1. Butyrate is a naturally occurring fatty acid, (the name butyrate comes from the word 'butter') that is also a product of digestion of soluble fiber in our colon.
Sodium butyrate is the sodium salt of butyric acid. Butyric acid is a saturated short chain fatty acid, consisting of 4 carbon atoms.
Butyrate is a "major end-product of the microbial digestion of carbohydrates in the alimentary canal." Occurrence, absorption and metabolism of short chain fatty acids in the digestive tract of mammals.
"Butyric acid is a SCFA (short chain fatty acid) produced during the fermentation of fiber by endogenous intestinal bacteria and is also present in fruits, vegetables, and milk fat". Heerdt, 1998
When three butyrate molecules are connected together, we get Tributyrin (glyceryl tributyrate). Tributyrin is an approved food additive in Canada and the US, and more importantly, is included on the Codex list of food additives. so it should be internationally accepted.
2. Butyrate has numerous anti-cancer actions
Butyrate is effective due to its structure and utilization. Potentiation by Specific Short-Chain Fatty Acids of Differentiation and Apoptosis in Human Colonic Carcinoma Cell Lines
"Although each of the SCFA (short chain fatty acids) tested significantly reduced primary cell invasion, butyrate was the most potent, inhibiting primary invasive human colon cancer invasion" Short-chain fatty acids inhibit invasive human colon cancer
"these are exciting times for the dietary micronutrient butyrate and HDAC inhibitors in the challenge of preventing and treating cancer." Inhibition of Histone Deacetylase Activity by Butyrate
"Beyond their nutritional effect, short-chain fatty acids, especially butyrate, modulate cell differentiation, proliferation, motility, and in particular, they induce cell cycle arrest and apoptosis." Butyrate-induced apoptosis and cell cycle arrest in bovine kidney epithelial cells: Involvement of caspase and proteasome pathways (pdf)
"Butyrate is a short-chain fatty acid (SCFA) that acts as a HDAC inhibitor and is being clinically evaluated as an anti-neoplastic therapeutic, primarily because of its ability to impose cell cycle arrest, differentiation, and/or apoptosis in many tumor cell types, and its favorable safety profile in humans." Short-chain fatty acid inhibitors of histone deacetylases: promising anticancer therapeutics?
"butyrate induces different biological effects on different cell types" and "the results showed that butyrate treatment activates caspase-3 activities and induces accumulation of acetylated histone. At least two proteins, cdc6 and cdk1, become targeted for destruction on butyrate treatment." and "these results functionally locate the proteasome pathway upstream of the caspase pathway. All these results indicate that butyrate functions as both a nutrient and signaling molecule regulating cell growth and proliferation." Butyrate-induced apoptosis and cell cycle arrest in bovine kidney epithelial cells: Involvement of caspase and proteasome pathways
Butyrate (as tributyrin) works against breast cancer cells. Dietary Butyrate Inhibits NMU-Induced Mammary Cancer in Rats and Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cells
Butyrate affects the regulation of 98 genes. It downregulates 16 cytokine genes. The differential regulation of metastasis-associated genes by NaB provides explanation for the antiinvasive properties of NaB." Expression proﬁling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB
These results indicate that NaB, besides regulating other fundamental cellular processes, is able to modulate the expression of two important angiogenesis-related molecules and suggested a further possible clinical application of this short-chain fatty acid as an anti-angiogenic compound in association with conventional chemotherapeutic agents." Modulation of angiogenesis-related proteins synthesis by sodium butyrate in colon cancer cell line HT29
Butyrate inhibits inflammation. Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-κB and PPARα
"Aromatase inhibitors are the most commonly used and effective means of nontoxic treatment of estrogen-responsive breast cancer." "sodium butyrate profoundly decreased" expression of genes related to aromatase. A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts.
3. Mitochondrial Membrane Action
Butyrate's actions are connected to mitochondrial action. Short-Chain Fatty Acid-initiated Cell Cycle Arrest and Apoptosis of Colonic Epithelial Cells Is Linked to Mitochondrial Function
In work very reminiscent of the Michelakis paper, Dr. Heerdt et al show the importance of the mitochondrial membrane potential as it relates to cancer: "Here, we showed the profound relationship between differences in
the MMP(=mitochondrial membrane potential) and important tumorigenic properties. Cells with higher intrinsic MMP have an enhanced capacity to (a) respond to hypoxia
by avoiding apoptosis and initiating angiogenesis, (b) escape anoikis and grow under anchorage-independent conditions, and (c)
invade the basement membrane. Therefore, these data establish that differences in the intrinsic MMP of colonic carcinoma cells are
likely linked to shifts in biochemical pathways and/or cell composition that play fundamental roles in determining the
probability of colonic tumor progression." They used butyrate in their research.
