An investigation of the effects of fulvic and humic acids on the absorption of selected drugs ...

180 Pages·2014·2.79 MB·English

An investigation of the effects of fulvic and humic acids on
the absorption of selected drugs, vitamins and minerals
using the everted mouse gut model.
by
Kirsten Willis
Submitted in fulfilment of the requirements for the degree
Magister Scientiae in Pharmacology
in the
Department of Pharmacology
Faculty of Health Sciences
University of Pretoria
Supervisor:
Prof A.D. Cromarty
Date of Submission:
February 2015
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Acknowledgements
Acknowledgements
I would like to acknowledge the following people for their part in the completion
of this study:
 My supervisor, Prof A.D. Cromarty, for his constant guidance, support and
patience. Your invaluable advice as well as the many hours spent teaching
and training me throughout my time as a post-graduate student will never
be forgotten. I appreciate all you have done for me!
 The staff at the University of Pretoria Biomedical Research Centre
(UPBRC) for their assistance with the animal work involved in this study.
 My fellow students of the Department of Pharmacology, University of
Pretoria, for being there to lend a helping hand or to give advice.
 My family, for giving me the opportunity to study further and for
supporting me throughout my years as post-graduate student.
Lastly, it is not without the efforts of many others that I completed this study.
Even though they have not been personally mentioned, I would like to
acknowledge their efforts and assistance during this study.
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Declaration
Declaration by candidate
The experimental work contained in this dissertation was carried out by Kirsten
Willis (author) from the Department of Pharmacology, Faculty of Health Sciences,
University of Pretoria under the supervision of Prof A.D. Cromarty.
I declare that the work contained in this dissertation is my own work and has not
been previously submitted for a degree at this, or any other tertiary institution.
_______________________ _______________________
Kirsten Willis Date
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Abstract
Abstract
Humic substances, such as the closely related humic and fulvic acids are
ubiquitous, naturally occurring organic macromolecules of complex but undefined
structure. These compounds are known complexing agents due to their
supramolecular like structures and are capable of binding a wide variety of
compounds.
Numerous studies have confirmed that humic and fulvic acids exhibit diverse
medicinal and therapeutic properties. For this reason, alternative or “natural”
medicinal preparations rich in these substances are being self-administered, often
concomitantly with conventional drugs. The possibility exists that these humic
substances, found in the alternative medicinal products, may result in drug-drug
interactions and bind to simultaneously ingested drugs. Complex formation may
affect absorption and alter overall bioavailability. Changes in these parameters
may lead to reduced therapeutic effect or toxic side effects of prescribed drugs in
patients.
Similarly, these humic substances may bind to and alter the uptake of ingested
nutrients, such as vitamins and minerals, obtained from food sources as well as
dietary supplements. Changes in absorption may result in a loss of proper
physiological functioning in the body or in unwanted effects of overdose.
This study investigated the effect of fulvic and humic acids on the absorption of
commonly administered classes of drugs, vitamins and minerals using the everted
mouse gut model that was successfully used to assess the membrane transport of
the test compounds. This model made use of everted segments of excised intestinal
tissue placed in Krebs Ringer Buffer (pH7.4), where physiological functioning of
the tissue is maintained for up to two hours after excision. The amount of test
compound which crossed through the intestinal membrane without and in the
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Abstract
presence of each humic substance was quantified using LC-MS/MS methods
developed for each of the drugs and vitamins, and ICP-MS, in the case of the
minerals.
The amount of test compound absorbed alone was compared to the amount
absorbed when in the presence of each humic substance. Changes in the uptake,
for each test compound was noted, the extent of the absorption increase or
decrease was compound specific. The changes in absorption observed could be
attributed to changes in compound solubility and mechanism of transport across
the intestinal membrane once in complex.
Drugs and vitamins were seen to be more prone to decreases in absorption in the
presence of the humic substances, whereas the majority of the minerals showed
significantly increased absorption. Binding of the minerals to the humic
substances through chelation, and not complex formation, could have a greater
effect on compound solubility.
