous death of each animal was computed. The percentage of parasitemia inhibition was calculated as follows: 100 − [(mean parasitemia for the treated group/mean parasitemia for the control group) × 100] (14). For comparisons of average parasitemias at different time points, analysis of variance was performed with a post hoc Mann-Whitney test for comparisons of the means. [1]

Absorption, Distribution, and Excretion
Absorption
Oral bioavailability is 0.63%, with peak absorption occurring approximately 1 hour after administration. Administration 30 minutes before a meal reduces bioavailability by 55% compared to fasting, and administration 1 hour before a meal reduces bioavailability by 30%.
Excretion Route
Risedronate sodium is excreted via the kidneys; unabsorbed doses are excreted in the feces.
Volume of Distribution
13.8 L/kg.
Clearance
Mean renal clearance is 52 mL/min, and the mean total clearance is 73 mL/min.
Absorption is rapid and dose-independent, occurring throughout the upper gastrointestinal tract. The mean oral bioavailability is 0.63%, and co-administration with food reduces bioavailability. Administration 0.5 hours before breakfast or 2 hours after dinner reduces absorption by 55% compared to fasting (no food or water for 10 hours before or 4 hours after administration). Compared to fasting, administration 1 hour before breakfast reduces absorption by 30%. /Risedronate Sodium/
Studies in rats and dogs showed that after a single intravenous dose of radiolabeled risedronate sodium, approximately 60% of the dose was distributed in the bones. In humans, the mean steady-state volume of distribution is 6.3 L/kg body weight. /Risedronate Sodium/
In rats, after multiple oral administrations, the absorption rate of risedronate sodium in soft tissues ranged from 0.001% to 0.01%.
Risedronate sodium was detected in pups exposed to lactating rats within 24 hours of administration, indicating a small amount of milk transport. Risedronate Sodium
Excretion: Excreted in feces; the drug is not absorbed (unaltered). Excreted by the kidneys; the drug is unaltered; approximately 50% of the absorbed dose is excreted within 24 hours, and 85% is excreted within 28 days. The mean renal clearance was 105 mL/min, and the mean total clearance was 122 mL/min. The difference between the two mainly reflects non-renal clearance or bone resorption clearance. Note: Renal clearance is independent of drug concentration, and renal clearance is linearly related to creatinine clearance. Risedronate Sodium Metabolites/Metabolites
Risedronate is unlikely to be metabolized before excretion. PCP bisphosphonates are resistant to chemical and enzymatic hydrolysis, thus preventing the metabolism of this molecule.
There is no evidence that risedronate sodium is metabolized in humans or animals.
No evidence has been found that risedronate sodium is metabolized in humans or mammals. Excretion Route: Risedronate sodium is primarily excreted unchanged via the kidneys. In rats, only a very small amount (<0.1% of the intravenous dose) of the drug is excreted via bile. Half-life: 1.5 hours

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Biological half-life
The initial half-life of risedronate is approximately 1.5 hours, and the terminal half-life is 561 hours.
Initial half-life: approximately 1.5 hours; terminal exponential half-life: 480 hours (this may represent the dissociation of risedronate from the bone surface).

Toxicity Overview
Risedronate sodium's effects on bone tissue are partly based on its affinity for hydroxyapatite, a component of the bone mineral matrix. Risedronate sodium also targets farnesyl pyrophosphate (FPP) synthase. Nitrogen-containing bisphosphonates (such as pamidronate sodium, alendronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid sodium) appear to act as analogs of isoprene diphosphate lipids, thereby inhibiting an enzyme in the mevalonate pathway—FPP synthase. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprene lipids (FPP and GGPP), which are crucial for the post-translational farnesylation and geranylation of small GTPase signaling proteins. This activity inhibits osteoclast activity and reduces bone resorption and bone turnover. In postmenopausal women, it reduces elevated bone turnover and, on average, results in a net increase in bone mass.

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Effects during pregnancy and lactation
◉Overview of use during lactation
Since there is currently no information regarding the use of risedronate sodium during lactation, alternative medications are recommended, especially for breastfed newborns or premature infants. However, breastfed infants are unlikely to absorb risedronate sodium.
◉Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉Effects on breastfeeding and breast milk
As of the revision date, no relevant published information was found.

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Route of exposure
Rapidly absorbed after oral administration (approximately 1 hour), primarily via the upper gastrointestinal tract.

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Symptoms
Side effects include abdominal pain, anxiety, back pain, hiccups, bladder irritation, bone problems and pain, bronchitis, bursitis, cataracts, chest pain, colitis, constipation, depression, diarrhea, difficulty breathing, dizziness, dry eyes, eye infections, flu-like symptoms, bloating, headache, high blood pressure, infections, insomnia, itching, joint problems and pain, leg cramps, muscle pain, muscle weakness, nausea, neck pain, neuralgia, pain, pneumonia, rash, tinnitus, sinus problems, sore throat, stomach bleeding, nasal congestion or runny nose, swelling, tendon problems, tumors, ulcers, urinary tract infections, dizziness, vision problems, and weakness.

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Drug Interactions
Antacids or mineral supplements containing divalent cations: When risedronate sodium is used concomitantly with antacids or mineral supplements containing divalent cations (e.g., aluminum, calcium, magnesium), a pharmacokinetic interaction occurs (reducing the absorption of risedronate sodium).
Nonsteroidal anti-inflammatory drugs (NSAIDs): No evidence of increased upper gastrointestinal adverse reactions was found.

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Protein binding rate: Approximately 24%.

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Oral TDLo 1800 ug/kg/3D in women - Sensory organs and special senses: Iritis: Ocular Lancet., 341(436), 1993

Antimicrob Agents Chemother. 2011 May;55(5):2026-31.

Risedronate sodium is a 1,1-bisphosphonic acid. Risedronate sodium is the sodium salt of risedronate sodium, a synthetic pyridyl bisphosphonate. Risedronate binds to hydroxyapatite crystals in bone, inhibiting osteoclast-dependent bone resorption. It is a pyridine and bisphosphonic acid derivative that acts as a calcium channel blocker, inhibiting bone resorption.

C7H9NNA2O7P2

327.0759

304.983

C, 27.56; H, 3.30; N, 4.59; Na, 7.54; O, 36.71; P, 20.30

115436-72-1

105462-24-6 (free acid);115436-72-1 (sodium);

4194514

White to off-white solid powder

692.3ºC at 760 mmHg

252-262°C

372.5ºC

4.03E-20mmHg at 25°C

0.063

4

8

4

18

375

0

DRFDPXKCEWYIAW-UHFFFAOYSA-M

InChI=1S/C7H11NO7P2.Na/c9-7(16(10,11)12,17(13,14)15)4-6-2-1-3-8-5-6;/h1-3,5,9H,4H2,(H2,10,11,12)(H2,13,14,15);/q;+1/p-1

sodium hydrogen (1-hydroxy-1-phosphono-2-(pyridin-3-yl)ethyl)phosphonate

Risedronic Acid Sodium; NE-58095; NE 58095; Risedronate sodium; 115436-72-1; Sodium risedronate; Actonel; Atelvia; Risedronic acid sodium salt; Risedronate (sodium); Risedronic acid monosodium salt; NE58095; Risedronate, Risedronic acid, Risedronate sodium, Actonel, Atelvia, Benet

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~8.33 mg/mL (~27.30 mM)
DMSO : ~1 mg/mL (~3.28 mM)

Solubility in Formulation 1: 16.67 mg/mL (54.64 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)