A COMPARATIVE STUDY TO EVALUATE THE EFFICACY AND SAFETY OF IROCHEL (UNANI CODED FORMULATION) FOR THE MANAGEMENT OF HUMORAL IMBALANCE (SU'E AKHLAT) CAUSED BY TRANSFUSION INDUCED IRON OVERLOAD IN THALASSEMIA PATIENTS IN COMPARISON WITH DEFEROXAMINE (ALLOPATHIC MEDICINES)

This research study was an experimental, randomized, controlled clinical trials to evaluate the safety and efficacy of coded Unani formulation "Irochel" for the management of iron overload due to repetitive blood transfusion in comparison with Deferoxamine in already diagnosed 132 transfusion-dependent thalassemia patients of both genders of 10 years to 16 years' ages. The study was conducted at Hamdard Matab, Araam Bagh where patients came from multiple thalassemia centers such as the National Institute of Blood Diseases (NIBD), Fatimid Foundation, Umair Sana Thalassemia Center, Kashif Iqbal Thalassemia Center, Civil Hospital, and Hussaini Blood Bank. The data was collected by Clinical Record Form (CRF) and then analyzed by SPSS version 23. The study was conducted in the years 2014-2020. The study comprises two phases: 1) Development of Irochel and 2) Clinical Trials.

Irochel is a polyherbal coded formulation comprising of six herbs which has excellent phytotherapeutic and nutraceutical value associated with tremendous amount of bioactive compounds present in these herbs. Researches have shown remarkable ethnobotanical and scientific evidences for the management of IOL, thalassemia and iron chelating ability of these novel herbs which are; Fagonia cretica L., Triticum aestivum L., Emblica officinalis L., Cucurbita pepo L., Momordica charanti L., and Tribulus terristris L. This polyherbal formulation is a round, shiny black colored and bitter taste tablet which is weighted as per dose 500 mg.

In the present study, IOL which is the major health problem in TDT, has been treated by the oral administration of IRC in comparison with DFO therapy. The efficacy of this polyherbal formulation was significantly proved by pharmacognostical evaluation of phytochemicals and antioxidant studies. As well as the toxicological studies proved that it is a safe drug for human use. The macroscopic phytochemical analysis showed that all herbs included in this product has astounding phytochemical profile which was also supported by the literature search in chapter 2 of this dissertation.

The study was divided in to two phases; 1) Development of Irochel and 2) Clinical trials of Irochel to analyze the safety and efficacy on human subjects.For the development of the herbal compound formulation, identification and organoleptic evaluation of herbs were carried out by Miss Taba Rauf, taxonomist, FEM, HU. Thereafter, the extract of each herb was obtain and phytochemical analysis proved presence of alkaloids, amino acids, carbohydrates, proteins, flavonoids, glycosides, saponin, tannins and resin (See Table-16). Thereafter, the product was undergoing for the evaluation of cytotoxicity, antioxidant and antimicrobial activities (See Tables-

. 17-21) The results of cytotoxicity by BSLA proven that test drug has a significant cytotoxicity showing LC50 124.327 μ gm per ml in dose dependent manner. This indicated that herbs included in this formulation has rich bioactive principals that can play a significant role in maintaining and preservation of health. As well as, DPPH free Radical Scavenging activity of the product was carried out. In a concentration of 0.5 mM, polyherbal extract exhibited 85.7 % significant radical scavenging activity.

In addition to this, antimicrobial activity of test drug was conducted, results exhibited moderate activity against S. aureus. This pathogen is found to be the most dangerous bacteria among all of the common staphylococcal bacteria. However, IOL has also been reported as a cause of escalated infectious diseases in TDT.

The toxicological studies (acute and subacute toxicity) showed that the test drug is a safe drug for human use. In acute and sub-acute toxicity, LD50 value was found to be greater than 5000 mg/kg of body weight. There were no signs of toxicity observed in animal's behavior, weight, food and water intake. In addition, necroscopic and histopathological examination showed no signs of toxicity. However, some parameters of hematological and biochemical parameters showed significant changes.

