FKBP12; calcineurin; macrocyclic lactone

FK-506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit in T lymphocytes.[1] K-506 inhibits a Ca(2+)-dependent process necessary for the induction of interleukin-2 transcription, which stops T-cell proliferation. [2] Cyclophilins and FK 506-binding proteins (FKBPs) are two different families of intracellular proteins (immunophilins) that FK 506 binds to. At drug concentrations that prevent activated T cells from producing interleukin 2, FK-506 specifically inhibits cellular calcineurin. [3] By blocking the same subset of early calcium-associated events involved in lymphokine expression, apoptosis, and degranulation, FK-506 and CsA have nearly identical biological effects on cells. The FK-506 binding proteins (FKBPs), a family of intracellular receptors, are where FK-506 binds. [4]

FK-506 results in increase in the paw and tail withdrawal threshold as revealed by behavioral pain assessment in rats against hyperalgesic and allodynic stimuli. Additionally, FK-506 lowers serum nitrate and thiobarbituric acid reactive substance (TBARS) levels. It also lowers tissue myeloperoxidase (MPO) and total calcium levels, while raising tissue reduced glutathione levels in rats. In rats with ischemia reperfusion (I/R), FK-506 reduces the progression of neuronal edema and axonal degeneration. [5]

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The aim of this study was to elucidate the effect of tacrolimus (FK506) and of C-X-C chemokine receptor type 4 (CXCR4), which is a receptor specific to the stromal cell-derived factor-1α (SDF‑1α), on growth and metastasis of hepatocellular carcinoma (HCC). Following treatment with different concentrations of FK506, AMD3100 or normal saline (NS), the proliferation of Morris rat hepatoma 3924A (MH3924A) cells was measured by the MTT assay, the expression of CXCR4 was analyzed with immunohistochemistry, and the morphological changes and the invasiveness of cells were studied with a transwell assay and under a scanning electron microscope, respectively. In addition, August Copenhagen Irish rat models implanted with tumor were used to examine the pathological changes and invasiveness of tumor in vivo, the expression of CXCR4 in tumor tissues and the expression of SDF‑1α in the adjacent tissues to the HCC ones, using immunohistochemistry. In vitro, FK506 (100‑1,000 µg/l) significantly promoted the proliferation of MH3924A cells (P<0.01), and increased the expression of CXCR4 in MH3924A cells, albeit with no significance (P>0.05). By contrast, AMD3100 had no effect on the proliferation of MH3924A cells, but significantly reduced the expression of CXCR4 (P<0.05). The invasiveness of MH3924A cells was significantly (P<0.01) enhanced following treatment with FK506, SDF‑1α, FK506 + AMD3100, FK506 + SDF‑1α or FK506 + AMD3100 + SDF‑1α. In vivo, tumor weight (P=0.041), lymph node metastasis (P=0.002), the number of pulmonary nodules (P=0.012), the expression of CXCR4 in tumor tissues (P=0.048) and that of SDF‑1α in adjacent tissues (P=0.026) were significantly different between the FK506-treated and the NS group. Our results suggest that FK506 promotes the proliferation of MH3924A cells and the expression of CXCR4 and SDF‑1α in vivo. Therefore, inhibiting the formation of the CXCR4/SDF‑1α complex may partly reduce the promoting effect of FK506 on HCC [4].

Tacrolimus (FK506) inhibits calcium-dependent events, such as IL-2 gene transcription, NO synthase activation, cell degranulation, and apoptosis. Tacrolimus also potentiates the actions of glucocorticoids and progesterone by binding to FKBPs contained within the hormone receptor complex, preventing degradation. The agent may enhance expression of the TGFβ-1 gene in a fashion analogous to that demonstrated for CsA. T cell proliferation in response to ligation of the T cell receptor is inhibited by Tacrolimus. Treatment with a low concentration of Tacrolimus (FK506,10 μg/L) does not significantly affect the proliferation of MH3924A cells (P=0.135). Upon treatment with higher concentrations of Tacrolimus (100-1,000 μg/L), the proliferation of MH3924A cells is significantly enhanced (P<0.01). However, when different concentrations of AMD3100 are combined with 100 μg/L Tacrolimus, the in vitro proliferation of MH3924A cells is increased (P<0.01).