Health care professionals, as well as individuals ingesting these and other
substances concurrently, should be aware of the potential effects on absorption
that may occur due to drug-drug interactions in order to avoid a loss of
therapeutic/physiological activity or negative toxic symptoms.
Key words: Humic substances, humic acid, carbohydrate-derived fulvic acid,
complex, everted mouse gut model, drug absorption, bioavailability
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Table of contents
Acknowledgements…………………………………………………………………………ii
Declaration …………………...…………………………………………..………………..iii
Abstract ………………………………………………………………...………………..…iv
Table of contents……………………………...……………………………………………vi
List of figures……………………………………………………………………..………..ix
List of tables………………………………………………………………………………xiv
Glossary of abbreviations…………………………………………………..…………xviii
1. Literature review…………………………………………………………………..……1
1.1 Background………………………………..……………………………………1
1.2 Drug interactions………………………………………………………………2
1.2.1 Types of drug interactions………………………………………….3
1.2.2 The effects of direct pharmacokinetic interactions on the
absorption and therapeutic activity of drugs………………..…………4
1.3 Assessing intestinal absorption of substances………………………….…6
1.3.1 Transcellular transport………………………………………..……8
1.3.2 Paracellular transport………………………..………………..……8
1.3.3 Carrier mediated transport……………………………………...…9
1.3.4 Transcytosis……………………………………………………..……9
1.4 Factors affecting the absorption of orally administered drugs……....…9
1.4.1 Drug-related physicochemical factors ………..…………...……10
1.4.2 Systemic factors………..……………………………..…………… 10
1.5 Nutrient absorption in the GIT……………………..…..………………… 10
1.6 Methods for assessing intestinal absorption of substances……………12
1.6.1 The everted gut sack technique……………………..…..........…12
1.6.2 Cultured cell monolayers………………………………………….14
1.6.3 Ussing chamber…………………………………………………….16
1.6.4 PAMPA…………………………………………………………...….17
1.7 Humic and fulvic acids…………………………………………...………….18
1.7.1 Humic and fulvic acids as binding agents…………………..….19
1.7.2 Humic and fulvic acid structure………………………………….20
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Table of contents
1.7.3 Applications of humic substances………………………………..21
1.7.3.1 Applications in agriculture……………………………..21
1.7.3.2 Applications in medicine………………………………..22
1.8 Study motivation……………………………………………………………..23
1.8.1 Rationale for the choice of absorption model…………………..24
1.9 Aims…………………………………………………………………...………..26
1.10 Objectives…………………………………..………………………….……..26
2. Materials and methods………………………………………………………………..27
2.1 Animal work ……………………………………………………...…………..27
2.1.1 Animal ethics……………………………………...………………..27
2.2 Experimental design ……………………………………...…….…………..29
2.2.1 Test compounds …………………………………...………..……..29
2.2.1.1 Dugs……………………………………...……………......29
2.2.1.2 Vitamins……………………………………...……….......30
2.2.1.3 Minerals .……………………………………...…..….......31
2.3 Materials……………………………………...…………………...………......32
2.3.1 Preparation of solutions…………………………………….........32
2.4 Methods……………………………………...……………………………......33
2.4.1 Everted mouse gut model…………………………………….......33
3. Sample analysis……………………………………...……………………………......36
3.1 Sample analysis of test compounds……...……………………………......36
3.1.1 Sample preparation ……………...……………………………......36
3.1.1.1 Sample preparation for drugs and vitamins…………36
3.1.1.2 Sample preparation for minerals………………………37
3.1.2 Sample analysis of drugs and vitamins using LC-MS/MS…..37
3.1.2.1 LC-MS/MS validation……………...…………………....38
3.1.3 Analysis of minerals using ICP-MS……………...………..…....40
3.2 Statistical analysis of samples……………………………...………..…....40
4. Drugs……………………………...…………...…………...…………...………..……..41
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Table of contents
4.1 Diclofenac………………...…………...…………...…………...………..……42
4.2 Penicillin V………………...…………...…………...…………...……………49
4.3 Warfarin………………...…………...…………...……………...……………56
