Theoretical Analysis of Anticancer Cellular Effects of Glycoside Amides

Vasil Tsanov; Hristo Tsanov

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THEORETICAL ANALYSIS FOR THE SAFE FORM AND DOSAGE OF AMYGDALIN

PRODUCT

Vasil Tsanov, Hristo Tsanov

ABSTRACT

This article presents a theoretical analysis of the safe dosage form and its dosage of the amygdalin

derivative. By making a precise socio-anthropological analysis of the life of the ancient people of Botra [1]

(Hunza people, Burusho / Brusho people), we come to the hypothesis, which is confirmed by two proofs,

through a number of modern quantum-mechanical, molecular-topological and bio-analytical checks. A

convenient, harmless, form of amygdalin derivative is available that has the same biological and chemical

activity and could be used in conservative clinical oncology. The article also presents a theoretical comparative

analysis of biochemical reactivity in in vivo and in vitro media, by which we also determine the recommended

dosage for patient administration. Based on a comparative analysis of the data, obtained in published clinical

studies of amygdalin, is presented and summarized a scheme of the anti-tumor activity of proposed by the

author’s molecular form.

KEYWORDS

oncology, amygdalin, PM6, PM7, TD-DFT, pharmacological activity

1. Introduction

The apricot is a fruit known to people for millennia. Archaeological excavations in the ancient Armenian

city of Shenchovit near Yerevan revealed overlaid apricot excavations dating back to 6,000 years BC. The

first written mention of apricot was 4,000 years ago in a letter from a Chinese resident. The well-known apricot

comes from a variety of the high-mountainous region of Hindu Kush - Central Asia, where the borders of

China, Tajikistan, Afghanistan and Pakistan meet today. Natural forest and very old apricot trees can still be

found in northeast China and the Caucasus.

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It is a well-known fact by derontologists that the Hunzi people that inhabited the highlands of northern

Pakistan, not far from where the apricot originates, are the healthiest and longest-lived people in the world.

According to researchers and medical scientists who studied the life of the Huns in their natural environment

in the 1950s and 1960s, one hundred percent of them had perfect vision, and cancer, heart attack, high blood

pressure, high cholesterol and even appendicitis and gout were unknown states for them.

Throughout the year, their diet was rich in dried fruits and nuts, with apricots and apricot kernels

predominating, and their main source of fat was apricot seeds. Apricots were an important part of the Hunzi

life.

The apricot kernels contain an average of 21% protein and 52% vegetable oil and are widely used as a

substitute for almonds in the food, cosmetic and pharmaceutical industries. Due to its high content of

amygdalin, apricot seeds are a source of vitamin B17 and are used in alternative medicine for cancer therapy.

The American Cancer Society notes that apricots, as well as other carotene-rich fruits, reduce the risk

of cancer of the larynx, esophagus and lungs.

1.1. Pharmacological activity of amygdalin.

Amygdalin is a nitrile containing a diglycoside compound of the general formula C

, molecular

weight 457.42, with the structure D-mandelonitrile-β-D-glucoside-6-β-glucoside [2] and the structural formula

fig.1.

Fig. 1. Structural formula and full chemical name of Amygdalin

Amygdalin is non-toxic, but under the action of digestive juices and enzymes in the blood it releases

HCN, which, even at relatively low concentrations, is even deadly.

Numerous studies have been performed to prove its antitussive [2] and antiasthmatic [3] effects, analgesic

[4÷9], gastro-enterologic [10,11], promoting apoptosis of human renal fibroblast [12], boosting immunity

synthesis [13÷15] (including to increase polyhydroxyalkanoates in induced human peripheral blood T-

lymphocyte proliferation), anti-diabetic properties [16,17] (including inhibiting alloxan in hyperglycemia),

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potential in the treatment of Hansen's disease, atherosclerosis, immune suppression and most of all antitumor

effect [18÷50] and etc.

1.2. Clinical trial of amygdalin in the treatment of human cancer

1.2.1. Conducting research

In summary: A group of authors [33] published a detailed clinical trial of amygdalin (and its Laetrile

derivative). For the purposes of our research, the exact methodology of clinical trial must be studied in great

detail.