Cell Treatment and Lysis. Immunosuppressive agents were dissolved in ethanol at concentrations 1000-fold more than the concentration desired for cell treatments. Cells (106) were suspended in 1 ml of complete medium in microcentrifuge tubes; 1 Al of ethanol or of the ethanolic solution of FK 506, CsA, or rapamycin was added, and the cells were incubated at 37°C for 1 hr. Cells were washed twice with 1 ml of phosphate-buffered saline (PBS) on ice and lysed in 50 ,u of hypotonic buffer containing 50 mM Tris (pH 7.5); 0.1 mM EGTA; 1 mM EDTA; 0.5 mM dithiothreitol; and 50 ,ug of phenylmethylsulfonyl fluoride, 50 ,g of soybean trypsin inhibitor, 5 ,g of leupeptin, and 5 ,ug of aprotinin per ml. Lysates were subjected to three cycles of freezing in liquid nitrogen followed by thawing at 30°C and then were centrifuged at 4°C for 10 min at 12,000 x g.[3]

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Interleukin 2 (IL-2) Assay. Jurkat cells were cultured in complete medium at 106 cells per ml in 96-well flat-bottom plates. Cells were stimulated with OKT3 monoclonal antibody (1:4000 dilution of ascites) and 2 ng of phorbol 12- myristate 13-acetate (PMA) per ml for 24 hr in the presence or absence of FK 506 or CsA. IL-2 production was quantitated by measuring the ability of serial dilutions of cell supernatants to support the proliferation of the IL-2- dependent cell line CTLL-20 as described. One unit is defined as the amount of recombinant human IL-2 required to induce half-maximal proliferation of the CTLL-20 cells. FK 506 and CsA added directly to CTLL-20 cells do not inhibit IL-2-dependent proliferation. [3]

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The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.[3]

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Cells were cultured in the presence of 10 nM FK 506 for 1 hr and washed, and phosphatase activity was measured in lysates.

Mice; Six-week-old male C57BL/6J mice are maintained in a temperature- and humidity-controlled room with a 12-h light-dark cycle. FTacrolimus 30 mg/kg is given orally to colitic mice (n=10) for either 7 or 14 days (Days 10 to 23) as part of the multiple dosing study. The same regimen is used to administer placebos to the control group (n = 10) and the normal group (n = 5). 10 mL/kg of placebo or tacrolimus is given. On the day after the final dose, mice are put to death by CO2 inhalation. For the single-dose study, colitic mice are given Tacrolimus or a placebo (n=8) orally once on Days 7, 10, 17, or 24. The same procedure is used to administer a placebo to normal mice (n = 4). Eight hours after dosing, mice are put to death by CO2 inhalation.

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We investigated the effect of tacrolimus, a calcineurin inhibitor, on dextran sulfate sodium (DSS)-induced colitis. After inducing colitis in C57BL/6 mice by administering DSS solution as drinking water for 7 d, the animals were treated with tacrolimus. Severity of colonic inflammation was evaluated based on colon weight per unit length. Levels of cytokines (interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-12, and tumor necrosis factor (TNF)-α) released from isolated inflamed colons of mice treated with tacrolimus or vehicle were also measured. Treatment with tacrolimus for 14 d reduced the colon weight per unit length and suppressed the release of IFN-γ and IL-1β, but not other cytokines, in inflamed colons of colitic mice compared with vehicle-treated mice. A positive correlation was noted between colon weight per unit length and released level of IFN-γ or IL-1β. The release of IFN-γ and IL-1β was also suppressed after single dosing with tacrolimus to colitic mice. Taken together, these results suggested that tacrolimus ameliorated DSS-induced colitis by suppressing release of IFN-γ and IL-1β from inflamed colon.[4]