In this paper Dr. Heerdt et. al. used tributyrin: Initiation of Growth Arrest and Apoptosis of Mammary Carcinoma Cells
by Tributyrin Is Associated with Mitochondrial Activity
Butyrate alters mitochondrial membrane potential. Mitochondrial Membrane Potential in the Coordination of p53-independent Proliferation and Apoptosis Pathways in Human Colonic Carcinoma Cells
"cells with lower MMP (mitochondrial membrane potential) were more likely to respond to the chemopreventive activities of butyrate, including MMP dissipation, growth arrest, and apoptosis, than cells with higher MMP". The Intrinsic Mitochondrial Membrane Potential of Colonic Carcinoma Cells Is Linked to the Probability of Tumor Progression
"Butyrate incubation caused an overall decrease in mitochondrial membrane potential" Effects Of Fish Oil And Butyrate On Diet-mediated Apoptosis At The Promotion Stage Of Colon Carcinogenesis
"In an extension of our findings, we show for the first time that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+-dependent mitochondrial-intrinsic pathway. Notably, DHA-enriched mitochondria were sensitized to rapidly sequester Ca2+, which served to trigger apoptosis in the presence of butyrate." Docosahexaenoic Acid and Butyrate Synergistically Induce Colonocyte Apoptosis by Enhancing Mitochondrial Ca2+ Accumulation (June 2007)
4. Butyrate Synergies Many Therapies
Butyrate by itself is a modest anti-cancer agent, but serves to increase the action of many other anti-cancer agents. I give examples of the variety of agents that butyrate can synergize. I hope you see the incredible variety of therapies butyrate synergizes.
"...human and rat colon cancer cells undergo massive apoptosis when they are exposed to soluble Fas ligand in the presence of sodium butyrate, an agent that induces by itself
only a low rate of apoptosis." and "The results presented here demonstrate that sodium butyrate synergistically increases Fas ligand-induced apoptosis in the tested cell lines" and "Because the clinical toxicity of butyrate is low, its ability to enhance Fas-signal delivery in cancer cells could be of therapeutic interest." Cancer cell sensitization to Fas-mediated apoptosis by sodium butyrate
"In comparing gene expression profiles from mucosa, the fish oil/pectin diet vs the corn oil/cellulose diet showed significant changes in the expression of genes involved in lipid metabolism, nutrient transport and xenobiotic metabolism." from A Combination Of Omega-3 Fatty Acids And A Butyrate-producing Fiber Mitigates Colon Cancer Development (pdf)
"... these results,
induced only by the combination of butyrate with fish oil, may lead to increased
apoptosis at the promotion stage of colon carcinogenesis via a mitochondria-mediated
Effects Of Fish Oil And Butyrate On Diet-mediated Apoptosis At The Promotion Stage Of Colon Carcinogenesis
"We have previously shown that butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. Interestingly, fermentable fiber when combined with fish oil containing docosahexaenoic acid (DHA, 22:6n-3) exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis". and "the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+-mediated intrinsic mitochondrial pathway" Docosahexaenoic Acid and Butyrate Synergistically Induce Colonocyte Apoptosis by Enhancing Mitochondrial Ca2+ Accumulation
" butyrate (in the form of its prodrug, tributyrin) upregulates the expression of the vitamin D receptor and enhances the antiproliferative action of (OH)2D3 in human colon cancer cells,"
Tributyrin, a Stable and Rapidly Absorbed Prodrug of Butyric Acid, Enhances Antiproliferative Effects of Dihydroxycholecalciferol in Human Colon Cancer Cells
"Numerous studies have demonstrated in vitro synergism of butyrate and retinoids The availability of retinoid compounds with differential effects on the various retinoid receptors including all-trans-retinoic acid (RAR receptors), 9-cis-retinoic acid (RAR and RXR) and bexarotene (RXR) allows exploration of several potentially beneficial effects. In addition, the combination of butyrate drugs with other biological response modifiers such as the interleukins also holds the promise of improved therapeutics." Clinical and pharmacologic study of tributyrin: an oral butyrate prodrug
Butyrate-hexosamine molecular hybrids show great promise as anti-cancer agents. Targeting Glycosylation Pathways and the Cell
Cycle: Sugar-Dependent Activity of Butyrate-Carbohydrate Cancer Prodrugs
Butyrate synergized three leukemia treatments. And it did so by caspase activation .Butyrate Increases Apoptosis Induced by Different Antineoplastic Drugs in Monocytic Leukemia Cells
"sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats." Neither product worked alone, but in combination they caused amazingly good results: "We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells." Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line
"Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in particular, the treatment of retinoblastoma" .The Apoptotic Effects and Synergistic Interaction of Sodium Butyrate and MG132 in Human Retinoblastoma Y79 Cells
Butyrate and Artemisinin, a compound extracted from the wormwood Artemisia annua, synergize. Synergistic cytotoxicity of artemisinin and sodium butyrate on human cancer cells.
