​​​​Dr. Neeraj Kumar

Summary of Ph.D Dissertation: 

Development and evaluation of different formulations for heavy metal/ radiation poisoning.

• Nano DPI of Calcium-disodium EDTA

• In-situ ophthalmic gel of Calcium-disodium EDTA

• Effervescing floating tablet of Calcium - disodium EDTA.

 

Heavy metal poisoning is a widespread problem in many of the industrial nations. Exposure to these toxicants is not only confined to occupational workers but also to the general population at large. A phenomenal increase in the use of radionuclides in healthcare, food processing and energy production, has greatly increased vulnerability to radionuclide accidents, such as that seen in Fukushima (2011), Japan recently. Another major concern with respect to heavy metal toxicity that cannot be overlooked is the increased threat of a radiological device being used by rouge states or terrorist organizations that may lead to significant radio-metal contamination of the affected population, mainly through inhalation and ingestion. The challenge of removing such contaminants from the body assumes greater significance considering that, in vivo radiometal deposition even in miniscule quantities may impart significant radiation dose to body’s target organs depending upon their half-lives.

 

Although the use of chelating agents against metal toxicity was employed decades back, introduction of ethylene diamine tetra-acetic acid (EDTA) as a chelator is considered as a breakthrough in chelation therapy. Use ofEDTA and other chelating agents are the generally accepted treatment for lead and other heavy metal poisonings. Ca-Na2EDTA was selected for present studies due to its cost effectiveness, FDA approval and good chelation efficiency against various heavy metals.

 

Aim of the present study was to provide an easy and effective chelation therapy against heavy metal toxicants using Ca-Na2EDTA formulations. Route and dosage form were optimized on the bases of entry and deposition of heavy metals/ radio-isotopes during heavy metal/ radio-isotope exposure. Nano-particle based Dry powder inhaler for inhalation route, in-situ ophthalmic gel for eye and floating tablet for ingested heavy metal decorporation.The first objective of the study was achieved by developing analytical methods to determine Ca-Na2EDTA in formulations and experimental conditions. To quantify Ca-Na2EDTA various methods were developed and validated. UPLC for biological samples,

 

UV method for in-vitro studies, and radiometry methods for in-vivo samples (rabbit aqueous humor samples and scintigraphy studies). A novel UPLC method was developed and validated for Ca-Na2EDTA, reversed phase ionpairliquid chromatography was used to analyze traces of Ca-Na2EDTA in pharmaceutical formulations and natural samples. A simple less time consuming ultra-performance liquid chromatographic (RP-UPLC) method was developed, optimized and validated for determination of Ca-Na2EDTA. The method was validated for accuracy, precision, specificity, linearity, and sensitivity. The LOD and LOQ were found out to be 283.13ng/ml and 857.96 ng/ml respectively.

 

Similarly UV method was developed and validate for in-vitro experimentation using cupric nitrate solution as a coloring agent. Developed UV method was also validated for accuracy, precision, specificity, linearity, and sensitivity. The LOD and LOQ were found out to be 2.772 μg/ml and 8.42μg/ml respectively by U.V method.A novel radiolabeling method was optimized using stannous salt as a reducing agent and 99m- technetium pertechnetate as radioisotopes for non–invasive, in-vivo estimation of Ca-Na2EDTA. The optimized protocol for radiolabeling of Ca-Na2EDTA showed more than 94.40 ± 0.45% radiolabeling efficiency at an incubation time of 30min with 4.52 ± 0.25 % R/H 99mTc and1.05%, free 99mTc. In-vitroSaline and serum stability studies confirmed that99mTc-Ca-Na2EDTA was stable in saline and serum with more than 90.25±1.05% and 92.45 ±2.25% radiolabeling efficiencyrespectivelyat the end of 24hrs. The radiolabeled complex was found to be stable and there, was no significant breaking or leaching of technetium from radiolabeled drug complex.

 

Preformulation studies and drug incompatability studies showed that there was no chemical interaction or physical between and the polymers/ excipients used and drug (ca-Na2EDTA). The first objective of formulation development was to develop and characterize nano-edetate calcium disodium (Ca-Na2EDTA) dry powder inhaler (DPI) and assess its in-vitro and in-vivo deposition by pharmacoscintigraphy techniques. Factors influencing nanoparticle formation including concentration of drug, polymer solution and stirring rate were also determined. Optimized formulation was characterized with the help of SEM, TEM and Malvern Zetasizer studies. Any change in physical characteristics after nanosizing was determined by FT-IR, XRD and DSC studies. Anderson cascade impacter (ACI) showed that:

 