Absorption, Distribution and Excretion
Following oral administration, tacrolimus is not completely absorbed in the gastrointestinal tract, and there is significant individual variability. The absolute bioavailability in adult kidney transplant patients was 17±10%; in adult liver transplant patients, it was 22±6%; and in healthy subjects, it was 18±5%. The absolute bioavailability in pediatric liver transplant patients was 31±24%. In 18 fasting healthy volunteers, after a single oral dose of 3, 7, and 10 mg tacrolimus, the peak plasma concentration (Cmax) and area under the curve (AUC) increased proportionally to the dose. Absorption rate and extent were highest when taken on an empty stomach. Meal timing also affects bioavailability. Compared to a fasting state, immediate administration after a meal reduced the average Cmax by 71% and the average AUC by 39%. Administration 1.5 hours after a meal reduced the average Cmax by 63% and the average AUC by 39% compared to a fasting state. In the human body, less than 1% of the administered dose is excreted unchanged in the urine. When administered intravenously, fecal excretion accounted for 92.6±30.7%, and urinary excretion accounted for 2.3±1.1%. 2.6 ± 2.1 L/kg [Pediatric liver transplant patients]
1.07 ± 0.20 L/kg [Patients with renal insufficiency, 0.02 mg/kg/4 hours, intravenous]
3.1 ± 1.6 L/kg [Mild hepatic insufficiency, 0.02 mg/kg/4 hours, intravenous]
3.7 ± 4.7 L/kg [Mild hepatic insufficiency, 7.7 mg, oral]
3.9 ± 1.0 L/kg [Severe hepatic insufficiency, 0.02 mg/kg/4 hours, intravenous]
3.1 ± 3.4 L/kg [Severe hepatic insufficiency, 8 mg, oral]
0.040 L/hr/kg [Healthy subjects, intravenous]
0.172 ± 0.088 L/hr/kg [Healthy subjects, oral]
0.083 L/hr/kg [Adult kidney transplant patient, intravenous injection]
0.053 L/hr/kg [Adult liver transplant patient, intravenous injection]
0.051 L/hr/kg [Adult heart transplant patient, intravenous injection]
0.138 ± 0.071 L/hr/kg [Pediatric liver transplant patient]
0.12 ± 0.04 (range 0.06-0.17) L/hr/kg [Pediatric kidney transplant patient]
0.038 ± 0.014 L/hr/kg [Patients with renal insufficiency, 0.02 mg/kg/4 hours, intravenous injection]
0.042 ± 0.02 L/hr/kg [Mild hepatic impairment, 0.02 mg/kg/4 hours, intravenous injection]
0.034 ± 0.019 L/hr/kg [Mild hepatic impairment, 7.7 mg, oral]
0.017 ± 0.013 L/hr/kg [Severe hepatic impairment, 0.02 mg/kg/4 hours, intravenous injection]
0.016 ± 0.011 L/hr/kg [Severe hepatic impairment, 8 mg, oral]
This study aimed to assess the concentration of tacrolimus in breast milk and neonatal exposure during breastfeeding. This observational cohort study was conducted at two tertiary referral high-risk obstetric clinics. The study included 14 women who took tacrolimus during pregnancy and lactation and their 15 infants, 11 of whom were exclusively breastfed. Tacrolimus levels were analyzed using liquid chromatography-tandem mass spectrometry. Maternal and umbilical cord blood samples were collected at delivery, and maternal, infant, and breast milk samples were collected postpartum, where possible. Tacrolimus levels decreased in all infants who underwent continuous sampling, with a daily decrease of approximately 15% (geometric mean concentration ratio 0.85; 95% confidence interval 0.82–0.88; P < 0.001). Tacrolimus levels were not elevated in breastfed infants compared to bottle-fed infants (median 1.3 μg/L [range 0.0–4.0] vs. 1.0 μg/L [range 0.0–2.3]; P = 0.91). The maximum absorption of tacrolimus in breast milk was estimated to be 0.23% of the maternal dose (adjusted for body weight). The amount of tacrolimus ingested by infants through breast milk was negligible. Breastfeeding did not appear to slow the decline in high tacrolimus blood concentrations at birth in infants. Maternal and umbilical cord (venous and arterial) blood samples were collected at delivery from eight solid organ transplant recipients to measure the bound and free concentrations of tacrolimus and its metabolites in blood and plasma. Pharmacokinetics of tacrolimus in breast milk was assessed in one of the subjects. At delivery, the mean concentration (± standard deviation) of tacrolimus in umbilical cord blood was 6.6 ± 1.8 ng/ml, equivalent to 71 ± 18% (range 45–99%) of the maternal concentration (9.0 ± 3.4 ng/ml). The mean concentration (0.09 ± 0.04 ng/ml) and free drug concentration (0.003 ± 0.001 ng/ml) of tacrolimus in umbilical cord plasma were approximately one-fifth of the corresponding maternal concentration. The concentration of tacrolimus in umbilical cord artery blood was 100 ± 12% of the concentration in umbilical cord blood. Furthermore, the infant received less than 0.3% of the mother's weight-adjusted dose of tacrolimus through breast milk. The difference in tacrolimus concentrations between maternal and cord blood may be partly attributed to placental P-gp function, higher erythrocyte distribution, and higher hematocrit in umbilical cord blood. Ten colostrum samples were collected from six women in the early postpartum period (0-3 days), with an average drug concentration of 0.79 ng/mL (range 0.3-1.9 ng/mL). The median ratio of breast milk to maternal plasma was 0.5. Tacrolimus has a plasma protein binding rate of approximately 99%, which is concentration-independent within the range of 5-50 ng/mL. Tacrolimus primarily binds to albumin and α-1-acid glycoprotein and is highly bound to erythrocytes. The distribution of tacrolimus in whole blood and plasma depends on various factors, such as hematocrit, temperature during plasma separation, drug concentration, and plasma protein concentration. In a US study, the average ratio of whole blood concentration to plasma concentration was 35 (range 12-67). Based on blood drug concentrations, there is no evidence that intermittent topical application of tacrolimus for up to one year results in accumulation in the body. As with other topical calcineurin inhibitors, it is currently unclear whether tacrolimus distributes to the lymphatic system. For more complete data on the absorption, distribution, and excretion of tacrolimus (9 metabolites in total), please visit the HSDB record page.