After fully informing the patients about the type and manner of the study, the test begins (Table 1),

constantly monitoring the concentration of total cyanide in the blood. The first analysis was performed 2 hours

after the start of oral drug administration. If the cyanide level is higher than 2 µg/ml but less than 3 µg/ml,

amygdalin is discontinued for up to 48 hours or until all symptoms suggesting toxicity are discontinued.

Therapy was continued in all patients at least until they had irrefutable evidence of progressive

malignancy or until severe clinical deterioration allowed further treatment and follow-up.

Tabl. 1. Amygdalin and “Metabolic Therapy” Regimens

AGENT

STANDARD DOSE

HIGH DOSE

Amygdalin

intravenous course

4.5 g/m2 of body-surface

7 g/m2/day X 21 days

Oral maintenance

area/day x 21 days 0.5 g 3

times daily

0.5 g 4 times daily

Vitamins

25,000 U/day

100,000 U/day

2 g/day

10 g/day

400 U/day

1200 U/day

B complex and minerals

1 capsule/day

Pancreatic enzymes

(Viokase)

12 tablets/day

Koch, M.D., Violante E. Currie, M.D., Charles W. Young, M.D., Stephen E. Jones, M.D., And J. Paul Davignon, Ph.D.

In short, the author's research team points out that the methods of this study are completely comparable

to those used in the studies of every new agent being developed and tested for cancer treatment through more

traditional channels. They are designed to maximize the ability of amygdalin to exhibit therapeutic activity, if

such potential exists.

1.2.2. Clinical results

The characteristics of all patients undergoing experimental treatment are listed in Table 2. Types of

tumors include predominance of colorectal, lung, and breast cancer with a standard dose regimen.

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Tabl. 2. Characteristics of eligible patients

Characteristic

Standard-Dose

Regimen

High-Dose

Regimen

No. of patients

All Patients

Sex

Male

100

Female

Age (yr)

Median

Range

18-84

39-73

18-84

Primary tumor

Colorectal *

Lung §

—

Breast *

—

Melanoma

—

Sarcoma ,

Pancreas *

—

Stomach *

—

Kidney *

—

Lymphoma

—

Ovary *

—

Other (1 or 2 each)

—

Prior radiation therapy ֍

Yes

106(60)

Prior chemotherapy ֍

Yes

109

118

60(34)

Performance status ֍ ○

0-1

116

127 (71)

2-3

Institution

University of Arizona

—

UCLA

—

Mayo Clinic

New York Memorial

—

* Adenocarcinoma

§ Non-small-cell carcinoma

֍ Figures in parentheses denote percentages.

○ Eastern Cooperative Oncology Group score: 0, fully active, to 4, totally

disabled.

Sarna, M.D., Robert Koch, M.D., Violante E. Currie, M.D., Charles W. Young, M.D., Stephen E. Jones, M.D.,

And J. Paul Davignon, Ph.D.

All patients selected for the high-dose regimen had colorectal cancer. Particularly remarkable is that

over one third of all patients did not receive prior chemotherapy. In addition, 71% of the patients had a good

working condition - that is, they were able to work full-time or part-time.

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1.2.3. Toxic reactions and symptoms

Data on the possible toxicity of intravenous and oral amygdalin are shown in Table . 3.

Tabl. 3. Toxicity of amygdalin therapy

Toxic REACTION

ROUTE

INTRAVENOUS

ORAL

% of 178 patients

% of 132 patients

Nausea

Vomiting

Headache

Dizziness

Mental obtundation

Dermatitis

Kvols, M.D., Gregory Sarna, M.D., Robert Koch, M.D., Violante E. Currie, M.D., Charles

W. Young, M.D., Stephen E. Jones, M.D., And J. Paul Davignon, Ph.D.

The authors point out that adverse reactions are rare and also inherent in the oncologic diseases involved.

The side effects of increased cyanide concentration (greater than 3 μg/ml) in the blood disappear after the

amygdalin discontinuation.

Higher concentrations of amygdalin intake are also investigated, but then adverse reactions increase

significantly.

This is precisely the purpose of this article: To provide a dosage form that provides the necessary

concentration of active molecules in vivo to patients in need of anti-tumor therapy.