The quality control pharmaceutical analysis of Irochel comprising physical characteristics of tablets which showed that it is a disk shaped round, shiny black colored and bitter taste tablet having characteristic odor. The tablet average weight is 500.83 ± 2.245 mg with diameter of 4.830 ± 0.320 mm, thickness 11.17 ± 0.123 mm, hardness 4.70 ± 0.923 kg, friability 0.929 % and disintegration time was 32 minutes. WHO guidelines (2003) and drug regulatory authority of Pakistan have also mentioned standard measures for polyherbal formulations same as of USP and B.P. The results exhibited that the size of the granules was uniformed, the flow of granules from hopper was uninterrupted, lubricant was mixed well and particles was evenly distributed and dies, punches as well as speed of machine was normal during manufacturing. The disintegration time indicates that Irochel will be disintegrated in alkaline flora of intestine.

Hence, these pre-clinical studies; pharmacognostical, pharmacological, toxicological and pharmaceutical analysis proves that this product is a safe, effective and standardize polyherbal formulation for human use.

The randomized clinical trial was conducted to evaluate the efficacy and safety of IRC on TDT subjects with IOL, in comparison with DFO. In the present study, 66 subjects of both genders between the ages of 10-16 years in each group (control and test) were registered and evaluated.

The DFO was administered subcutaneously via portable infusion device for about 8-12 hours per day at the dose of 40-60 mg/kg bw. While IRC was administered orally 500 mg b.d. with lukewarm glass of milk.

The age, gender and temperament of all subjects of both groups were evaluated. In which, test group comprise 30 males and 36 females had mean ages as 14.56 ± 1.612 and 14.58 ± 1.518 respectively. Similarly, in the control group 34 males and 32 females were registered, their mean ages were 14.82 ±1.141 and 14.26 ± 1.445 respectively.

The Vitals including temperature, pulse, respiratory rate and blood pressure as well as BMI of the subjects showed means of normal values of both groups.

In the general physical examination of all subjects in both groups shows changes in face; most of the subjects having Mongolian face as well as the signs of anemia, jaundice and dehydration was positive before the commencement of study in both groups. After the administration of IRC the improvement in these signs shows that this product has good effects in maintenance of Hb, decreasing hemolysis of RBCs, lead to decreases hyperbilirubinemia which results in clear clera, skin and tongue, as well as recover hydration. Most of the subjects was showed moderate to severe dehydration before treatment in both group.

As herbs in IRC have vast spectrum of phyto-therapeutic effects, that is the reason due to which there is a significant improvement in systemic complaints of the subjects included in test group such as psychoneurological, respiratory, cardiovascular, gastrointestinal, hepatobiliary, urogenital, musculoskeletal and dermatological problems. While in the control group, as the DFO is only a chelating agent and it has no effects on other systems of the body, therefore, in control group there was no significant effects in systemic complaints.

The complexion of skin was analyzed by using visual skin color chart. The polyherbal formulation showed significant results in lightening the complexion. This may be due to decreasing SF levels and removing of free radicals from the body. The excess iron in β-Tm is also deposited in the skin which causes dark coloration or bronze skin or other change in complexion, hyperbilirubinemia also contribute in changing of skin complexion. Similarly, the skin moisture, sebum, elasticity and pigmentation was showed significant improvement in skin of subjects with hemochromatosis in β-Tm. Phytochemicals and vitamins i.e ascorbic acid and vitamin E present in the IRC is responsible for the lightening in the complexion, moisturizing and glowing effect of skin, improve tonicity and decrease pigmentation due to Hb or melanin or both. The results were contrary in control group.

In laboratory investigations; CBC, Iron Profile, Lft, Lipid Profile, and Urinalysis was performed. The results of CBC parameters shows significant improvement after treating with IRC for three months. There was superlative improvement in mean RBCs count, although subjects in test group were only taking 1 unit of blood. As well as their Hb level was also increased exceptionally. The results exhibited that the mean and SD of RBCs (10^6/ul) at baseline in the test group before treatment was 3.27 ± 0.549 (10^6/ul) whereas after three months of treatment with IRC the mean and SD was 3.67 ± 0.533 (10^6/ul). As far as control group is concerned, in the commencement of study as baseline before treatment RBCs showed mean and SD as 3.32 ± 0.486 (10^6/ul) and after three months' treatment with control drug mean and SD showed 3.39 ± 0.472 (10^6/ul). The p value was obtained through paired sample t-test which exhibited significant difference in before and after treatment with IRC as found to be 0.000. Whereas, in case of Control group p value found to be as 0.321 which is considered as insignificant. In addition, RBcs indices also showed significant results in test group. Similarly, The Hb (gm/dl) of test group in the commencement of medication at baseline was showed mean and SD as 8.04 ± 0.709 (gm/dl). After three months of treatment mean and SD displayed as 9.59 ± 1.124 (gm/dl). In the control group the mean and SD of Hb before treatment was observed as 8.56 ± 1.186 (gm/dl) and after treatment with DFO the mean and SD showed 8.90 ± 1.289 (gm/dl). The p-value in test group before and after treatment found to be as 0.000 which exhibited highly significant difference. While in control group it was 0.121 which is > 0.05 and considered as insignificant.