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Metabolism/Metabolites
Tacrolimus metabolism is primarily mediated by CYP3A4, and secondarily by CYP3A5.Tacrolimus is metabolized into eight metabolites: 13-demethyltacrolimus, 31-demethyltacrolimus, 15-demethyltacrolimus, 12-hydroxytacrolimus, 15,31-didemethyltacrolimus, 13,31-didemethyltacrolimus, 13,15-didemethyltacrolimus, and a final metabolite involving O-demethylation and fused ring formation. In human liver microsomal incubation assays, the major metabolite identified was 13-demethyltacrolimus. In vitro studies have shown that the 31-demethyl metabolite has the same activity as tacrolimus. Tacrolimus is primarily metabolized via a mixed-function oxidase system, particularly the cytochrome P-450 system (CYP3A). A metabolic pathway generating eight possible metabolites has been proposed. In vitro experiments have shown that demethylation and hydroxylation are the main biotransformation mechanisms. The major metabolite identified in human liver microsomal incubation experiments is 13-demethyltacrolimus. In vitro studies have shown that the 31-demethyl metabolite has the same activity as tacrolimus. Known human metabolites include 13-O-demethyltacrolimus and 15-O-demethyltacrolimus. The elimination half-lives in healthy adult volunteers, kidney transplant recipients, liver transplant recipients, and heart transplant recipients are approximately 35, 19, 12, and 24 hours, respectively. The elimination half-life in pediatric liver transplant patients was 11.5 ± 3.8 hours, and in pediatric kidney transplant patients it was 10.2 ± 5.0 hours (range 3.4–25 hours). In a mass balance study of intravenously administered radiolabeled tacrolimus in 6 healthy volunteers, the elimination half-life calculated based on radioactivity was 48.1 ± 15.9 hours, while the elimination half-life calculated based on tacrolimus concentration was 43.5 ± 11.6 hours. With oral administration, the elimination half-life calculated based on radioactivity was 31.9 ± 10.5 hours, while the elimination half-life calculated based on tacrolimus concentration was 48.4 ± 12.3 hours… This article reports a case of tacrolimus toxicity in a non-transplant patient. The patient received 2.1 mg/kg/day of tacrolimus for 4 consecutive days (therapeutic doses range from 0.03 to 0.05 mg/kg/day). Her tacrolimus elimination half-life was 16.5 hours, while the mean half-life in healthy volunteers was 34.2 ± 7.7 hours.