"The present data show that quercetin produced synergistic effect in terms of cell killing in association with NaB. Both curcumin and ferulic acid potentiated NaB-induced reduction of cell number". Efficacies of plant phenolic compounds on sodium butyrate induced anti-tumour activity.
Butyrate typically synergizes with retinoids. For example, butyrate with trans-retinoic acid was synergistic. (source) and (source)
but was antagonistic with N-(4-hydroxyphenyl)-retinamide (4-HPR). Source
"Some success in overcoming retinoic acid (RA)-resistance has been reported for acute promyelocytic leukemia in cell lines and the clinic by combining histone deacetylase inhibitors, like sodium butyrate (NaB), with RA." A Retinoid/Butyric Acid Prodrug Overcomes Retinoic Acid
Resistance in Leukemias by Induction of Apoptosis
For a detailed examination of how DHA and tributyrin synergize, see the synergy page.
5. Tributyrin as a Therapy
The half-life of sodium butyrate in mice and rabbits is less than 5 minutes. Pharmacokinetic study of butyric acid administered in vivo as sodium and arginine butyrate salts
Tributyrin is " a natural component of anhydrous milk fat (AMF)" Dietary Butyrate Inhibits NMU-Induced Mammary Cancer in Rats
French researchers create tributyrin for testing and found it more stable than butyrate. Differential elimination of synthetic butyric triglycerides in vivo: a pharmacokinetic study
Tributyrin proves to be the best prodrug of butyrate. Effect of butyrate analogues on proliferation and differentiation in human neuroblastoma cell lines
"Tributyrin proved to be the most potent agent in its ability to induce these phenotypic changes," Tributyrin, an oral butyrate analogue, induces apoptosis through the activation of caspase-3
Early work on oral tributyrin shows ability to achieve pharmacological doses in rodents. It reveals an upper limit to tributyrin at between 8 and 10 grams per kilogram. Plasma pharmacokinetics of butyrate
Tributyrin is detectable in plasma after oral administration. Tributyrin (TB) is detectable as free drug in plasma after oral administration
"A butyrate prodrug, the triglyceride tributyrin, shows great promise in achieving effective and prolonged serum levels when given orally to mice and rats, and has been recommended for human trial". "Particular disease targets would include certain leukemias, thalassemia, and sickle cell anemia." Butyrate and phenylacetate as differentiating agents: Practical problems and opportunities
That above article is followed by a patent:by the author. http://www.patentstorm.us/patents/5645852-claims.html. Highlights include:
Tributyrin gave a plasma half-life of 40 minutes, compared to 6 minutes using butyrate.
Tributyin can be used in a carrier, "Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stensic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients."
3. "In a preferred embodiment of the invention, an effective amount of one or more butyryl glycerides effective to treat leukemia is an amount totaling between 1 and 50 grams per dose."
4. Adding vitamin D with tributyrin is strongly suggested.
"Overall, tributyrin had a 2.5- to 3-fold growth inhibitory and apoptosis-inducing potency compared with equimolar concentrations of sodium butyrate. Our results demonstrate that tributyrin is more potent than butyrate in regard to cell growth inhibition and apoptosis induction at pharmacologically relevant concentrations. Hence, tributyrin may be a promising candidate for clinical protocols in prostate cancer" Tributyrin induces differentiation, growth arrest and apoptosis in androgen-sensitive and androgen-resistant human prostate cancer cell lines
Both butyrate and tributyrin show significant anti-tumor in vivo action in models of human prostate cancer. Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer.
