Amygdalin, quackery or cure?
Background: The cyanogenic diglucoside, amygdalin, has gained high popularity among cancer patients together with, or in place of, conventional therapy. Still, evidence based research on amygdalin is sparse and its benefit controversial.
Purpose: Since so many cancer patients consume amygdalin, and many clinicians administer it without clear knowledge of its mode of action, current knowledge has been summarized and the pros and cons of its use weighed.
Methods: A retrospective analysis was conducted for amygdalin relevant reports using the PubMed database with the main search term "Amygdalin" or "laetrile", at times combined with "cancer", "patient", "cyanide" or "toxic". We did not exclude any "unwanted" articles. Additionally, internet sources authorized by governmental or national institutions have also been included.
Sections: Individual chapters summarize pharmacokinetics, preclinical and clinical studies and toxicity. Conclusion: No convincing evidence showing that amygdalin induces rapid, distinct tumor regression in cancer patients, particularly in those with late-stage disease, is apparent. However, there is also no evidence that purified amygdalin, administered in "therapeutic" dosage, causes toxicity. Multiple aspects of amygdalin administration have not yet been adequately explored, making further investigation necessary to evaluate its actual therapeutic potential.
Complementary and alternative medicine
The use of complementary and alternative medicine (CAM) has steadily increased over the past decades. CAM includes nonconventional therapy such as homeopathy, vitamin therapy, phytomedicine and traditional Chinese medicine, acupuncture and yoga (Fisher et al. 2014). The ingestion of natural products is the most wide spread CAM practice. Up to 80% of cancer patients in the United States (Saghatchian et al. 2014), and more than 50% of cancer patients in Europe use CAM together with or in place of conventional therapy (Huebner et al. 2014a). Dissatisfaction with conventional treatment and reduction of chemotherapeutic side effects are the most commonly given reasons for using CAM (Gillett et al. 2012; Citrin et al. 2012). Patients also wish to actively contribute to their therapy, hoping to omit no chance of cure (Huebner et al. 2014b).
Information on CAM is mainly obtained from family, friends and increasingly from the internet. These sources are of unknown quality regarding evidence and reliability (Huebner et al. 2014a). Physicians are generally not trained to discuss CAM with their patients (Frenkel et al. 2010) and have little knowledge about CAM themselves (Parker et al. 2013), so that helpful communication between patients and physicians rarely occurs. Reports on the therapeutic efficacy of particular CAM associated compounds are sparse and well-designed, evidence based clinical studies are lacking.
The discrepancy between the use of a natural product and knowledge about a hypothesized anti-tumor property is notably apparent for amygdalin. Amygdalin is a cyanogenic diglucoside (D -mandelonitrile-[beta]- d -gentiobioside; syn: d -mandelonitrile-[beta]- D -glucosido-6-[beta]- D -glucoside) highly concentrated in fruit kernels from Rosaceae species such as Prunus persica (peach), Prunus armeniaca (apricot) and Prunus amygdalus var. amara (bitter almond). Amygdalin is naturally found in the dextrorotatory configuration (R-amygdalin), which is considered the active form. The (inactive) S-isomer does not occur naturally (Milazzo et al. 2007). Approximately 50g/kg (3-5%) amygdalin is found in bitter almond kernels (Lee et al. 2013), between 2.7 and 3.1% in Semen Persicae and between 3.6 and 5.2% in Semen Armeniacae (Tanaka et al. 2014). In contrast, the amygdalin-content of seeds from apples (Malus domestica) range from 1 to 4g/kg (Bolarinwa et al. 2015).
Proponents of amygdalin consider it a natural cancer cure, based on the unproven theory that amygdalin is specifically broken down to cytotoxic cyanide by the hydrolytic enzyme [beta]-glucosidase, which is supposed to be enriched in tumor cells. It has further been speculated that the mitochondrial enzyme, rhodanese, that detoxifies cyanide by conversion to thiocyanate is not as abundant in tumor cells as in normal cells, leading to selective tumor cell cyanide poisoning. Opponents of amygdalin, however, warn that amygdalin is ineffective and even toxic, since [beta]-glucosidase may not be enriched in tumor cells. Rather, cyanide might systemically accumulate, leading to severe cyanide poisoning.
Since so many cancer patients use amygdalin, and many clinicians administer it without clear knowledge of its mode of action, this overview aims to present current knowledge about amygdalin and discuss the pros and cons of its use.
History of amygdalin
Amygdalin was initially isolated from bitter almonds (Prunus dulcis) in the 1830 s by Robiquet and Boutron-Charlard (Wisniak and Robiquet 2013) and further investigated by Liebig and Wohler. Based on animal as well as self-experimentation by Widtmann and Denk (not clearly described in their publication), amygdalin was designated non-toxic. Liebig and Wohler concluded that pure amygdalin was vital for general use (Riecke 1840). As early as 1845, amygdalin was used as an anticancer-compound in Russia (Moss 1996). First reports on amygdalin application in the United States date from the 1920 s (Curt 1990). The oral formulation available at that time, however, was judged too toxic and, therefore, abandoned (National Cancer Institute 2015a).
In the 1950 s, a semi-synthetic, injectable form of amygdalin was developed and patented as Laetrile (LAEvorotatory mandeloniTRlLE) by Ernst T. Krebs (Dorr and Paxinos 1978). Although the term "laetrile" is frequently used as a synonym for amygdalin, laetrile is structurally different from the natural compound with the chemical composition d -mandelonitrile-[beta]-glucuronide. To avoid confusion, the term amygdalin will be used in the present article. However, the distinction will be made between amygdalin and laetrile, when necessary. Amygdalin became one of the most popular, non-conventional, anti-cancer treatments in the 1970 s and by 1978, 70,000 US cancer patients had used it (Moss 2005). Evaluation of amygdalin produced by a Mexican company revealed that both the oral and injectable forms of amygdalin did not comply with US pharmaceutical product standards, and several ampules were found to be contaminated with bacteria (Davignon et al. 1978). Amygdalin was then banned from transport into the US or across state lines. Nevertheless, use of this substance for terminally ill cancer patients remained legal in 23 states in the USA (Curran 1980).
