Study of bioavailability of oral iron preparationsKusum C Gupta1, Teresa Paul1, JM Mehta2, NK Desai1, Bharati Sanghvi3, UK Sheth1
1 Clinical Pharmacology Unit and the Clinical Drug Trial Unit, Council of Scientific & Industrial Research, Department of Pharmacology, Seth G. S. Medical College, & K.E.M. Hospital, Parel, Bombay 400 012., India
2 Department of Medicine, K.E.M. Hospital, Bombay 400012., India
3 Department of Statistics, University of Bombay, Bombay., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 802126
Source of Support: None, Conflict of Interest: None
Two preparations o f oral iron, were evaluated in normal volunteers and in patients of iron deficiency anemia. The study in 10 normal volunteers measured the bioavailability of the two preparations. The study in 20 patients assessed the therapeutic equivalence and tolerance. In the volunteer study, serum iron levels at 4 and 8 hours were significantly higher with the iron vitamin-mineral combination than with the plain iron. The AUC was also significantly higher. In patients there was no difference in the effect of the two formulations. With both there was a rapid rise in Hb, PCV and reticulocyte count in the first week, after that the rate of increase was slow. Both preparations were equally well tolerated.
Oral iron remains the therapy of choice in treatment of a majority of cases of iron deficiency anaemias. A number of preparations of different iron salts are available either as single drug preparation or as formulations with added vitamins, minerals etc. Each preparation is claimed to be better tolerated with a fewer side effects and more effective. We have investigated two oral iron preparations for bioavailability of iron, tolerance and therapeutic equivalence. One preparation contained only ferrous fumarate and this was compared to a combination containing an equivalent amount of ferrous fumarate with additional vitamins and other metals. The implication of such studies, differ between bioavailability and therapeutic equivalence and their interrelationship as evident from the results obtained in these studies, as discussed.
Preparations used were,
Drug A: Ferrous Fumarate 135 mg.
per tablet Vitamin C 300 mg.
Folic Acid 3 mg.
Vitamin B 12 10 µg.
Vitamin B 6 , 5 mg.
Copper Sulphate 0.2 mg.
Sodium Molybdate 6 mg.
Mang. Sulphate 1 mg.
Drug B: Ferrous Fumarate 135 mg/tab.
Both the preparations were identical in size, colour and shape and identity of composition was only checked after the analysis of the results.
Part I-Bioavailability studies
This study was carried out in 10 healthy volunteers with minimum haemoglobin levels of 11 gm% and PCV 40%. All the subjects included in the study had not received any iron or multivitamin preparation for at least two weeks prior to their inclusion in this trial. Informed consent was obtained for the study.
Two tablets of Drug A or Drug B both of which contain 90 mg of elemental iron (as ferrous fumarate 135 mg) were administered on any empty stomach, to each of the subject in randomized double blind cross over fashion. 5 ml of blood samples were collected at 0 hr. and 2, 4, and 8 hours after administration of the drug. The subjects received standardised breakfast containing toast, two eggs and coffee two hours after the drug administration. The cross over study using the second preparation was carried out after an interval of 7 days. Blood samples were collected as before. Serum iron levels were estimated by the method of Peters et al  The area under the curve for both the drugs was also calculated.
Part II-Therapeutic Equivalence studies
20 adult patients with proven iron deficiency anaemia were included in this study. Patients suffering from acute or chronic haemorrhagic episode were not included. Those suffering from helminth infection, received appropriate treatment before their inclusion in the trial. Patients were matched for age and sex. They were divided into two groups of 10 each. The drugs A and B were administered in randomised double blind fashion so that there were 10 subjects for each drug study. The dose of the drug was 2 tablets twice a day. The average haemoglobin of patient in drug A group was 3.72 gm% ± 0.31.
The average haemoglobin of patients receiving drug B was 5.10 gm%. All the patients had haemoglobin below 8 gm%. All the patients were hospitalized during the study and received identical diet. The following investigations were carried out before beginning the trial, on the 4th day and at weekly intervals for a period of 8 weeks.
1. Haemoglobin estimation. (Cyan-methamoglobin method).
2. Packed cell volume (P.C.V.)
3. Reticulocyte count.
After the first 4 weeks of treatment as the haemoglobin improved and the patients felt better, some of them did not continue with the study as these patients were mostly farmers and wanted to go back to work in the fields. The number of patients remaining in the study after the 4th week is shown in [Table 3]. The drop out rate both in drugs A and B was not significantly different.
Side effects like pain in abdomen, diarrhoea or constipation if any were inquired into:
Part I-Bioavailability: Serum iron levels (mcg/100 ml) in the 10 volunteer subjects after administration of single dose of Drug A and drug B at an interval of one week is shown in [Table 1].
Average serum iron levels in mcg/100 ml with both preparations A and B and their comparison at different time intervals with statistical significance of difference is shown in [Table 2].
The average serum values at 0 hr. are almost the same. Thus, the fasting serum levels of the volunteers on the days they received the 2 preparations did not differ significantly. For both the drugs, the serum iron levels are maximum at 4 hours and decline at 8 hours. At 2 hours, there is no significant difference between serum iron levels for the 2 preparations. The 4 hour and 8 hour serum iron levels for Drug A are higher than those of Drug B and these levels are statistically significant at 5% level of significance.