"Tributyrin mediated cell death was linked to up-regulation of caspases 3 and 8" Tributyrin-induced differentiation promotes apoptosis of LS 174T colon cancer cells in vitro (abstract) (full text,pdf)
"Although the mechanisms linking cell cycle arrest and apoptotic cascades in MCF-7 cells are yet to be fully understood, we have established that the triglyceride analogue of the SCFA butyrate, tributyrin, a nontoxic, well-tolerated food additive, initiates entry of mammary carcinoma cells into both pathways." Heerdt 1998
Tributyrin is shown to control gastric cancer cells. Tributyrin inhibits human gastric cancer SGC-7901 cell growth inducing apoptosis and DNA synthesis arrest
Tributyrin is shown to be a potential treatment for blood cancers. Tributyrin plus all-trans-retinoic acid efficiently induces fetal hemoglobin expression in human erythroleukemia cells. and Butyrate_induced_erythroid_differentiation_of_human_leukemia_cells
Tributyrin is effective against pancreatic cancer. Tributyrin, a butyrate precursor, impairs growth and induces apoptosis and differentiation in pancreatic cancer cells.
The Problem with Butyrate: "The sodium salt of butyric acid, sodium butyrate,
has potent effects on a variety of cell lines in vitro, including both malignant and normal mammary epithelial cells Despite its potency in vitro, butyric acid is rapidly metabolized in vivo, making it
difficult to achieve and maintain effective levels in the serum, even when its sodium or arginine salts are administered by continuous i.v. infusion. Moreover, i.v. infusion carries with it the possibility of
complications due to concomitant elevations in serum sodium or arginine. Therefore, the effectiveness of butyric acid salts as a chemotherapeutic or chemopreventive agents is compromised." Heerdt 1998
Tributyrin is the Solution: "Tributyrin, a triglyceride analogue of butyric acid (butyryl triglyceride), is similar in structure to 95% of the fatty acids found in foods, can be p.o.(orally) administered, is well tolerated even by young children, and is an approved food additive in the United States. Moreover, because tributyrin contains three butyric acid moieties esterified to glycerol, when completely hydrolyzed by cellular lipases or esterases, it yields 3-fold more butyric acid than sodium butyrate, producing and maintaining higher serum butyrate levels than p.o.-administered butyric acid salts" Heerdt 1998
6. Using Tributyrin. Researching the Clinical Trials
Fortunately we have clinical trials for tributyrin.
Phase I Study of the Orally Administered Butyrate Prodrug Tributyrin in Patients with Solid Tumors (1997)
1. They used doses from 50 to 400 mg/kg/day
2. No objective tumor responses seen. One patient with renal cancer had cancer stabilized, and was at a 400 mg/kg/day dose without apparent toxicity.
Phase I and Pharmacokinetic Study of Tributyrin: a Butyrate Prodrug and Possible Inhibitor of Histone Deacetylase. (2001)
1. The goal was to achieve levels above 500 mM. to replicate doses shown active in in vitro studies.
Oral administration of tributyrin was able to briefly achieve levels 500 mM with daily administration
A three times a day dosing was used. Levels of 500 mM were routinely obtainable.
There was no dose limiting toxicity.
Six of the 28 patients (21%) showed prolonged disease stabilization.
6. Tributyrate is well tolerated and levels associated with in vitro activity are easily achieved with tid (thrice a day) dosing.
Clinical and pharmacologic study of tributyrin: an oral butyrate prodrug (2003) (Abstract)
1. A three times daily dose schedule was used.
2. 20 patients with advanced solid tumors in the study.
3. They were treated with tributyrin at doses from 150 to 200 mg/kg three times daily.
4. There was no dose-limiting toxicity.
5. Escalation was halted at the 200 mg/kg three times daily level due to the number of capsules required.
6. A median butyrate concentration of 52 wM was obtained but there was considerable interpatient variability.
7. No objective responses were seen.
8. There were four patients with prolonged disease stabilization (note that is 20%, almost identical to their previous study) Two patients with non-small-cell lung cancer had survived for >1 year at the time of this report without evidence of progression.
9. Conclusion. Tributyrin is well tolerated and levels associated with in vitro activity are achieved with three times daily dosing.