As far as DFO is concerned, as it is evident that it has only chelating effects therefore, it does not affect RBCs count or Hb level in the body, rather than this, the subjects in control group required more BT and mostly transfused either more than 1 unit of blood or having shorten duration between transfusions. This leads to increase SF and SI level in these subjects but also does not have significant improve RBCs count or Hb level. The mean and SD, confidence interval and p value of total leucocytes count com (WBCs) and differential leukocytes count (DLC) comprises on Neutrophils (%), Lymphocytes (%), Eosinophils (%), Monocytes (%) and Basophils (%) was analyzed by paired sample t test. The results exhibited that the IRC showed remarkable effect on WBCs count by increasing these cells. These results were also supported by acute and subacute toxicity study on this product as well. It clearly exhibited that this polyherbal formulation has excellent defensive capability against infections and inflammation. The p value was obtained to signify the results which exhibited 0.031 as moderately significant difference for test group and 0.270 for control group which considered as insignificant.

The mean and SD of Platelets count of test and control groups and p value was obtained by applying paired sample t-test. The p value of test group was found to be as 0.001which showed the significant difference among before and after treatment. In comparison with test group, the p value of control group obtained as 0.849 which exhibited no significant difference among the before and after treatment with DFO.

The mean and SD of erythrocyte sedimentation rate (ESR) (mm/1st hr) was analyzed. The p value was obtained 0.125 for test group which >0.05 thus, found to be insignificant. Whilst, the p value for control group found to be as 0.002 which exhibited significant difference before and after treatment.

Iron profile is the key marker for the assessment of IOL in subjects with β-Tm. Irochel manifested superlative improvement in decreasing SI level. The p value was found to be as 0.030 and 0.000 respectively. Similarly, the serum ferritin (ng/ml) before commencement of treatment with IRC at baseline showed mean and SD as 6133.98 ± 2906.506 (ng/ml) and after three months' administration serum iron (ng/ml) exhibited mean and SD as 4084.19 ± 2334.710 (ng/ml). In the same way, control group before initiation of the treatment SF (ng/ml) showed mean and SD as 5056.12 ± 2359.235 (ng/ml). After three months of treatment with control drug SF (ng/ml) manifested mean and SD as 6354.62 ± 3002.043 (ng/ml). The p value obtained as 0.000 and 0.006 for test and control groups respectively, which displayed highly significant difference for both groups. Likewise, for TIBC (ug/dl) the p value acquired as 0.038 for test group which is considered as significant and for control group as 0.635 which is found to be insignificant.

In addition to above mentioned results, this polyherbal formulation represent remarkable improvement in Lft, as the TB, SGPT, GGT and ALP levels was significantly decreased after treatment with this polyherbal formulation. Comparably, after treatment with DFO there was higher levels of lft parameters. In lipid profile, IRC has slight decreasing effect on total cholesterol, HDL and TGs but significantly lowering LDL levels as p value found to be as 0.000. It indicates that this herbal product having good effects on cardiac health by maintaining TC, HDL, LDL and TGs levels in the body. Conversely, in control group there was significant increase in TC, LDL and TGs levels, p value obtained as 0.003 for TC and 0.000 for LDL and TGs both. This shows that DFO has no effect on lipid profile and elevated SI and SF levels causes these subjects more prone to cardiac diseases.

The Urinalysis of all subjects were carried out by both conventional and traditional method. Traditional analysis of Urine was performed by using Avicenna MUST KIT. By analyzing conventional method there was no difference in quantity, specific gravity and pH of urine, the color of the urine showed that most of the subject in both groups having dark yellow, dark orange and dark brown color that indicates that IRC and DFO both eliminating excess iron from the body. Microscopic examination of urine showed that both groups having mild to moderate urinary tract infections, but the percent decrease in presence of RBCs, pus cells, crystals and bacteria were observed in test group without taking any other medications. Whereas, control group shows slight decrease in these parameters by taking proper medication. There was no complaint of renal colic, dysuria, difficulty in passing urine, incontinence etc. By analyzing traditional method, the parameters which were observed were odor, clarity, turbidity and sediments other than above mentioned parameters.