During this controversial time, the National Cancer Institute (NCI) decided to evaluate the efficacy of amygdalin treatment. A clinical trial, sponsored by the NCI with approval of the US Food and Drug Administration (FDA), failed to demonstrate anticancer activity (Moertel et al. 1982). Since then, amygdalin has been banned by the FDA and not authorized for sale as a medicinal product in the USA or Europe, with some exceptions (Milazzo et al. 2007). In the UK, amygdalin is considered "prescription medicine only" and can only be prescribed under medical supervision (Milazzo et al. 2007). The German Federal Institute for Drugs and Medical Devices (BfArM) has classified amygdalin as a questionable drug (BfArM 2015a). Despite widespread federal limitation, the compound continues to be manufactured and administered as an anticancer drug worldwide. Many websites promote and market amygdalin and many physicians administer it. At least 35 clinics or medical practices in Germany offer an amygdalin based tumor therapy, as shown by an incomplete list compiled by the BfArM (BfArM 2015b). Information is not available about how many persons presently use amygdalin.
In 1986 Strugala et al. identified two different metabolic pathways for orally administered amygdalin. The first pathway was described as "first pass" metabolism of amygdalin to prunasin (D-mandelonitrile [beta]- D -glucoside) by cleavage of the terminal glucose residue via enzymatic [beta](1-6)-glucosidase activity in the proximal small intestine. Due to limited analytical methods, earlier studies did not discriminate between amygdalin and prunasin, and tracing amygdalin's metabolic route was not possible. The second pathway was the [beta]-glucosidase driven total hydrolysis of amygdalin to glucose, benzaldehyde and cyanide by microflora in the colon (Strugala et al. 1986) (Fig. 1).
Freese et al. proposed that mammalian [beta]-glucosidase, responsible for the "first-pass" effect, might be different from bacterial [beta]-glucosidase responsible for final hydrolyzation. The reasoning was that human [beta]-glucosidase is primarily localized in the neutral pH cytosol of mammalian tissue, whereas bacterial [beta]-glucosidase is localized in acidic lysosomes (Freese et al. 1980). Corroborating this, reduction of gut flora activity following antibiotic treatment was shown not to influence the hydrolytic cleavage of amygdalin into prunasin, but suppressed total hydrolysis of amygdalin into benzaldehyde and cyanide (Strugala et al. 1986). Still, a preferred metabolic pathway cannot be established since amygdalin degrades to prunasin in both acidic and neutral environments (Strugala et al. 1986). Freese, himself, failed to demonstrate hydrolysis of gentiobiose, the integral part of amygdalin, in mammalian tissue. Several normal and neoplastic tissues have also shown no [beta]-glucosidase activity, which precludes the existence of intracellular [beta]-glucosidase (Newmark et al. 1981) and favors [beta](1-6) glucosidase localization in humans in the gut wall (Strugala et al. 1986). Other digestive enzymes in the upper gastrointestinal tract that may act like [beta](1-6) glucosidase have been identified, hydrolyzing amygdalin into glucose and prunasin, as well (Shim and Kwon 2010).
Disregarding enzymatic digestion, experiments in a rat model have shown that prunasin is actively transported in the small intestine by a glucose carrier system, without formation of benzaldehyde or cyanide during passage from mucosa to serosa (Strugala et al. 1995). The carrier system has been identified as the epithelial sodium-dependent monosaccharide transporter SGLT1 (Wagner and Galey 2003). Absorbed prunasin is finally cleared by the kidney without formation of benzaldehyde or cyanide (Rauws et al. 1982). However, isomers of prunasin have been detected by a highly sensitive liquid chromatography-tandem mass spectrometrie method (Li et al. 2014), showing that configurational modifications might occur following absorption. Based on a gastrointestinal digestion model combined with a human intestinal cell culture, prunasin has been shown to be degraded into mandelonitrile by [beta]-glucosidase in the small intestine. Prunasin is then taken up as hydroxymandelonitrile into the cells and not further metabolized through mucosal passage (Shim and Kwon 2010). Further investigation is required to prove whether this process takes place in humans or whether it is restricted to an in vitro response.
The oral bioavailability of prunasin is 50% and that of amygdalin 1% (evaluated in a hamster model (Frakes et al. 1986)). Apparently, prunasin, a monosaccharide, is specifically transferred, whereas amygdalin, a disaccharide, requires prior hydrolysis by [beta]-glucosidase at the mucosal brush border (Strugala et al. 1986). There is no doubt that [beta]-glucosidase from gut bacteria plays a dominant role in amygdalin metabolism since reduction of gut flora activity following antibiotic treatment drastically decreases hydrolysis of amygdalin into benzaldehyde and cyanide (Strugala et al. 1986). The hydrolytic role of bacteria has been confirmed, since intravenously administered amygdalin does not result in cyanide or benzaldehyde production (Newton et al. 1981). Bacteroides fragilis has been identified as the most active bacterial species in human feces breaking down amygdalin. Administering amygdalin together with food rich in [beta]-glucosidase may also contribute to benzaldehyde and cyanide synthesis (Newton et al. 1981).
Prunasin, remaining in the gastrointestinal tract, can further be degraded into mandelonitrile by [beta]-glucosidase. Mandelonitrile is unstable and finally dissociates into cyanide and benzaldehyde. Cyanide, which reaches maximum blood levels 1.5-2 h after amygdalin administration (Hill et al. 1980), is rapidly converted by sulfuration into thiocyanate (SCN-), with an elimination rate of about 17 [micro]g/kg body weight per minute. Thiocyanate is excreted mainly in the urine (Nagahara et al. 1999), while a small amount is exhaled as formate (HCOO-) and C[O.sub.2] (Singh et al. 2013).
Sulfuration is catalyzed by rhodanese (syn.: thiosulfate sulfur transferase) and [lambda]-mercaptopyruvate sulfur transferase (Zagrobelny et al. 2004). Cystathionine [lambda]-lyase (syn.: [lambda]-cystathionase) activity is elevated during cyanide intoxication, indicating that this enzyme may also be involved in cyanide detoxification (Singh et al. 2013). The exact role of [lambda]-cystathionase requires further investigation. Amounts of cyanide degrading enzymes vary, depending on the tissue, but highest concentrations are found in liver and kidney (Sylvester and Sander 1990). Subcellular enzyme distribution is not homogeneous. 3-mercaptopyruvate sulfur transferase occurs in cytoplasm and mitochondria (cytoplasm > mitochondria), while rhodanese is located only in mitochondria (Nagahara et al. 1999). The enzymatic composition in mitochondria is critical, since cyanide poisoning occurs via inhibition of the mitochondrial respiratory chain. Different enzymatic distribution could make some organs more sensitive to cyanide than others.