The area under the curve for Drug A is 3563.5 mcg/100ml/hr. While for Drug B it is 2764.0 mcg/ 100ml/hr. This difference of 799.5 mcg/100ml/hr. is also statistically significant at 5% level of significance [Figure 1].
Thus it was concluded that taking into consideration the serum iron levels at 4 and 8 hours and also the area under the curve, Drug A (combination of ferrous fumarate with vit. C, Vit. B 12 etc.) had a higher bio-availability as compared to Drug B (ferrous fumarate only) when both these preparations were given orally in a single dose containing 90 mg of elemental iron.
With Drug A the average Hb was initially 3.72 Gm/100ml but after a week it increased to 6.17 Gm% which is a significant rise. At the end of 8 weeks, the average Hb reached 11.10 Gm%.
With Drug B the initial average Hb was 5.19% and it gradually increased to 11.70 Gm% at the end of 8 weeks.
The initial average Hb for the two drugs are significantly different, so analysis of covariance technique was used to compare the averages for the 2 drugs at different points of time. The averages were not statistically different at any point of time.
The 2 groups of patients do not differ significantly as far as PCV readings are concerned, so Student's t-test was used. From an initial average of 18.2 for Drug A the average rose to 27.1 at 1 week, and for Drug B the corresponding figures were 21.7 and 26.7 respectively. For both the drugs, the averages slowly increased upto 41.3 for A and 42.2 for B at the end of 8 weeks.
There is no statistical difference between the 2 drugs at any point of time as far as PCV is concerned.
With drug A, the initial average is 2.95 and at the end of 1 week it is 6.2, peak of 8.6 is reached at 3rd week and then it falls, for B the initial average is 3.9, peak 5.9 at 4th week, and thereafter almost the same level is maintained.
There is no statistical difference between the 2 drugs at any point of time as far as reticulocyte count is concerned. The level of statistical significance is 5%.
So, it can be concluded that considering Hb, PCV and reticulocyte count for both Drug A and B, there is no difference in the effect of the 2 drugs. With either of these drugs, the averages increase rapidly at the end of 1st week and after that the rate of increase is slow.
Both the preparations were equally well tolerated, without any complaints of abdominal pain, or changes in bowel habits like diarrhea or constipation. No other side effects were noted.
Systemic drugs administered orally or parenterally have to reach, through the general circulation, the target tissue in an active form, and in an adequate amount to exert therapeutic effects. Since it is not possible to measure the amount of drug reaching the target tissue, the drug reaching the general circulation is usually measured. Hence, bioavailability of a drug is defined as the percentage of a drug contained in a drug product that enters the systemic circulation in an unchanged form after administration of the drug product. The concept also includes the rate at which the entry occurs.
For the purpose of most bioavailability studies, it is necessary to determine, (i) the peak of serum concentration curve (ii) the time of maximum drug serum concentration and (iii) the area under the drug serum concentration time curve. The area under the curve is closely related to the amount of drug that enters the circulation. 
The present results in volunteers on amounts of iron absorbed at 8 hours, as measured by rise in serum iron, compared to basal levels, showed significantly higher levels with combination of preparation of iron, as compared to tablets containing equivalent amounts of iron alone. These were cross over studies in the same subjects; hence the rate of absorption of both the preparations given orally could be compared, as the individual variations would remain the same with both preparations. In drugs like iron which is to be used for 8 weeks or more for treatment, bioavailability results of single dose treatment may be misleading. Iron from plasma gets stored in tissues, and is then released for formation of haemoglobin. Rate of absorption, storage and release will depend upon the deficiency of iron in the body. A single dose of one preparation may raise serum iron level considerably as compared to another one-but both preparations may raise it sufficiently high when given repeatedly over long periods and total body response may be similar with both preparations. This is particularly true where normal range of plasma levels may be very wide, as it is seen with serum iron levels. Therapeutic trial in patients with iron deficiency anaemia with two oral iron preparations studied for bioavailability in Part I, have shown that both preparations have comparable therapeutic equivalence. Total response in 8 weeks, rate of rise in haemoglobin per week and reticulocyte response was similar with both iron preparations. Patient acceptance and tolerance were also same with both preparations. In both groups nearly equal number of patients did not finish 8 weeks of trial, as they felt quite well and did not want to stay in hospital any further.
This study brings out clearly the importance of studying therapeutic equivalence and not merely bioavailability in drugs which have a wide range of serum levels and have to be used for long periods for treatment of chronic conditions. Evaluating products on the basis of comparative rate of absorption alone can be risky and unrealistic because the assessment of bioequivalence or inequivalence under these conditions will not be predictive of therapeutic equivalence or in equivalence. Bio-availability in a mathematical way could be considered merely as a calculation by which the efficacy of absorption of a test drug is compared to a reference product. In 'cases of drugs which have a wide range in plasma levels, and are to be used by repeated administration over long periods of time, projection of merely peak blood levels and area under curve having rate and amount of absorption after a sizeable dose, may be misleading, to the practitioners when presented in promotional literature.
We are thankful to Warner Hindustan Limited for the supply of the drugs (Rediplex and Ferrous Fumarate tablets) and their part financial support for this project.[Table 4],[Table 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]