Toxicity Summary
Identification and Uses: Tacrolimus is a white to off-white crystalline powder. It is a calcineurin inhibitor, an immunosuppressant available in various formulations. Tacrolimus oral capsules and intravenous solutions are used to prevent organ rejection in patients who have received liver, kidney, or heart transplants. Tacrolimus topical ointment is available as a second-line therapy for short-term and non-continuous chronic treatment of moderate to severe atopic dermatitis in non-immunely compromised adults and children. Human Exposure and Toxicity: While most acute tacrolimus overdose (up to 30 times the intended dose) is asymptomatic and all patients recover without sequelae, some acute overdose cases have resulted in adverse reactions, including tremor, renal dysfunction, hypertension, and peripheral edema. Patients receiving tacrolimus at therapeutic doses have an increased risk of developing lymphoma and other malignancies, particularly cutaneous malignancies, as well as an increased risk of bacterial, viral, fungal, and protozoan infections, including opportunistic infections. These infections can lead to serious consequences and even be life-threatening. Although there are currently no adequate and well-controlled studies in pregnant women, tacrolimus use during pregnancy in humans has been associated with neonatal hyperkalemia and renal dysfunction. Animal studies: Rats and baboons showed similar toxicological characteristics after oral or intravenous administration of tacrolimus. In both rats and baboons, the dose at which toxicity occurred after intravenous administration was lower than that after oral administration. The dose at which toxicity occurred in rats was lower than that in baboons. The main target organs were the kidneys, islets of Langerhans and exocrine pancreas, spleen, thymus, gastrointestinal tract, and lymph nodes. In addition, a decrease in erythrocyte parameters was observed. Tacrolimus is reproductively and developmentally toxic in both rats and rabbits. In rats, long-term oral administration of high doses of tacrolimus led to changes in reproductive organs and glaucoma/ocular lesions. Daily oral administration of 1 and 3.2 mg/kg tacrolimus caused significant parental toxicity symptoms and resulted in alterations in fertility and overall reproductive function in rats. Effects on reproduction included partial embryonic death, reduced implantation number, increased post-implantation loss rate, and decreased embryo and offspring survival. In a rabbit teratogenicity study, all oral doses of tacrolimus (0.1, 0.32, or 1 mg/kg/day) resulted in maternal toxicity, including weight loss. The 0.32 and 1 mg/kg/day doses also caused developmental toxicity, such as increased post-implantation embryo loss, reduced number of surviving fetuses, and increased morphological variations. In a rat teratogenicity study, a dose of 3.2 mg/kg/day was observed to increase post-implantation embryo loss. A maternal dose of 1 mg/kg/day resulted in weight loss in the F1 generation. A maternal dose of 3.2 mg/kg/day resulted in weight loss, reduced number of surviving fetuses, and some skeletal deformities in the F1 generation. Tacrolimus did not show genotoxicity in in vitro Salmonella and Escherichia coli assays or in the Chinese hamster lung cell mammalian assay. No in vitro mutagenicity was observed in the CHO/HGPRT assay (Chinese hamster ovary cell assay, used to detect positive mutations at the HGPRT locus), nor in the in vivo chromosome breakage assay in mice. Tacrolimus does not induce unplanned DNA synthesis in rodent hepatocytes. Interactions In a given dose of mycophenolic acid (MPA) products, MPA exposure is higher when co-administered with Prograf compared to cyclosporine because cyclosporine blocks the enterohepatic circulation of MPA, while tacrolimus does not. Clinicians should note that MPA exposure may increase when switching from cyclosporine to Prograf in patients concurrently taking mycophenolic acid (MPA)-containing medications. Grapefruit juice inhibits CYP3A enzymes, leading to elevated whole blood trough tacrolimus concentrations; therefore, patients should avoid consuming grapefruit or grapefruit juice while taking tacrolimus. Since tacrolimus is primarily metabolized by CYP3A enzymes, drugs or substances that inhibit these enzymes are known to increase whole blood tacrolimus concentrations. Drugs that induce CYP3A enzymes are known to decrease whole blood tacrolimus concentrations. When tacrolimus is used concomitantly with CYP3A inhibitors or inducers, dose adjustments and frequent monitoring of tacrolimus whole blood trough concentrations may be necessary. Additionally, patients should be monitored for adverse reactions, including changes in renal function and QT prolongation. Verapamil, diltiazem, nifedipine, and nicardipine inhibit CYP3A metabolism of tacrolimus, potentially increasing whole blood concentrations. When these calcium channel blockers are used concomitantly with tacrolimus, monitoring of whole blood concentrations and appropriate dose adjustments of tacrolimus are recommended. For more complete data on tacrolimus interactions (18 items in total), please visit the HSDB record page. Non-human toxicity values: Rat intravenous LD50: 23,600 μg/kg /tacrolimus hydrate/ Rat oral LD50: 134 mg/kg /tacrolimus hydrate/