Earliest studies date from the late 1970 s, when the FDA pronounced amygdalin ineffective and toxic, even though it was one of the most popular non-conventional treatment options used by cancer patients. Ardenne and Reitnauer observed a cancerostatic effect and tumor regression in DS carcinosarcoma-bearing rats after simultaneous glucose, amygdalin and [beta]-glucosidase infusion. Detailed information about the underlying mode of action was not provided. However, the authors reported a pH reduction and a "somewhat toxic" process, which might indicate [beta]-glucosidase driven amygdalin metabolization and cyanide synthesis (Ardenne and Reitnauer 1975). The described anti-tumor effects were not confirmed by others. Tumor shrinkage was not observed when melanoma or lymphatic leukemia cells were implanted intraperitoneally (i.p.) into mice, followed by a single dose or daily i.p. amygdalin treatment (50-5000 mg/kg/day) over 4 days (Hill et al. 1976). In two further mouse models with transplantable tumors, amygdalin did not induce tumor regression for Ridgway osteogenic sarcoma, Walker 256 carcinosarcoma, melanoma, Lewis lung carcinoma or leukemia cells. However, a slight increase in life span was observed in some animal groups treated with amygdalin, compared to controls (Laster and Schabel 1975; Wodinsky and Swiniarski 1975). Extending the animal studies by transplanting mammary and liver tumor cells i.p. followed by amygdalin i.p. treatment daily over 6 days (500 and 1000 mg/kg/day) also did not lead to growth inhibition (Stock et al. 1978a).
In early investigations (cited above) therapeutic protocols did not reflect the clinical situation, since patients consume amygdalin over long time periods. Stock et al. (1978b), therefore, modified the experimental design to investigate long-term amygdalin effects using a mouse model with spontaneous mammary carcinoma. The resulting publication will be dealt with in detail, since it imparts the controversy connected to amygdalin. In a first trial, amygdalin was injected i.p. in [CD.sub.8][F.sub.1] mice 6 times a week until death or sacrifice of the animals. Amygdalin applied at 1000 and 2000mg/kg did not destroy the primary mammary tumors. However, differences were noted with respect to the final tumor volume, which became particularly evident in the 1000mg/kg group (11.78[cm.sup.3]; control versus 5.46[cm.sup.3], i.e. 54% reduction). The 2000mg/kg group also revealed a diminished final tumor volume, however to a lesser extent than in the 1000mg/kg group (reduction: 33%, compared to untreated controls). Notably, tumor growth was temporarily stopped for 7-21 days during amygdalin treatment, beginning in the first week of treatment. In addition, metastatic tumor dissemination into the lung was observed in nearly 90% of the controls but in only 22% of amygdalin treated animals. Treated animals "appeared to be in better health" than untreated animals. An anti-tumor effect of amygdalin was presumed.
For an unexplained reason, the experiments were repeated in an identical fashion with the 2000, but not the more effective 1000mg/kg/day schedule. Final tumor volume and metastases were again recorded. The reason given for repetition was that "the experiments (of the first trial) were given unauthorized circulation before it was possible to obtain independent confirmation" and that the authors Stock and Martin questioned the results from the initial trial, carried out under the auspices of the co-author Sugiura. Why the 1000mg/kg schedule was excluded here, although it was superior to the 2000 mg/kg application protocol, is unclear. In this second trial, there was no difference between the tumor volume in treated and untreated animals. Data pertinent to metastatic formation were discrepant in the two studies since amygdalin diminished the number of lung metastases in the first study (54% controls versus 30% amygdalin) but not in the second study (67% versus 65%). Two further experimental approaches led to conflicting results, lung metastases being reduced under amygdalin treatment in one study but elevated in the other.
In a third trial, the authors investigated whether amygdalin prevents tumor development using the same mouse strain as before, but before tumors had developed. Application of 1000mg/kg/day amygdalin (2000mg/kg amygdalin was not evaluated) reduced tumor development from 82 (controls) to 72% and lung metastases from 81 to 17%. Histologic examination revealed "many mitotic figures" in the control tumors, whereas "degenerated" tumor cells with "fewer mitotic figures" were observed after amygdalin application.
Results from the second and third trial were not commented upon in detail. Instead, further studies were undertaken on Swiss albino mice with spontaneous mammary adenocarcinomas. According to the early CD8F] mouse experiments, amygdalin (10003000 mg/kg/day) did not destroy the tumor but induced a temporary growth stop. In addition, 91% of the control animals showed lung metastases compared to 22% in amygdalin-treated animals. General health was reported to be better in the treated than in the non-treated group. Transferring the 2000 mg/kg/day protocol to female AKR mice with advanced spontaneous leukemia, however, failed to demonstrate an effect of amygdalin on tumor development and progression, although there was a benefit 1-2 days after receiving amygdalin. Further experiments were then initiated to investigate lung metastization under amygdalin (1000 and 2000 mg/kg/day) treated versus control [CD.sub.8][F.sub.1] mice. Amygdalin was provided from Sidus, Germany, instead of from the previous Mexican supplier. Amygdalin injection i.p. and lung tissue sampling were carried out in two different institutions and the number of lung metastases was similar. A "blind" test with [CD.sub.8][F.sub.1] mice receiving 2000 mg/kg/day amygdalin also revealed no influence on tumor metastasis, although tumor growth inhibition was noted in the initial week of treatment. A second "blind" test was then initiated because "the prior blind experiment suffered a loss of assurance of blindness". In this experiment, no differences were found with respect to lung metastases, nor was a temporary tumor stop observed.
The principal investigator, Stock, finally concluded that "amygdalin possesses neither preventive nor tumor-regressant, nor antimetastatic nor curative anticancer activity". Not all members of the investigative group agreed with this since some of the data promised anticancer activity stemming from amygdalin application. A note was attached to the publication: "Sugiura continues to believe that amygdalin is a palliative agent".
Another in vivo investigation, initiated in the same year, demonstrated inactivity of DL-amygdalin against human breast and colon tumor xenografts in nude mice (Ovejera et al. 1978). The original article is not available. Therefore, details on the experimental strategy and results cannot be provided. It is not clear why the authors used DL-amygdalin instead of the natural dextrorotatory configuration, which is considered to be the active compound (Milazzo et al. 2007).