Nano Ca-Na2EDTA exhibited significantly higher respirable fraction of 67.35 ± 2.27% and 66.40 ± 2.87% by scintigraphic and spectroscopic analysis respectively, as compared to 10.08 ± 1.17% and 9.36 ± 1.02% respectively for micronized form. Optimized formulation was evaluated for its safety and efficacy against various heavy metal (lead, cadmium, cesium, arsenic etc.) exposures to animal models. Ventilation lung scintigraphy done in 12 volunteers showed significant increase in drug delivery till, alveolar region was administered with nano Ca-Na2EDTA via inhalation. An in-vitro, in-vivo correlation was also established on the basis of respirable fraction achieved by ACI experiments and scintigraphy studies done on human volunteers. The stability studies of optimized nano-Ca-Na2EDTA DPI were performed and found to be stable for six months on the basis of particle size and 5.34 year as per ICH guidelines for powder formulation based drug content, hence developed formulation may have a role in neutralizing heavy metal toxicity through inhalation route, including radiometal contamination.Our second aim of product development was to developin-situ ophthalmic formulation to prevent the eyes from heavy metal exposure. Acute and chronic exposure of heavy metal to eyes may cause burning, brown stains, discoloration of the cornea and disturbances of vision. A major problem in ocular therapeutics is the attainment of optimal chelating agent concentration at the site of action, which is comprised mainly due to pre-corneal loss resulting in only a small fraction of the drug being available at the ocular site. The effective dose administered may be altered by increasing the retention time of chelating agent in the eye. Hence our objective was to formulate a chitosan based long acting, mucoadhesive in-situ gel of Ca-Na2EDTAto provide localized and prolong release of drug against heavy metal exposure.

 

Gamma scintigraphy and other nuclear medicine techniques were employed in the development of this ocular formulation. The radiolabeled complex of Ca-Na2EDTA was added to the optimized in-situ ophthalmic gel. In-situ gel was prepared with 1.0% Chitosan and other standard excipients. Drug uniformity, in-situ gelation and mucoadhesive properties of the novel formulation were investigated in artificial tear fluid. Gamma scintigraphy was performed in six healthy human volunteers, to assess and compare the retention of the optimized in-situ ophthalmic gel and conventional eye drop. Dissolution profile of optimized in-situ ophthalmic gel showed 45% release of Ca- Na2EDTA in artificial tear fluid at 6 hours, while in case of normal ophthalmic solution100% drug was released within 4 hrs. Pre-corneal retention data in New Zealand white rabbits showed that, the retention of in-situ gel formulation was nearly double at 4 hours in comparison to the conventional eye drops. Ocular toxicity studies Draize test and HETCAM test on animal models confirm its safety and absence of irritancy on the ocular surface. Gamma scintigraphy studies in healthy human volunteers were confirmed that, the retention of optimized in-situ gel at six hours were three times more retention in comparison to conventional eye drop. Ex-vivo–in-vivo correlation was also studied for mucoadhesion or retention of Ca-Na2EDTA on the ocular surface. Accelerated stability studies as per ICH guide line show that optimized formulation is stable for up to 19 monthsat 5ºC.

 

Our last objective of the investigation was focused on the optimization of floating tablets containing Calcium disodium edetate (Ca-Na2EDTA) a model drug for chelation therapy against heavy metal exposure. Formulations were optimized for filler (sodium chloride) and Hydroxyl propyl methyl cellulose (HPMC K30). Sodium bicarbonate was used as a gas generating agent. A formulating floating drug delivery system (FDDS) with content of sodium chloride and HPMC K-30 were selected as independent variables. Ca-Na2EDTA matrix tablets were prepared by direct compression technique. Dissolution profiles were subjected to various kinetic modeling and drug release equations. It was found that drug release occurred, in proration to transport, diffusion mechanism and erosion, followed by in-vitro floating characterization statistically. Effect of hardness on floating properties and dissolution medium on drug release from tablet were examined. Floating properties such as FLT, TFT, and Swelling index were also determined. A novel method was opted for noninvasive in vivo estimation. Optimized formulation was shown to have floating efficiency till the end of four hours in animal and six hours in human subjects. From the results, it was theoretically calculated that the drug concentration in gastric milieu shall be enough for pharmacological action for 24 hours indicating once a day dose. Hydrophilic matrix floating tablets of Ca-Na2EDTA were developed to increase the gastric residence time, which leads to increased bioavailability by giving sufficient time to release the drug in the GI tract. Optimized formulation was subjected to curve fitting analysis, and its followed Korsmeyer and Peppas model of kinetics and the mechanism was found to be non Fickian/anomalous. Efficacy studies of optimized floating tablet show less accumulation in the organ or fast excretion of heavy metal/ radio-isotope from. Significant fall in uptake of technetium pertechnetate by salivary gland and thyroid was seen in presence of optimized tablet, which indicates the effectiveness of prepared formulation against technetium pertechnetate exposure. The stability according to ICH guideline indicates that the optimized formulation was stable up to 5.2 years at room temperature (25ºC).

 

Overall, this research work highlighted the usefulness of inhalation, ocular and oral route of delivery for Ca-Na2EDTA formulation DPI, in situ gel and floating tablet respectively for prevention from heavy metal exposure.Finally, our findings suggest that, Ca-Na2EDTA formulations can be used to neutralize/chelate internalized heavy metal ions at the portal of entry, so that they can be excreted without absorption. The novel formulations can probably be a medium for fast and sustained systemic absorption of the drug and may be developed as an alternative to intravenous administration, especially in an emergency or field conditions. The formulation has the potential to be used as a preventive or therapeutic approach for members of rescue teams, as well as victims in a radioactive fallout zone and people working in mining and other such industries having a high probability of metal ion exposure.