Interesting quotes from the full text of 2003 study:
1. "Most importantly, the drug can be administered orally. Though a large number of capsules were required the agent was well tolerated with several patients remaining on therapy for months. Toxicity was minimal, making it an excellent candidate for combination therapy with other agents. Interestingly, many of the patients treated reported an improved sense of well-being, appetite and pain control. "
2. "Interestingly, these (plasma) levels are quite comparable and in fact higher than the levels achieved with intravenous administration of butyrate and several derivatives "
3." Although tributyrin rapidly hydrolyses in plasma the doses administered were sufficient to produce measurable concentrations up to 4 hours after administration."
In a clinical trial using a different prodrug of butyrate, the authors reported an interesting tumor response. "A partial response occurred in a 41-year-old female with metastatic squamous cell carcinoma of the lung and a history of laryngeal papillomatosis, who had not received any prior systemic therapy. The patient experienced a 54% reduction in the size of her pulmonary metastases after treatment with two courses of AN-9 at the 0.376 g/m2/day-dose level. Thereafter, the patient received escalating doses of AN-9 to a maximum of 1.5 g/m2/day and experienced a 90% reduction in the size of her measurable disease after eight courses. The
patient developed progressive disease after receiving 13 total courses over 14 months." and "Six other patients with various malignancies experienced stable disease lasting 4–10 months as their best response." Interestingly, the researchers behind the 2003 clinical trial reported two patients with non small cell lung cancer showed exceptional response. Also of interest is the 7 out of 28 patients showing response to butyrate, an almost identical response rate as shown in the tributyrin studies.
In the clinical trials, tributyrin showed no dose-limiting toxicity. (however, in one rodent study discussed above, toxicity was reported at 10 grams/kg. The highest dosage used in the clinical trials was 400 milligrams/kg, or 1/25 of the lethal dose).
Toxicity reported (from 1997 study): Grade 3 nausea and vomiting were observed in two patients at the initial dose level. Grade 3 myalgia was also seen in a patient at the 150 mg/kg dose. Other, grades 1 and 2 toxicities were observed without respect to dose and included anemia, constipation, anorexia, abopecia, azotemia, abdominal cramps, diarrhea or soft stool, headache, fatigue, nausea, flatulence,
lightheadedness, dizziness, rash, odor (at the highest dose), dysphoria, and clumsiness.
Toxicity reported from the 2001 study. "There was no dose limiting toxicity. 3 patients had Grade III nausea/vomiting (none at the highest dose levels), 1 patient had Grade III myalgias. Pk : At the tid dosing (three times a day) schedule (n=14) preliminary data indicate that levels 500 mM are routinely obtainable. Response: No objective responses were seen. There were six patients with prolonged disease stabilization ranging from 3-23 months, median progression free survival was 57 days. Levels of histone acetylation are pending.
Toxicity reported from the 2003 study "was minimal throughout the study and is summarized in Table 2. Only two patients experienced a grade 3 toxicity. One patient had nausea and vomiting which resolved with de-escalation to 150 mg/kg three times daily. A second patient who had had prior therapy with cisplatin, docetaxel and vinorelbine and had a history of diabetes mellitus experienced worsening neuropathy and hyperglycemia. A dose-limiting toxicity was not identified and the escalation was halted at 200 mg/kg three times daily as further increases were not felt to be practical due to the large number of capsules which would need to be administered (28 capsules [(soft gelatin capsules containing 500 mg of tributyrin without additives] three times daily for a typical 70-kg patient). Compliance was not formally monitored. However, patients were questioned and uniformly reported that they were able to swallow the capsules and tolerated them well."
7. In Summary:
Tributyrin is a food additive, with low toxicity and low cost. It and related butyrate prodrugs have been used successfully in numerous clinical trials. Research shows it to be a stand-alone anti-cancer agent that can synergize a number of other cancer-fighting agents.
When butyrate is used with DHA, the long chain fish oil, the rate of apoptosis leaps. Anti-oxidants counter many of the anti-cancer effects of DHA.
Tributyrin's very low toxicity and great ability to synergize many therapies makes it a very logical addition to almost any cancer treatment plan.
Tributyrin tastes bad. What Paul does to solve this issue is place a tablespoon of Tributyrin (TB) in a shot glass, add one to two tablespoons of red wine, then gulp it down. He says it works very well. Another tributyrin user says that adding TB to plum juice works great.
Questions? Email Jim