One of the first positive assessments of amygdalin from 1978 show that cyanide released by enzymatic hydrolysis of amygdalin enhanced .the radiation response of V79 lung fibroblasts. Although direct anti-tumor effects of amygdalin were not assessed, the authors ascribed anti-tumor potential to amygdalin, since cyanide reached the tumor cells (Biaglow and Durant 1978). This evaluation was adopted by others, separately assessing the effects of amygdalin (1.76 g/kg, i.p.) and its metabolites, cyanide (injected as sodium cyanate) and thiocyanate on normal and neoplastic tissue in a rat model (Lea and Koch 1979). Both cyanate and thiocyanate exerted inhibitory effects on transplanted hepatomas and a colon tumor, not observed in the host tissue (liver, colon).
Cyanate uptake was similar in the colon tumor and the host colon mucosa, so that the different sensitivities to cyanate could not be attributed to different entry paths in normal and neoplastic tissue. Another investigation showed that amino acid incorporation into protein was selectively inhibited by cyanate/thiocyanate in tumors, when compared to that in surrounding normal tissue (Allfrey et al. 1977). This difference between normal and neoplastic tissue, however, was not further investigated.
In contrast to cyanate/thiocyanate, the parent compound, amygdalin, has been shown to block [[sup.3]H]thymidine incorporation into normal and tumor DNA to a similar extent (Lea et al. 1979), indicating proliferation blocking activity of amygdalin in both normal and tumor cells. Amygdalin itself, therefore, evokes a response, possibly not dependent on [beta]-glucosidase metabolization of amygdalin to cyanate/thiocyanate. The question of whether tumorinhibiting effects of amygdalin are due to the parent drug or to its metabolites or in different tumors to differing components remains open.
In vitro effects of amygdalin on leukemia cells were subsequently reported, evidenced by a 50% inhibition of colony formation by KG-1 and HL-60 cell lines at an amygdalin concentration of 3.5 mg/ml (Koeffler et al. 1980). Similar to the investigation of Biaglow and Durand (1978), [beta]-glucosidase was added to promote amygdalin hydrolysis. Data from amygdalin alone were not provided, making it difficult to assess whether the growth blocking effects were caused by cyanide or amygdalin. A further in vitro trial using HL-60 cells demonstrated antiproliferative and apoptotic effects of amygdalin (applied in the presence of [beta]-glucosidase) with an [IC.sub.50] of 6.4 mg/ml (Kwon et al. 2003), whereas amygdalin therapy (200-2000 mg/kg, i.p. injection on day 1, 5 and 9), directed toward experimental murine tumor models P388 lymphocytic leukemia and P815 mast-cell leukemia, failed to increase life span (Chitnis et al. 1985).
In vitro systems allow immediate delivery of amygdalin and its metabolites to the target cell, in contrast to in vivo models where immediate delivery to the target cell is unlikely. Therefore, an antibody-directed enzyme prodrug therapy was developed to activate amygdalin specifically at the targeted tumor site. Amygdalin was cytotoxic to bladder cancer cells at high concentrations (without [beta]-glucosidase) but enormously increased its anti-tumor potential when tumor cells were tagged with a tumor-associated monoclonal antibody conjugated with [beta]-glucosidase (Syrigos et al. 1998). Very recently, Li and co-workers state "that amygdalin and [beta]-glucosidase combined with an antibody-enzyme-prodrug system may be a future targeted antitumor therapy with considerable promise" (Li et al. 2015). Still, this strategy has not been employed in an animal model, precluding a final conclusion as to therapeutic use.
Although cyanide had been hypothesized as responsible for the anti-tumor activity of amygdalin, investigations had also been carried out indicating that amygdalin itself and not its metabolite cyanide was responsible for anti-tumor activity. Amygdalin and prunasin, isolated from Prunus persica seeds, both inhibited Epstein-Barr virus early antigen activation in vitro and delayed carcinogenesis on mouse skin in vivo (Fukuda et al. 2003). The same inhibition was found when the extracted glycosides amygdalic acid, mandelic acid [beta]-D-glucopyranoside, benzyl [beta]-gentiobioside or benzyl [beta]-D-glucopyranoside were applied. Based on structureactivity relationships, the authors proposed that the CN-substituent was of minor importance to tumor inhibition and pointed to the gentiobioside structure as the relevant inhibitory factor. Amygdalin's action was apparent without [beta]-glucosidase addition, making a cyanide-independent mechanism likely.
Other investigations support a cyanide-independent amygdalin mechanism. A significantly reduced cell number and downregulation of cell cycle associated genes was achieved with 5 mg/mi amygdalin, administered without [beta]-glucosidase, to SNU-C4 colon cancer cells, (Park et al. 2005). Similarly, amygdalin (0.1-10mg/ml) exhibited dose-dependent cytotoxicity to DU-145 and LNCaP prostate cancer cells and induced apoptosis, evidenced by DNA strand breaks, caspase-3 activation, down-regulation of antiapoptotic Bcl-2 and up-regulation of the pro-apoptotic Bax protein (Chang et ai. 2006). The same mechanism is seen in human cervical cancer HeLa cells, whereby in vitro results were additionally confirmed by an in vivo xenograft model (Chen et al. 2013). Amygdalin (0.05-0.4 mg/ml) also suppresses primary rat kidney fibroblast proliferation and attenuates the process of renal interstitial fibrosis in rats (3 and 5 mg/kg/day) (Guo et al. 2013).
Two novel publications have meanwhile become available dealing with the influence of amygdalin on bladder cancer cell growth and invasion. Significantly reduced growth, proliferation and cell cycle progression of UMUC-3, RT112 and TCCSUP cell lines was found after administering 10 mg/ml amygdalin. Molecular analysis revealed evident influence on the growth regulating proteins akt and rictor and on the cdk2-cyclin A axis (Makarevic et al. 2014a). Adhesion and migratory behavior was also modulated by amygdalin, primarily by altering the expression profile of integrin a and [beta] subtype receptors (Makarevic et al. 2014b). The bladder cancer cell model has now been transferred to a lung cancer cell system. According to the bladder cancer study, amygdalin stopped lung cancer cell growth with an [IC.sub.50] of 12.2 mg/ml and reduced tumor cell metastasis by acting on integrin adhesion receptors as well as on akt and rictor signaling pathways (Qian et al. 2015).