[1]. Nature. 1991 Aug 29;352(6338):803-7.

[2]. Nature. 1992 Jun 25;357(6380):692-4.

[3]. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3686-90.

[4]. Tacrolimus promotes hepatocellular carcinoma and enhances CXCR4/SDF 1α expression in vivo. Mol Med Rep. 2014 Aug;10(2):585-92.

Therapeutic Uses

Immunosuppressants

Prograf is indicated for the prevention of organ rejection in patients receiving allogeneic kidney transplants. Prograf is recommended for use in combination with azathioprine or mycophenolate mofetil (MMF) and corticosteroids. /US Product Label/
Prograf is indicated for the prevention of organ rejection in patients receiving allogeneic liver transplants. Prograf is recommended for use in combination with corticosteroids. Treatment monitoring is recommended for all patients receiving Prograf. /US Product Label/
Prograf is indicated for the prevention of organ rejection in patients receiving allogeneic heart transplants. Prograf is recommended for use in combination with azathioprine or mycophenolate mofetil (MMF) and corticosteroids. /Included in US Product Label/
For more complete data on the therapeutic uses of tacrolimus (13 in total), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Malignancy and serious infections. Due to immunosuppression, there is an increased risk of developing lymphoma and other malignancies, especially cutaneous malignancies. Susceptibility to bacterial, viral, fungal, and protozoan infections, including opportunistic infections, is also increased. Prescription of Prograf should only be given by physicians experienced in immunosuppressive therapy and the management of organ transplant patients. Patients receiving this medication should be treated in facilities with adequate laboratory and ancillary medical resources. The physician responsible for maintenance therapy should have all the information necessary for patient follow-up.
/Black Box Warning/ Warning: The long-term safety of topical calcineurin inhibitors has not been established. Although causality has not been established, rare malignancies (such as skin cancer and lymphoma) have been reported in patients treated with topical calcineurin inhibitors (including Protopic ointment). Therefore: Prolonged continuous use of topical calcineurin inhibitors (including Protopic ointment) should be avoided in any age group and should only be applied to areas of atopic dermatitis; Protopic ointment is not suitable for children under 2 years of age; only 0.03% Protopic ointment is suitable for children aged 2–15 years.
Topical tacrolimus should be avoided for malignant or precancerous skin conditions (such as cutaneous T-cell lymphoma (CTCL)) because the clinical presentation of these conditions may be similar to dermatitis.
Due to the potential increase in skin cancer risk, patients using topical tacrolimus are advised to limit sun or other UV exposure by wearing protective clothing and using a broad-spectrum sunscreen with a high SPF.
For more complete (42) data on drug warnings for tacrolimus, please visit the HSDB record page.