2. Methodology

This study is based on a hypothesis that undergoes theoretical and logical analysis to confirm and/or

reject it. The methodological scheme is:

1) Determining the optimal chemical formula - after definition, the hypothesis is subjected to two logical

proofs depending on the environment:

- in vivo - this evidence is based on a unique chemical property of the nitrile group to catalytically

convert to an amide in acidity an HCl acid. The conditions are close to those of the digestive juices

in the stomach, and the catalyst is an ion that is available under certain conditions;

- in vitro - an enzymatic reaction is analyzed to achieve the same result, i.e., modification of the

amygdalin molecule to form overlapping with the input hypothesis.

It comes with a conclusion from the part.

2) Determination of the pharmaceutical molecular form:

After confirming the defined hypothesis, it is necessary to compare the physical and chemical

relationships of the modified molecule with those of the parent amygdalin. With the help of mathematical

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chemical methods, a number of comparative methodologies are carried out, which aim to compare each of the

studied parameters between the obtained molecular forms. For this purpose, the indicators are analyzed:

- molecular topology - Molecular Topological Index, Shape Coefficient, Topological Diameter, Total

Connectivity;

- molecular networks - distribution coefficients: LogP, LogS, PKa, etc .;

- electronic reference in the atomic molecular system - Number of HBond Donors, Number of HBond

Acceptors, Formal Charge, etc .;

- molecular properties obtained by semi-empirical methods, such as: dipole moment, thermal capacity,

thermodynamic energy, etc.

Immediately after determining the average deviation between the parent and modified molecules, a

check is carried out by comparing with a well-studied, close to experimental, modification obtained by the

same methods and conditions.

It comes with a conclusion from the part.

3) Determination of the drug dose:

In order to determine the dosage, it is necessary to consider the most likely active forms of amide,

already introduced into the in vivo medium. Knowing the conditions (pH, temperature, etc.) in the human

body and comparing them with concentrations of close in molecular structure and the eventual chemical

treatment of medicinal products that have already passed clinical trials. Stoichiometric calculations are applied

and a relatively accurate dosage is obtained for the administration of the new pharmaceutical formulation in

order to maximize their anti-tumor activity.

3. Determination of the optimal chemical formula

3.1.Hypothesis

The bioactive form of amygdalin is it’s hydrolyzed to amide nitrile group.

3.2. Evidence

3.2.1. PROOF 1

Based on the chemical deposition of nitriles fig.2.

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Fig. 2. Catalytic chemical hydrolysis of nitrile to amide in an acidic environment [51]

The catalyst for this reaction can only be non-coordinatively bonded Co. Cobalt in the human body is in

the order of 22÷59 nmol/l (in the age group 30-56 years for men and women) [52]. Its daily requirement is

0.08 mg/day (for 1÷2-year-old) and reaches 0.30 mg/day (for over 60-year-old). Let us clarify that cobalamin

(known as vitamin B12) is made up of a corin ring that associates cobalt in its complexing form - therefore

cobalt in cobalamin may NOT be a catalyst for the reaction.

Based on the fact that the total content of cobalt in urine is from 3.9 to 30 nmol/d, and that of cobalamin

is 23.3 to 44.5 pmol/d, therefore there is a constant exchange of non-coordinated cobalt in the body.

After analyzing the foods consumed by Hunza people, those containing cobalt were clearly identified,

but there were no traces of vitamin B12: spinach (0.07÷1.20 mg/kg); beet leaves (0.39÷0.41 mg/kg); onions

(0.13 mg/kg); carrots (0.02 mg/kg) [53], etc.

Entered into the stomach uncoordinated cobalt (regardless of its form) will quickly dissolve under the

action of hydrochloric acid and thus enter the blood, therefore, the times of admission of amygdalin and cobalt

will not coincide. The amygdalin will enter the blood with its nitrile group from which it will release cyanide

ion.

The spinach also contains significant amounts of oxalic acid (320 mg/kg). Oxalic acid forms insoluble

cobalt oxalate (3.15 × 10

−9

gr/100ml at 37 degrees of Celsius) with cobalt ions and therefore will not pass

through the gastric walls to the blood.

After analysis and simulations (based on average enthalpy and potential energy of the molecule) by TD-

DFT [54], an extremely stable retention cycle of the non-coordinated cobalt in the stomach is shown in

GAMESS US [55], shown in fig. 3:

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Fig. 3. Schematic depicting the physiological retention of cobalt in the stomach

However, at pH<2, the oxalic salts are partially hydrolyzed and metal anionic (in this case cobalt)

separated. The reaction is reversible and displaced in the direction of the starting material (cobalt oxalate).