All investigations, except the earliest, employed cell cultures treated with amygdalin alone, without [beta]-glucosidase. None of these in vitro studies included comparative tests with both amygdalin and [beta]-glucosidase. Hence, how relevant this enzyme is for amygdalin's anti-tumor properties can only be speculated upon. Either amygdalin exerts its effects on cell cultures without [beta]-glucosidase, traces of [beta]-glucosidase are present in the cell culture medium (unlikely, since tumor cells are cultivated in heat inactivated fetal bovine serum) or [beta]-glucosidase is present inside the tumor cells, governing amygdalin metabolism and subsequent cyanide appearance. Although [beta]-glucosidase was not found in cancer cells in earlier investigations (Newmark et al. 1981), figlucosidase has meanwhile been detected in several tumor cell lines (Arafa 2009; Oliveri et al. 2013), making this aspect worthy of further evaluation.
All available cell culture investigations demonstrate amygdalin's anti-tumor properties. Regardless of the cell line, characteristic molecular alterations take place. In early studies (e.g. Kwon et al. 2003) amygdalin was added to tumor cells together with [beta]-glucosidase. Newer investigations report similar amygdalin induced anti-tumor efficacy without adding [beta]-glucosidase (e.g. Makarevic et al. 2014a], Since amygdalin's anti-tumor effects are apparent with or without [beta]-glucosidase, different modes of action are possible. Amygdalin may rapidly be absorbed by tumor cells and then degraded by intracellular [beta]-glucosidase. Or, extracellularly added [beta]-glucosidase might degrade amygdalin into prunasin, which then enters the cell via a glucose carrier (Strugala et al. 1995). However, neither an amygdalin nor a prunasin carrier system has been identified. Should such carrier systems exist, amygdalin combined with extracellular [beta]-glucosidase might favor the prunasin route, whereas amygdalin without extracellular [beta]-glucosidase might favor an amygdalin-sensitive carrier. In vitro studies must be planned to evaluate the amygdalin influence with and without [beta]-glucosidase on a panel of tumor and normal cells. Indeed, it is also not clear, whether amygdalin acts differently in cancer cells than in normal cells, as has been hypothesized by amygdalin proponents. Irrespective of how amygdalin crosses the cell membrane, the fates of amygdalin and prunasin inside the cell remain unexplored. Cyanide and/or other metabolites, even amygdalin itself, might modulate intracellular pathways. First and foremost, the chemical component responsible for the anti-tumor effects must be identified.
In contrast to in vitro investigation, amygdalin effects in animal studies are inhomogeneous. Different models, different amygdalin sources and different treatment strategies may account for the diverse in vivo outcome. Statistical analysis is often missing, particularly in older investigations, making interpretation difficult. Nevertheless, one aspect parallels in vitro accounts in as much as early publications differ from the new. Studies published in the late 1970 s concluded that no therapeutic benefit of amygdalin was apparent, whereas reports later than 2000 propagate amygdalin as a cancer cure. The cause of this switch is unclear, but it has contributed to deepening the gap between proponents and opponents of amygdalin as a useful cancer treatment. To judge amygdalin's value in vivo a larger cohort of animals is required to attain enough data to permit a knowledgeable estimate about its therapeutic potential.
Further important issues must be addressed. A recent publication demonstrated metastasis-inhibitory properties of amygdalin in vitro (Makarevic et al. 2014b). No analogous in vivo trials have been conducted, except for the controversial report from Stock et al. (1978). Temporary growth blockage by amygdalin has also been observed in a few cases (Stock et al. 1978). This finding has neither been commented upon nor subsequently re-evaluated. Due to lacking statistical evaluation, this response could be specific, could be an artifact or resistance might develop during chronic amygdalin application. Future work, therefore, should concentrate on time-dependent alterations in tumor growth during amygdalin therapy.
Clinical trials with amygdalin are sparse, conveying ambiguous results. A study from John Morrone including 10 cases of inoperable cancer with metastases indicated clinical benefit from laetrile (specified as 1-mandelonitrile-beta-glucuronide), using dosages of 1 g i.v., for 4-43 weeks (mean 17.5 weeks, mean 2 injections/week). Pain relief in 10 and possible regression of malignant lesions was reported for 8 patients (Morrone 1962). The analgesic effect was speculated to be produced by benzoic acid, possibly by oxidation of the metabolite, benzaldehyde. Manuel Navarro clinically administered laetrile, not further specified, in the Philippines and described prolonged survival in tumor patients taking this drug at a concentration of 0.1-0.5 g (i.v. and oral) (Navaro 1959; Navarro 1964). Contreras, running a clinic in Mexico, analyzed 500 patients administered laetrile, (not further specified; 1-10 g i.v. daily, followed by 1 g daily oral administration). A "significant" disease arrest or even regression became evident in 15% of very advanced cases, and palliative action "improving the comfort and well-being" of the patient, was seen in about 60% (American Cancer Society 1971). Hans Nieper has presented results from 30 cancer patients where amygdalin (0.1 -1 g oral daily) led to "subjective improvement" in 21 patients, with tumor regression in 5 of them (Weber 1975). During the 9th International Cancer Congress, Tokyo, Japan, 1966, Rossi et ai. (1966) reported on a 12 year study of 150 terminally ill cancer patients who had taken amygdalin (0.11 g/day; i.v. or i.m. application). Tumor regression or growth stop was reported in 21%.
Common to all these investigations is that detailed methodological and technical information were not provided. Source and chemical formula of amygdalin were often withheld and, when "laetrile" was used, it is unclear whether the semi-synthetic, patented form of amygdalin or natural amygdalin was employed. The same ambivalence applies to information about treatment duration, about the period of tumor regression and about the inclusion of conventional treatment strategies. Therefore, these reports present anecdotal, rather than scientifically sound information.