The Pearson correlation and linear regression analysis was obtained among dominant body temperament and SF levels before and after treatment of both groups. The results exhibited there was positive correlation between dominant body temperament and SF level before and after treatment with test and control groups. The transfusion dependency showed that all the subjects of both groups were getting transfusion before the start of the study between 12-14 days in which 48 (72.7 %) subjects were getting 1 unit of blood and 18 (27.3 %) were getting 2 units of blood one after another on a same day. After treating with Irochel for three months the transfusion dependency was decline and the duration was increases as shown in the table-82 that out of 66 subjects only 18 (27.3%) subjects getting transfusion between 12-14 days, 34 (51.5 %) was transfused between 15-20 days, 11 (16.7 %) having transfusion once in a month and 3 (4.5 %) are those who were getting BT between 31-35 days, and all the subjects were getting only 1 unit of blood during the course of study. This increase in transfusion duration and decrease in demand of blood are direct reflection of maintenance and improvement of RBCs count and Hb level in the body. Conversely, all the subjects of control groups were getting transfusion between 12-14 days and they were not showed any decline in transfusion dependency as well as 51 (77.3%) were transfusing by 1 unit of blood and 15 (22.7%) getting 2 units of blood before the start of the study, whereas, during and after the course of study for three months, 40 (60.6%) of subjects getting 1 unit of blood and 26 (39.4%) subjects were transfusing by 2 units of blood one after another on the same day. It clearly indicated that DFO has no effect on RBCs count and Hb level. Despite of this, due to mean increase SI and SF levels in control group more ROS formed which destroy RBCs and subsequently decreasing Hb levels.

Therefore, the demand of blood in control group was persistent and increased as it was in the beginning of study. All the subjects showed good compliance for polyherbal formulation, where as in control group 20 (30.3 %) subjects showed good compliance, and 39 (59.1 %) exhibited poor and 7 (10.6 %) compliance. The subjects in test group exhibited no adverse effect of this product, whereas all the subjects in control group having adverse effects relating to biological systems such as GIT, hepatobiliary, bone pain due to demineralization, skin itching, and swelling and hard lump at the site of injection.

Moreover, the subjects in test group were not taking any supportive medicine during the course of the study, while all the subjects in control group getting supportive medicine such as vitamin C and E, folic acid once daily as well as calcium, vitamin D, multivitamins and other medications as per need. This represent that IRC has invincible nutraceutical value, whereas DFO doesn't have this quality.

On the bases of above mentioned all results and general health of subjects in both groups exhibited that after treatment with Irochel for three months 43 (65.2%) showed marked improvement, 16 (24.2 %) showed good improvement, 7 (10.6 %) showed slight improvement and no one showed no improvement. Contrary to this, the control group showed 22 (33.3 %) slight improvement and 44 (66.7 %) exhibited no improvement.

It was estimated that in Pakistan, the annual cost of Desferal is ranging from 252,000 PKR to 756,000 PKR for one patient according to dose 20 mg/kg bw to 60 mg /kg bw. Whereas, the annual cost of Irochel estimated as 63, 691.2 PKR as per dose of 500 mg twice daily. In this study, Irochel found to be more potent and cost-effective in comparison with Desferal for the management of IOL in subjects with β-Tm. There were no adverse effects was observed during the course of study and it found to have excellent acceptability and compliance by all treated subjects as compared to Desferal. Thus, the alternate hypothesis is clarified that Irochel is a safe, potent and cost effective drug for the management of iron overload in transfusion dependent thalassemia patients.

Inclusion criteria

• The research was conducted in individuals suffering from transfusion-dependent thalassemia (TDT) and having iron overload (IOL) with the serum ferritin (SF) value greater than 1000 ng/ml of blood.
• Chronic disorders such as hepatosplenomegaly, diabetes mellitus, and bone pain are included.

Exclusion criteria

• Serious cardiac patients.
• Patients having a history of serious transfusion reactions.
• Patients having neurological disorders.
• Patients having a history of bad compliance.