Thus, the residence time of cobalt in the stomach increases significantly. In addition to spinach, other

cultivated plants typical of the area contain oxalic acid: tea (370 mg/kg), sorrel (360 mg/kg), rhubarb (240

mg/kg), fig (100 mg/kg), beet (40 mg/kg), plums (12 mg/kg), grapes (7 mg/kg), etc.

Under these conditions, the constantly consuming amygdalin has the conditions (concentration of

reagents, catalyst, pH, temperature, etc.) to react to catalytic hydrolysis to its corresponding amide.

3.2.2. PROOF 2

In addition to cobalt ions, the catalyst may also be cobalt linked to a covalent bond. For example, nitrile

hydratases (NHases; EC 4.2.1.84). They are metal-containing enzymes that convert the nitrile and / or cyano

group to amides, including the amygdalin fig.4.:

The optimum action of nitrile hydratase, for example from Rhodococcus rhodochrous, is from 5.5 to 8.5

pH. Their activity is highest in the range of 10÷80 degrees, and their effect is from 20 minutes to 8 hours. The

acidity in the stomach is much higher and from there these enzymes will be inhibited and even agglutinated.

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Fig. 4. Schematic diagram of the enzymatic hydrolysis of the amygdalin-nitrile group to its amides and

carboxylic acid

The peoples studied have consumed large quantities of apricot and other fruit oils. Much of the

prokaryotes and eukaryotes that produce nitrile hydratases live in lakes or in the moist environment around

them. There is a possibility, due to technological processes, to enter and live for a long time in these oils. Thus,

these insecurities may have lived in unprotected vessels and catalytically hydrolyze the amygdalin nitrile

group to amide and / or carboxylic acid.

3.3. Conclusion of the part

Regardless of the manner of preparation of the hydrolyzed nitrile group of amygdalin to amide and / or

carboxylic acid, its already modified form has a much safer chemical structure to the body. The absence of

the nitrile group, which readily converts to the cyano ion, makes it possible to increase its constant

concentration many times in the in vivo environment.

4. Determination of pharmaceutical form

All further calculations were made after optimization with respect to the minimum energy of the

molecules via MM2 [56] and MMFF94 [57] and followed by molecular dynamics responsible for 310 K, 1.02

bar in a medium of 0.9% solution of NaCl in water using CPCM [58] methodology.

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4.1. Mathematical Chemical Indicators

4.1.1. Molecular topology

For a more complete characterization of the hypothetical molecules, they were compared with the

starting one (amygdalin). Comparisons were made with respect to: molecular topology (Table 4), Molecular

Networks (Table 5), electronic reference in the atomic-molecular system (Table 6), and physical and

thermodynamic parameters (Table 7).

Tabl. 4. Comparative analysis of the molecular topology of pure amygdalin, its amide and carboxyl

derivatives, resulting from the hydrolysis of its nitrile group

-CN

-C(O)NH

-COOH

Balaban Index [59÷63]

1660564

1893740

Cluster Count

Molecular Topological Index

20391

21571

21342

Num Rotatable Bonds, [No. of bonds]

Polar Surface Area [Å

]

202.32

221.62

215.83

Radius, atoms

Shape Attribute

30.03125

31.03(03)

Shape Coefficient

Sum Of Degrees

Sum Of Valence Degrees

124

128

130

Topological Diameter

Total Connectivity

2.14E-05

1.75E-05

Total Valence Connectivity [64]

8.47E-10

4.46E-10

3.46E-10

Wiener Index [65,66]

3080

3308

4.1.2. Molecular Networks:

Tabl. 5 Partition coefficients of amygdalin and its hydrolysates of the nitrile group to amide and carboxylic

acid in the 0.9% NaCl phase in water

-CN

-C(O)NH

-COOH

LogP [67]

-2.81

-3.67

-2.93

LogS [68]

-0.047

0.216

-0.036

PKa [69]