A sound patient study was published in 1975 in Gottingen, Germany (Weber 1975). Twenty-five cancer patients, 10 with lymphoproliferative disease and 8 with mamma carcinoma, received amygdalin (provided from Jossa Arznei, Steinau, Germany) at a daily oral dosage of 0.5-2 g (mean 1.2 g/day) for 2-43 weeks (mean 17.2 weeks) together with a sulfur donor. One patient experienced transient improvement over 9 weeks, evidenced by a reduced tumor-derived esophageal stenosis, and two patients demonstrated improved well-being. No benefit was seen in the remaining 22 patients. The scanty benefit of amygdalin was disappointing, since earlier studies had suggested strong anti-tumor potential. Further trials to appraise amygdalin were recommended.
Previous to 1978, the National Cancer Institute (NCI) requested American physicians and other health professionals, as well as prolaetrile groups, to report the experience of patients taking laetrile to treat their disease. The response rate was very low, and only 68 cases were subjected to evaluation by a panel of oncologists (possibly because the majority of American patients went to Tijuana cancer clinics and were treated by Mexican physicians (Moss 2005)). Of these, six laetrile courses were judged to have produced a response (two complete and four partial) (Ellison et al. 1978). The term laetrile was not further specified in the publication and, therefore, could be related to natural amygdalin, to the semisynthetic compound or to both. In addition, specific information about the treatment regimen (i.v. or oral administration, drug concentration, treatment duration) and tumor history were not provided, making it difficult to assess the value of this retrospective analysis.
Recognizing insufficient documentation, the NCI agreed to sponsor phase I/II clinical trials, which might allow "definite conclusions supporting the anti-cancer activity of laetrile" (Ellison et al. 1978) to be drawn. In a first trial, reported upon in 1981, dosing, method and schedule of amygdalin administration were examined for six cancer patients. Although oral application of amygdalin (3x0.5 g/day) led to increased blood cyanide levels, an effect not seen under i.v. administration (4.5 g/[m.sup.2]/day), no toxic side effects were recorded (Moertel et al. 1981; Ames et al. 1981). A prospective, open-label evaluation of amygdalin plus "metabolic therapy" (vitamins and pancreatic enzymes, plus dietary changes) was then conducted on tumor patients, most having breast, colon or lung cancer. The study cohort was not homogenous since two-thirds of the patients had received conventional therapy and one-third had received no prior treatment. Natural amygdalin was given i.v. for 3 weeks followed by oral maintenance therapy for 7 weeks, according to a dosing schedule defined by a pilot study (Moertel et al. 1981; Ames et al. 1981). A subset of patients received higher amygdalin doses. An RS-epimer racemic mixture was used for i.v. therapy, whereas the R-epimer was used for oral treatment (Moertel et al. 1982). Tumor response and survival related to amygdalin treatment were documented for 175 patients in a study supported by grants from the NCI whose lead investigator was C.G. Moertel.
Results of the study were presented to the public in different forms, making it difficult to assess amygdalin's value. Moertel et al. summarized: "no substantive benefit was observed in terms of cure, improvement or stabilization of cancer, improvement of symptoms related to cancer, or extension of life span" (Moertel et al. 1982). In contrast, the NCI reported cancer stabilization in one patient, 7% temporary improvement in performance status and 20% symptomatic relief (temporary in "most patients"). 54% of patients experienced disease progression at the end of i.v. therapy, and all patients showed disease progression 7 months after completing i.v. therapy (National Cancer Institute 2015b). This information is partially different from another NCI report, as disease progression after 7 months was related to the end of both i.v. and oral amygdalin therapy, and symptomatic relief was now designated temporary in all patients (National Cancer Institute 2015c). The American Cancer Society reported on the same study that cancer had progressed in 91% of patients after 3 months, indicating that in 9% progression had not taken place. All patients experienced disease progression 5.5 months after finishing treatment (National Cancer Institute 2015d). Based on this study, the NCI concluded that amygdalin is ineffective against cancer, and the necessity for further investigation was nonexistent. No further clinical trials were carried out, and today's assessment of amygdalin's value as a treatment for cancer is based on the Moertel study from 1982.
After the NCI's announcement of amygdalin's ineffectiveness, the Moertel study design was criticized by amygdalin proponents. They found fault with inadequate statistics, control groups had not been included, follow up was not done and inactive amygdalin had been applied (Correspondence 1982). Criticism may have been exaggerated and trial repetition, this time controlled, might have been unnecessary in view of amygdalin's ineffectiveness. Still, some of the criticism remains justified. Why a racemic mixture was used for i.v. therapy, although the dextrorotatory configuration is considered the active form, is not clear. Information about the quality and purity of the R-amygdalin used for oral application was not provided, which alone severely limits the quality of the Moertel study. The administered amygdalin "corresponded with the products distributed by the major Mexican supplier" (Moertel et al. 1982), which had been found to be chemically sub-potent and of poor quality by JP Davignon (1978), the senior scientist of the clinical trial. However, pointing out that poor trial standards may have led to the conclusion that amygdalin was ineffective does not automatically prove that amygdalin is an effective antitumor drug.
On the other hand, poor trial standards do not automatically imply that amygdalin is ineffective either. Two Cochrane reviews, carried out 2011 and 2015, point to the total absence of randomized controlled trials (RCTs) including cluster and cross-over trials and quasi-RCTs (Milazzo and Horneber 2015; Milazzo et al. 2011). At first glance, the lack of such studies may not permit a judgment indicating that amygdalin is generally ineffective. In fact, the lack of studies may not permit any judgement about amygdalin effectivity at all. Another possibility to judge amygdalin effectivity is to use the only available study with patients from 1982, which is flawed since it does not meet RCT criteria. Based on this premise, Milazzo et al. note in 2015 that "nothing must be added to the conclusion" drawn in 1982 that the risk-benefit balance of amygdalin as a cancer treatment is overridden by risk.
Consequently, good quality trials, which to date have not been initiated, are essential to determining the effectiveness of amygdalin. Adding to the confusion about the effectivity of amygdalin, the metabolite benzaldehyde was administered i.v. as benzylideneglucose. It induced sensational response rates in tumor patients, 50% partial and 10% complete remission (Kochi et al. 1985; Tatsumura et al. 1990). These results could not be confirmed by others and the metabolite was assumed to be ineffective (Tanum et al. 1990).
The confusion is not least reflected by a systematic review on all types of clinical data, published in 2007. Although it was concluded that beneficial effects of amygdalin had not been proven, several studies were listed in the same article documenting partial or even complete remission after amygdalin administration (Milazzo et al. 2007).