 

Summary of M.Pharm. Dissertation: 

Development and evaluation of cyclodextrin complexation of atenolol to improve its dissolution rate and solubility.

 

Atenolol is an establish β- blocker, used in the treatment of hypertension, and offers the convenience of once daily administration. Although it is slightly soluble in water and its oral bioavailability is about 46% to 60%. Consequently, there is considerable interest in developing new formulations with better aqueous solubility and good bioavailability. Therefore, in the M.Pharm dissertation a novel physico-chemical approach has been used to combine the established β-blocker, atenolol with a cyclic macromolecule i.e. β-cyclodextrin or hydroxypropyl-β-cyclodextrin to improve the aqueous solubility of the drug, thus enhancing its dissolution rate, thereby showing a faster onset of action and Following conclusions can be drawn from the results obtained

Atenolol showed an AL type phase solubility curve with increasing concentration of β-cyclodextrin and hydroxypropyl-β-cyclodextrin suggesting the formation of 1:1 complex in solution state but we goes further and also prepared 1:2 complex.

In solid state the complexes of atenolol with β-cyclodextrin and Hydroxypropyl-β-cyclodextrin were prepared by three methods viz., Grinding, kneading and freeze drying technique in two molar ratio i.e., 1:1 arid 1:2. These complexes were characterized using Differential scanning calorimetry (DSC), Fourier transform infra red spectroscopy (FT-IR), Scanning electron microscopy (SEM) and Nuclear magnetic resonance (NMR) methods, it was concluded from the results of these characterization methods that maximum complex formation was achieved by freeze drying technique in a 1:2 (drug: cyclodextrin) molar ratio with both β-cyclodextrin and hydroxypropyl-β-cyclodextrin as complexing agents. Partial complex formation was achieved by grinding methods.

The complexes of atenolol with β-cyclodextrin and hydroxypropyl-β-cyclodextrin as complexing agent in the molar ratio of 1:1 and 1:2 prepared by different techniques were subjected to dissolution studies. The dissolution data indicated that all the complexes showed an increased rate of dissolution and dissolution was more in acidic medium, which may be due to ionization of the drug molecule as it is a weakly alkaline in nature. Technique used from making the complexes was found to have an influence on the dissolution rate. The complexes prepared by freeze drying method were found to yield a complex of higher rate of dissolution over kneading and the later over the grinding method. Supporting an earlier observation that maximum complex formation freeze drying technique in a molar ratio of 1:2 Solubility profile of complexes of atenolol prepared using β-cyclodextrin and hydroxypropyl-β-cyclodextrin as complexing agent in a molar ratio of 1:2 by freeze drying method in pH 1.2, pH 7.4 and distilled water indicated that the acid solubility of atenolol was enhanced considerably by formation of an inclusion complex with β-cyclodextrin and hydroxypropyl-β-cyclodextrin. Final optimized formulation contained atenolol-β-cyclodextrin and hydroxypropyl-β-cyclodextrin inclusion complex (FD, 1:2) equivalent to 50 mg of atenolol, starch, tale, magnesium stearate and micro crystalline cellulose. Comparative release studies also showed significant increased dissolution of the drug as compared to the marketed formulation “Aten-50” (Zydus health care).

The result of the release rate data indicated that the coefficient of variation was lower for first order rate constant than the corresponding zero order values thereby suggesting that the drug release followed first order kinetics. The tablets containing complexed drug were subjected to accelerated stability studies to ascertain the chemical and physical stability of the formulation. The tablets containing atenolol-β-cyclodextrin (FD, 1:2) inclusion complex and atenolol-hydroxypropyl-β-cyclodextrin (FD, 1:2) inclusion complex were kept 30 ± 2°C and 65% ± 5% RH. No significant changes in the properties like weight, hardness, thickness friability and disintegration time of the formulation was observed. The rate constant for the drug decomposition was 1.626x10-4 day-1 and 2.084x10-4 day-1 for β-cyclodextrin and hydroxypropyl-β-cyclodextrin complex respectively. On the basis of in vitro studies, pharmacokinetic studies were done on six albino wister rats for β-cyclodextrin and hydroxypropyl-β-cyclodextrin complexes of atenolol and atenolol alone after the approval from the institutional ethical committee. The bioavailability result shows that developed formulation of atenolol-hydroxypropyl-β-cyclodextrin complex faster absorption than atenolol-β-cyclodextrin and conventional atenolol tablet. Furthermore, higher the significant value of Cmax, Tmax and AUC0-15 were obtained.

It can be concluded that the developed formulated containing atenolol-β-cyclodextrin freeze dried complex (1:2) will have significant advantages in terms of a faster onset of action as compared to the presently available conventional atenolol tablet in the Indian Pharmaceutical market.