O-1

16.644

16.7357

18.794

O-2

17.4061

17.5938

O-3

12.889

12.9592

13.0704

O-4

13.0331

13.033

13.0254

O-5

18.9903

19.0068

16.983

O-6

20.6647

20.6646

20.6647

O-7

14.9367

14.9366

14.9367

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OH(COOH)

3.03401

Molar Refractivity [70]

10.525

10.915

10.701

4.1.3. Electronic treatment in atomic molecular system

Tabl. 6. Electron Assignment in the Atomolecular System [71,72] of amygdalin and its hydrolysates of the

nitrile group to amide and carboxylic acid

-CN

-C(O)NH

-COOH

Formal Charge

Number of HBond Acceptors

Number of HBond Donors

Ovality

NaN

Principal Moment

1901.916

6152.891

6968.502

2042.063

6108.058

7033.240

2100.609

6066.442

7045.560

Henry's Law Constant [73]

24.508

27.945

26.255

4.2. Molecular properties obtained by semi-empirical methods

Tabl. 7. Molecular and electron-configuration properties of amygdalin and its hydrolysates of the nitrile

group to amide and carboxylic acid obtained by semi-empirical methods [74]

method

-CN

-C(O)NH

-COOH

Core-Core Repulsion, [eV]

PM7 [75]

51868.25

56375.19

56412.48

COSMO Area, [Å

]

378.83

385.94

387.52

COSMO Volume, [Å

]

489.25

508.77

507.20

Dipole, [Debye]

4.991

5.84

5.71

Ionization Potential, [eV]

9.93

9.87

9.90

Total Energy, [eV]

-6254.95

-6578.48

-6674.00

Heat Capacity, [Cal/Mol-Kelvin]

PM6 [76]

/ 310.15 K,

1.02 bar /

96.27

100.19

97.03

Thermodynamic Energy, [Kcal/Mol]

283.95

297.77

289.42

Data in Tables 4, 5, 6, 7 show that values (including their equated statistical error) over 23% of the

indicators are the same. No drastic topological and molecular network deviations and electron-configuration

anomalies are observed. The differences represent up to 12% mean deviation of the amygdalin index with its

hydrolyzed derivatives.

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To test the tolerance of 12%-th still not jeopardize the normal physiology of the body, let us consider a

well-studied enzyme hydrolytic industrial process (fig.5) of a passage of 3-Cyanopyridine to Nicotinamide

[77] (vitamin B3) by means of nitrile hydratase. The thermodynamic quantities are compared: total energy,

thermal capacity, and thermodynamic energy, since these physical functions are the consequence of most

topological, morphological, and electron-configuration relationships of each molecule in tabl.5.

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Fig. 5. Enzyme hydrolysis of 3-Cyanopyridine to Nicotinamide and nicotinic acid

It is important to note that all three enzymes are almost always together, whether of micro-bacterial or

bio-physicochemical origin.

Tabl. 5. Total Energy, Heat Capacity and Thermodynamic Energy on 3-Cyanopyridine, Nicotinamide and

Nicotinic Acid by 310 K, 1.02 bar in medium of 0.9% NaCl solution in water using CPCM [78] methodology

3-Cyanopyridine

Nicotinamide

Nicotinic

acid

Total Energy PM7, [eV]

-1162.27

-1485.64

-1581.25

Heat Capacity, [Cal/Mol-Kelvin]

16.73

19.75

20.37

Thermodynamic Energy, [Kcal/Mol]

55.72

70.77

63.54

By comparing the data in Table 5 with the conclusions of Table 1, 2, 3, 4, the average standard deviation

is confirmed when comparing the molecular nature of the nitriles, the amide and their corresponding

carboxylic acid.

4.3. Conclusion of the part

Due to the theoretical molecular modification, namely the transition from condensed and / or

polymerized nitrile to carbohydrate in its amide and as a by-product and its acid, does not change the total

activity of the parent compound as well as its active groups not involved in hydrolysis. The chemical stability

and predictability of hydrolysis products are the basis for a pharmaceutical formulation that covers all

international requirements for a conservative medicinal product.

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5. Determine drug dose

In order to determine the dosage, it is necessary to consider the most likely active forms of amide already

in vivo.

In oral use (which is also recommended), the connection between the two glycosidic nuclei will be

attacked by the salivary gland. Due to the relatively short time for complete contact between the substance

and the enzyme, the probability of a reaction being below 3% and