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Pharmacodynamics
Tacrolimus reduces the activity of peptidyl prolyl isomerase by forming a new complex with the immunoaffinity FKBP-12 (FK506-binding protein). Tacrolimus inhibits T-lymphocyte signaling and IL-2 transcription. Tacrolimus has similar activity to cyclosporine but with a lower incidence of rejection. Tacrolimus has also been shown to be effective in the topical treatment of eczema, especially atopic eczema. It suppresses inflammation in a similar way to steroids, but with less potency. A significant advantage of tacrolimus in dermatology is that it can be applied directly to the face; topical steroids, on the other hand, cannot be used on the face because they significantly thin the facial skin. In other parts of the body, topical steroids are often a better treatment option. Cyclosporine A and FK506 inhibit T-cell and B-cell activation, as well as other processes crucial for an effective immune response. In T lymphocytes, these drugs interfere with an unknown step in the signaling process from T-cell antigen receptors to cytokine genes that coordinate the immune response. The presumed intracellular receptor for FK506 and cyclosporine is cis-trans-prolyl isomerase. Drug binding inhibits isomerase activity, but studies of other prolyl isomerase inhibitors and analysis of cyclosporine-resistant mutants in yeast suggest that the drug's effect stems not from the inhibition of isomerase activity, but from the formation of an inhibitory complex between the drug and the isomerase. The transcription factor NF-AT is crucial for early T cell gene activation and appears to be a specific target of cyclosporine A and FK506, as transcription mediated by this protein is blocked in T cells treated with these drugs, with little effect on other transcription factors such as AP-1 and NF-κB. This paper demonstrates that NF-AT is formed when an antigen receptor signals to induce the translocation of a pre-existing cytoplasmic subunit to the nucleus and bind to a newly synthesized nuclear subunit. FK506 and cyclosporine A block the translocation of cytoplasmic components without affecting the synthesis of nuclear subunits. [1]

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After the T cell receptor (TCR) recognizes an antigen, it initiates a series of events, including the transcription of lymphokine genes, especially the transcription of interleukin-2 (IL-2), which ultimately leads to T cell activation. The immunosuppressants cyclosporine A (CsA) and FK-506 prevent T cell proliferation by inhibiting the Ca(2+)-dependent events required to induce IL-2 transcription. The complex formed by FK-506 or CsA with their respective intracellular binding proteins can inhibit the calmodulin-dependent protein phosphatase calcineurin in vitro. The pharmacological significance of this observation in terms of immunosuppression or drug toxicity remains unclear. Although calcineurin is present in lymphocytes, it has not been previously thought to be involved in TCR-mediated activation of lymphokine genes or general transcriptional regulation. This article reports that transfection with the catalytic subunit of calcineurin increases the half-maximal inhibitory concentration (IC50) of the immunosuppressants FK-506 and CsA, and that the mutant subunit synergizes with phorbol ester to activate the interleukin-2 promoter in a drug-sensitive manner. These results suggest that calcineurin is an integral part of the T-cell receptor (TCR) signaling pathway, playing a role in drug-sensitive interleukin-2 promoter activation. [2]