4.4 Rifampicin………………...…………...…………...……………...……….…62
4.5 Valsartan……………...…………...…………...……………...……….……..69
4.6 Zidovudine……………...……...…………...……………...……….…………75
4.7 Combined results summary……………...…………...……………..……..82
4.8 Discussion……………...…………...…………...…………….....…….……..83
5. Vitamins……………...…………...…………...……………………......……….……..98
5.1 Vitamin B ……………...…………...…………...……………....…….……..99
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5.2 Vitamin E……………...…………...…………...……………...…………...106
5.3 Discussion……………...…………...…………...………….…...……….….111
6. Minerals……………...…………...…………...……………...…………….….……..113
6.1 Major minerals……………...………..……...……………...……….……..115
6.1.1 Calcium……………...……………...……………...……….……..115
6.1.2 Magnesium……………...…………...…………...……….………119
6.2 Trace minerals……………...………….……………...….….……..125
6.2.1 Iron……………...……...…………...……………...……….……..125
6.2.2 Zinc……………..………...…………...……………...……………134
6.3 Discussion………...…………...…………...……………...……….……….137
7. Concluding discussion ……………...…………...……………..…………………...145
8. References……………...…………...……………..…………………………..……...151
Addendum A
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List of figures
List of figures
Figure 1: Diagram showing different regions of the intestines…………7
Figure 2: Schematic diagram of intestinal epithelium…………...………8
Figure 3: Schematic diagram of the everted gut sack technique……...13
Figure 4: Schematic diagram of a Caco-2 cell monolayer………………15
Figure 5: Schematic diagram of the Ussing chamber…………………...17
Figure 6: Schematic diagram of the PAMPA method…………………...18
Figure 7: Properties of humic substances……………………………...…19
Figure 8: Molecular structure of diclofenac………………………….……43
Figure 9: Reversed phased chromatography of diclofenac…………...…44
Figure 10a-d: Comparison of diclofenac absorption in different regions of
the mouse intestine (10A: duodenum, 10B: jejunum, 10C:
ileum and 10D: colon) ……………………………………………46
Figure 10e: Combination of graphs 10A-D illustrating difference of
diclofenac absorption from different regions of the mouse
intestine (duodenum, jejunum, ileum and colon) ……………47
Figure 11: Molecular structure of penicillin V……………………………..50
Figure 12: Reversed phased chromatography of penicillin V……………51
Figure 13a-d: Comparison of penicillin V absorption in different regions of
the mouse intestine (13A: duodenum, 13B: jejunum, 13C:
ileum and 13D: colon) ……………………………………………53
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List of figures
Figure 13e: Combination of graphs 13 A-D showing comparison of
penicillin V absorption in different regions of the mouse
intestine (duodenum, jejunum, ileum and colon) ……………54
Figure 14: Molecular structure of warfarin…………………………...……57
Figure 15: Reversed phased chromatography of warfarin………….……58
Figure 16a-d: Comparison of warfarin absorption in different regions of the
mouse intestine (16A: duodenum, 16B: jejunum, 16C: ileum
and 16D: colon) ……………………………………………………59
Figure 16e: Combination of graphs 16A-D showing comparison of
warfarin absorption in different regions of the mouse
intestine (duodenum, jejunum, ileum and colon) ……………60
Figure 17: Molecular structure of rifampicin………………………………63
Figure 18: Reversed phased chromatography of rifampicin……………..65
Figure19a-d: Comparison of rifampicin absorption in different regions of
the mouse intestine (19A: duodenum, 19B: jejunum, 19C:
ileum and 19D: colon) ……………………………………………66
Figure 19e: Combination of graphs 19A-D showing comparison of
rifampicin absorption in different regions of the mouse
intestine (duodenum, jejunum, ileum and colon) ……………67
Figure 20: Molecular structure of valsartan……………………………….70
Figure 21: Reversed phased chromatography of valsartan……………...71
Figure 22a-d: Comparison of valsartan absorption in different regions of the
mouse intestine (22A: duodenum, 22B: jejunum, 22C: ileum
and 22D: colon) ………………………………………………..….72
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