Since the amygdalin trials, based on low standard study protocols, were undertaken more than 35 years ago, a well-designed controlled clinical trial would be worthwhile. Knowledge about the pathophysiology of cancer has increased, trial standards have significantly improved and the spectrum of analytical methods has broadened over the last decades. Particular attention should be paid to temporary amygdalin response, since time to progression has not yet been investigated. A critical evaluation of CAM research has come to the conclusion that, despite general documentary deficiencies, amygdalin is among those CAMs warranting follow-up trials (Nahin 2002). The suggestion is justified. The use of amygdalin in the 21 st century has not been paralleled by high-standard science. In view of the public interest for amygdalin, the controversy surrounding amygdalin should be resolved (Reiman 1982).
A serious drawback connected to oral, but not parenteral, use of amygdalin is the risk of cyanide poisoning, manifesting as headache, dizziness and confusion and, if severe, as paralysis, coma and death. It must be emphasized that a toxic HCN level related to purified amygdalin has not yet been defined. The Committee on Toxicity of Chemicals in Food has estimated 10 apricot kernels/day to result in a hazardous HCN level in adults with toxic signs becoming overt (Committee on Toxicity 2006). Chaouali et al. (2013) has proposed that ingestion of 50 bitter almonds eaten at once is lethal to adults, and 5-10 almonds is fatal for young children (based on a mean HCN level in bitter almonds of 1062 [+ or -] 149 mg/kg). Established blood cyanide levels correlating with toxicity or lethality after amygdalin consumption are also not available. Speijers sets a value of 500 [micro]g HCN/dl blood as being lethal (Speijers 2015). Sauer et al. (2015) claims 20 [micro]g HCN/dl serum as toxic and 300 [micro]g HCN/dl serum as lethal. Though these values are not directly related to amygdalin, but to the potentially toxic metabolite of amygdalin, they may provide rough guidelines in judging HCN toxicity in reported cases of amygdalin overdosing.
Several cases of toxicity after oral amygdalin consumption have been documented, among them children under 5 years of age. In one case, an 11 -month old girl accidentally swallowed 1-5 500 mg amygdalin tablets, became lethargic, fell into a coma and died 71 h post ingestion (Humbert et al. 1977). A 2 year-old patient, who was administered amygdalin 500 mg orally and an additional 3.5 g as an enema on a daily basis, showed signs of cyanide poisoning (vomiting, diarrhea, lethargy, tachypnea and cyanosis), not immediately but following the second rectal dose (Ortega and Creek 1978). Similar symptoms became obvious in a 4-year-old child ingesting 12x500 mg amygdalin tablets all at once with a whole blood cyanide level of 163 [micro]g/dl (Hall et al. 1986). Sauer et al. (2015) recently reported acute cyanide poisoning in a 4-year-old boy being administered "unquantified amounts" of amygdalin i.v. in addition to oral amygdalin (4 x 500 mg/day) plus apricot kernels (5-10/day). His serum cyanide level was 52 [micro]g/dl.
Although the use of CAM among pediatric cancer patients is popular worldwide, no data is available on the number of children receiving amygdalin. Presumably, more cases of amygdalin intoxication have occurred, but have not been reported in the last years. The daily oral dose of amygdalin set for adults lies between 500 and 2500 mg (Newton et al. 1981). Based on this dosage and that amygdalin derived HCN may not be efficiently detoxified and excreted by children (Miller et al. 1981), considerable overdosing may be responsible for the negative outcome in the four cited pediatric cases.
Another aspect concerning cyanide poisoning must also be considered. The microflora in human feces exhibit strong glycosidesplitting activity (Newmark et al. 1981). Nearly 50% of amygdalin can be hydrolyzed in the intestine, leading to rapid HCN release (Newton et al. 1981). This is important, since cyanide poisoning has been linked to the rectal administration of an amygdalin enema rather than to the oral mode of application (Ortega and Creek 1978; Morse et al. 1979).
Ingestion of apricot kernels or bitter almonds, either alone or in addition to pure amygdalin is thought (Sauer et al. 2015; Akil et al. 2013) to be the main risk for cyanide intoxication. Apricot kernels contain large amounts of cyanide and when crushed or chewed release their cyanide content. Along with the plant derived [beta]-glucosidase, which is released as well, toxic accumulation of HCN may occur (Newton et al. 1981). In cases where amygdalin has been administered together with apricot kernels and toxicity has occurred, it is not possible to determine whether the kernel derived [beta]-glucosidase accelerated the metabolization of the amygdalin tablets or whether it was the amygdalin summation from tablets and kernels causing toxicity. Regardless of the reason, as long as the usefulness of amygdalin has not clearly been proven and dosing for children has not been established, it seems unjustified to subject children to amygdalin therapy.
Amygdalin overdosing also occurs in adults. Shortly after swallowing about 12 g amygdalin, a patient developed dizziness, tetanic contractures of the hands, generalized convulsions, became comatose and died a day later (Sadoff et al. 1978). A dose of 9 g was ingested with suicidal intent by a woman, who later recovered. She developed severe metabolic acidosis and hypoxemia with a serum cyanide level of 143 [micro]mol/l (385 [micro]g/dl) (Moss et al. 1981). Acute cyanide poisoning became evident in a patient taking 12-18 amygdalin tablets (6-9 g) at once (Beamer et al. 1983).
However, a high dose and high cyanide levels in blood are not always toxic. A case has been reported of a patient taking twice his usual oral dose, having 600 [micro]g/dl cyanide in blood and showing no signs of toxicity (Maxwell 1978). These highly variable cases show that a toxic reaction to orally administered amygdalin may vary greatly from individual to individual.
Product differences complicate the problem of setting guidelines for a safe dose, as differentiated from an overdose. A case has been described in which the patient was taking the drug "in the manner and dosage prescribed" (1 g orally twice a day+3 g i.v. three times a week) (Barnett et al. 1981). The cyanide level in serum was 5 [micro]g/dl and in blood 11 [micro]g/dl, which is considerably less than the values measured in other toxic cases. The patient had completed a 10-month amygdalin course and started a new course for 1 month without any negative symptoms. Side effects became overt immediately after changing the amygdalin supplier. Therefore, use of amygdalin at "adequate" dosage might not implicitly be dangerous. Rather, this patient's history opens the question about purity, activity and biochemical properties of the compound used. Although this occurred 35 years ago, even now a standardized production protocol for amygdalin has not been established. Amygdalin can easily be ordered via internet, often without an internationally accepted certificate on its quality and often without any prior control of chemical or bacterial contamination. At least, in the future, regulations should be established and applied to manufacturing so that amygdalin does not place patients at risk due to inadequate safety and quality.