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The immunosuppressants cyclosporine A (CsA) and FK-506 bind to different families of intracellular proteins (immunavidins), referred to as cyclophilin and FK-506-binding protein (FKBP), respectively. Recent in vitro studies have shown that the CsA-cyclosporine complex and the FK 506-FKBP-12 complex can bind to and inhibit the activity of calcineurin (a calcium-dependent serine/threonine phosphatase). We investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibited intracellular calcineurin activity, and their drug concentrations were comparable to those that inhibited interleukin-2 (IL-2) production in activated T cells. Although rapamycin also binds to FKBP, its biological activity differs from that of FK 506, and it has no effect on calcineurin activity. In addition, excessive rapamycin inhibits the action of FK506, possibly because rapamycin displaces FK506 from FKBP. These results indicate that calcineurin is a target of drug-immunoaffin complexes in vivo and establish the physiological role of calcineurin in T cell activation. [3]

C44H69NO12

804.0182

803.481

C, 57.92; H, 5.69; Cl, 3.64; F, 5.85; N, 7.19; O, 9.85; S, 9.87

104987-11-3

Tacrolimus monohydrate;109581-93-3;Tacrolimus-13C,d2;1356841-89-8

445643

White to off-white solid powder

1.2±0.1 g/cm3

871.7±75.0 °C at 760 mmHg

113-115°C

481.0±37.1 °C

0.0±0.6 mmHg at 25°C

1.549

fungus Streptomyces tsukubaensis.

3.96

3

12

7

57

1480

14

O1[C@]2(C(C(N3C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@@]3([H])C(=O)O[C@]([H])(/C(/C([H])([H])[H])=C(\[H])/[C@]3([H])C([H])([H])C([H])([H])[C@]([H])([C@@]([H])(C3([H])[H])OC([H])([H])[H])O[H])[C@]([H])(C([H])([H])[H])[C@]([H])(C([H])([H])C([C@]([H])(C([H])([H])C([H])=C([H])[H])C([H])=C(C([H])([H])[H])C([H])([H])[C@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])([C@]1([H])[C@]([H])(C([H])([H])[C@@]2([H])C([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H])=O)O[H])=O)=O)O[H] |c:78|

QJJXYPPXXYFBGM-LFZNUXCKSA-N

InChI=1S/C44H69NO12/c1-10-13-31-19-25(2)18-26(3)20-37(54-8)40-38(55-9)22-28(5)44(52,57-40)41(49)42(50)45-17-12-11-14-32(45)43(51)56-39(29(6)34(47)24-35(31)48)27(4)21-30-15-16-33(46)36(23-30)53-7/h10,19,21,26,28-34,36-40,46-47,52H,1,11-18,20,22-24H2,2-9H3/b25-19+,27-21+/t26-,28+,29+,30-,31+,32-,33+,34-,36+,37-,38-,39+,40+,44+/m0/s1

(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1,14-dihydroxy-12-[(E)-1-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]prop-1-en-2-yl]-23,25-dimethoxy-13,19,21,27-tetramethyl-17-prop-2-enyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetrone

FR900506;FR 900506; FR-900506; FK 506; FK-506; FK506; fujimycin; Prograf; Protopic; Advagraf; Astagraf XL; Fujimycin; 104987-11-3; Prograf; Tsukubaenolide; Tacrolimus anhydrous; Protopic; Anhydrous Tacrolimus;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~94 mg/mL (~116.9 mM)
Water: <1 mg/mL
Ethanol: ~83 mg/mL (~103.2 mM)

Solubility in Formulation 1: 2.75 mg/mL (3.42 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 2.5 mg/mL (3.11 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (3.11 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 5% DMSO+corn oil: 15mg/mL

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