Most reports on amygdalin document that the purified drug itself does not cause cyanide poisoning, provided that a "normal" dose is applied. Cyanide poisoning was not observed in the German study with patients receiving a daily oral amygdalin dosage of 0.5-2 g (Weber 1975). A pilot study on six (Moertel et al. 1981) and then on 50-60 patients (Ames et al. 1981) with an oral amygdalin dose of 1.5 g/day produced blood cyanide levels up to 21 [micro]g/dl without signs of cyanide intoxication. The Moertel study from 1982 is difficult to interpret due to the lack of essential data. From the 178 cancer patients involved, "several" patients showed "symptoms of cyanide poisoning" or blood cyanide levels "approaching the lethal range" (Moertel et al. 1982). However, there was no dose-response relationship and high blood cyanide levels were only noted in three patients receiving low-dosed amygdalin and in no patient receiving high-dose amygdalin therapy (Milazzo et al. 2007). More patients with an i.v. regimen experienced a toxic reaction, compared to patients receiving oral amygdalin. This contrasts with pilot studies demonstrating no toxic effects under i.v. therapy (Moertel et al. 1981; Ames et al. 1981). Current knowledge about amygdalin shows that significantly greater cyanide levels are encountered following oral than i.v. application (Hill et al. 1980; Shragg et al. 1982).
In the Moertel trial, transient symptoms of cyanide toxicity became evident in one patient after additional ingestion of a large quantity of raw almonds (Moertel et al. 1981). In a similar case a patient ingested amygdalin over 8 months, only developing acute symptoms of cyanide poisoning on two occasions when she also ate bitter almonds (Shragg et al. 1982). Since no reports exist about toxicity when purified amygdalin is administered orally, it must be assumed that consuming amygdalin rich food carries severe risk of cyanide poisoning. Whether amygdalin exerts anti-tumor action or not, simultaneous use of the purified drug together with apricot kernels should be strictly avoided. Combining amygdalin and vitamin C should also be avoided. A 68-year-old cancer patient presented at hospital with reduced Glasgow Coma Score, seizures and severe lactic acidosis after consuming 3 g of amygdalin plus 4800 mg of vitamin C per day (Bromley et al. 2005). More than 20 years ago, evidence was provided that megadoses of vitamin C diminish body stores of cysteine, a sulfur containing amino acid, which is involved in detoxifying cyanide to thiocyanate (Basu 1983; Calabrese 1979). Based on this knowledge, it is not clear why a vitamin C-amygdalin combination is still recommended by some practitioners.
The overall risk of developing cyanide poisoning caused by purified amygdalin seems negligible when restricted to use in adults without any further "supportive" therapy. A retrospective analysis revealed only nine cases of cyanide poisoning from amygdalin ingestion between 1977 and 1983, during a time when amygdalin was highly popular among cancer patients (Hall and Rumack 1986). A case report from 1998 was the first in 20 years to report cyanide toxicity from apricot kernel ingestion in the United States (Suchard et al. 1998). Barwina et al. (2013) point to the "rarity of poisoning with amygdalin". Only four cases of HCN-intoxication have been documented between 2000 and 2015, whereby three of them were not directly related to the application of purified amygdalin (Sauer et al. 2015; Barwina et al. 2013). One case from 2005 was unclear, because the patient experienced symptoms of cyanide toxicity after consuming amygdalin and unspecified "vitamin supplements" (O'Brien et al. 2005).
Amygdalin gained high popularity among cancer patients in the 1970 s and experienced a renaissance in the first decade of the 21 st century. Driven by internet promotion and marketing, thousands purchase amygdalin, whereby the exact number of consumers remains unclear. Unfortunately, the newly awakened hope invested in amygdalin has not been accompanied by adequate scientific studies regarding its efficacy. Amygdalin's pros and cons are still based on clinical reports published nearly 40 years ago, which from today's perspective are all of limited scientific value. Critical, factual debate among scientists, with reference to contradictory publications, is nowhere to be found, leaving us in the same position as we were in the 1970 s. Methods to explore drug activity in vitro and in vivo have since been considerably widened, and knowledge about molecular events underlying tumor development and progression has been hugely increased. Based on available reports, highly purified amygdalin, applied in "therapeutic" concentrations does not cause toxic effects. However, amygdalin may also not induce rapid or even distinct tumor regression in cancer patients, particularly in those with late-stage disease. Up until now, a standard therapeutic regimen has not been established. We do not know which application form, oral or intravenous, induces the strongest response, if any. A correlation between an HCN increase in serum/blood and the blood lactate level to determine metabolic decompensation has never been investigated. Tumor markers have not been measured during amygdalin treatment. As long as these important issues have not been dealt with, the question of whether amygdalin is quackery or cure cannot satisfactorily be answered. What we can say with relative certainty is that an anti-tumor activity of amygdalin cannot be excluded but it is unlikely that amygdalin induces rapid, unmistakable tumor regression in patients with late-stage disease.
Abbreviations: CAM, complementary and alternative medicine; NCI, National Cancer Institute; BfArM, German Federal Institute for Drugs and Medical Devices; i.v., intravenously.
Received 21 October 2015
Revised 4 February 2016
Accepted 4 February 2016
Conflict of interest
The authors declare that there are no conflicts of interest.
This work was supported by the "Brigitta & Norbert Muth Foundation".
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Roman A. Blaheta (a) *, Karen Nelson (b), Axel Haferkamp (a), Eva Juengel (a)
(a) Department of Urology, Goethe-University, Building 25A, Room 404, Theodor-Stem-Kai 7, D-60590 Frankfurt am Main, Germany
(b) Department of Vascular and Endovascular Surgery, Goethe-University, Frankfurt am Main, Germany
* Corresponding author. Tel.: +49 69 6301 7109; fax: +49 69 6301 7108.
E-mail address: email@example.com (R.A. Blaheta).
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|Author:||Blahet, Roman A.; Nelson, Karen; Haferkamp, Axel; Juengel, Eva|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Apr 15, 2016|
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