Repurposing and Drug Development

### **Chapter 1**

## Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting

*Sunil T. Galatage, Arehalli S. Manjappa, Raghwendra R. Waghmode, Swapnil S. Harale, Rushikesh B. Katkar, Sujit A. Desai, Swapnil S. Chopade, Kranti S. Bille, Rubina U. Watangi, Shweta N. Kalebere, Aditya S. Hebalkar, Shradhey V. Dhobale, Harshada N. Gunjate, Poonam R. Dhenge, Purva S. Ikke, Saheblal A. Shaikh, Rutuja J. Patil, Sakshi B. Shinde, Rutuja V. Khatavakar, Anuja B. Patil, Prajakta N. Khatavakar, Sourabh S. Hegaje and Suresh G. Killedar*

### **Abstract**

Cancer is the leading cause of death, and incidences are increasing significantly and patients suffering from it desperately need a complete cure from it. The science of using an already-invented drug that has been approved by the FDA for a new application is known as "drug repurposing." Currently, scientists are drawn to drug repositioning science in order to investigate existing drugs for newer therapeutic uses and cancer treatment. Because of their unique ability to target cancer cells, recently repurposed drugs and the liposomal approach are effective in the treatment of cancer. Liposomes are nanovesicles that are drastically flexible, rapidly penetrate deeper layers of cells, and enhance intracellular uptake. More importantly, liposomes are biocompatible, biodegradable; entrap both hydrophobic and hydrophilic drugs. This chapter summarizes various approaches to drug repurposing, as well as drug repurposing methods, advantages and limitations of drug repurposing, and a liposomal approach to using repurposed drugs in cancer targeting. This chapter also summarizes liposomal structure, drug loading, and the mechanism of liposomes in targeted cancer treatment. The lipid-based liposomal approach is emerging as a powerful technique for improving drug solubility, bioavailability, reducing side effects, and improving the therapeutic efficacy of repurposed drugs for cancer treatment.

**Keywords:** cancer, drug repurposing, liposomes, drug targeting, enhanced permeability effect, Etc.

#### **1. Introduction**

During the treatment of cancer in a patient, it is necessary to follow certain principles, such as diagnosing the disease at an early stage, making efforts for its prevention, and completing the eradication of malignant cells. Whereas three modes of treatment are available to treat cancer, including surgery, chemotherapy (also called pharmacotherapy), and radiation therapy [1], Radiation therapy is nothing but the eradication of malignant cells by means of radiation.  In pharmacotherapy, various chemical entities are used to kill and disorganize an uncontrolled cell growth programme in a body [2]. Cancer does not only affect humans; it can also harm wildlife and other life forms. Tumour cells might break out from the initial bulk and begin the unregulated growth cycle all over again. The phenomenon of tumour cells leaving one location and developing cells that travel and proliferate over other body parts is known as metastasis. It was estimated by the WHO that cancer is the foremost cause of death in the world, and in the year 2018, it is expected that 9.6 million people died as a result of it. It is categorized by the development of osteocytes, bone lesions, anaemia, skeletal destruction, renal failure, and hypocalcaemia. It is a bone marrow cancer that affects both the marrow and the bones. It also affects different body locations; hence it is called multiple myeloma. Bone marrow-originating myeloid cells such as myeloid resultant suppressor cells, macrophages, myeloid dendritic cells, monocytes, osteoclasts, and lymphocytes are drafted to tumours, which can either increase antitumour immune function or encourage tumour growth [3]. Recent research indicates that anti-resorptive targeted therapies can have an impact on tumour-associated myeloid cells through direct or indirect pathways, indicating that anti-resorptives have an osteoclastin-dependent mechanism of action. As the cancer progresses, the signs and symptoms change dramatically. Symptoms can be entirely dissimilar from

**Figure 1.** *Schematic representation of cancer treatments.*

#### *Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting DOI: http://dx.doi.org/10.5772/intechopen.110105*

patient to patient. A few symptoms are most common, like fatigue, bone problems, kidney problems, and low blood counts. Some symptoms become severe; these are: osteoporosis, osteocytes, bone lesions, and skeletal destruction [4].

Targeted cancer treatment, in which the selected cancer cells are eliminated and healthy cells are left alone, is becoming extremely prevalent [5]. The arrival of nanostructures has resulted in the development of advanced materials and channels for cancer treatment targeting [6, 7]. Nanotechnology has opened up new possibilities for biological and biomedical applications, such as improving the targeted administration of anticancer drugs. Nanotechnology has a lot of benefits for treating cancer. In reality, tumour blood vessels are severely disordered, with an ineffective lymph capillary network and loose endothelial cells in comparison with normal tissue. Because of their improved permeability and high body retention, nanoparticles such as liposomes can be transported preferentially to the tumour location [8].

#### **2. Drug repurposing in cancer treatment**

This technique helps to destroy localized cancer cells. In pharmacotherapy, various chemical entities are used to kill and disorganize an uncontrolled cell growth programme in a body [8]. Chemotherapeutic agents pose the greatest risk to cancer patients because of the drugs' lethal effects and the possibility of cell damage to their bone marrow, which makes them more susceptible to other diseases. If we have not targeted the malignant cell only, then these chemotherapeutic agents also kill the normal cells in the same host, which creates more damage to the patient's body and its biological structure [9]. Different strategies of drug repurposing are denoted in **Figure 2**.

Extensive research is carried out to investigate and develop new therapeutic entities in the oncology field and drug research to achieve the maximum therapeutic effect with greater patient comfort and a lower toxicity profile. On the other hand it raises the cost of treatment for a patient, making it necessary to exert maximum effort to achieve desired treatment goals at the lowest possible cost of treatment. Drug repurposing is the most effective way to reduce the effort required to develop new drug molecules while also lowering treatment costs. The science of using an alreadyinvented drug that has been approved by the FDA for a newer application is known as drug purposing. Now a day drug repositioning science attracts the more researchers to investigate existing drugs for its newer therapeutic use. The drug being repositioned is already being used to treat diseases in humans, giving the manufacturer access to knowledge regarding its safety, effectiveness, therapeutic, and toxicity profiles. To reposition medications that are already approved for human use efficiently, rigorous selection is required, followed by a detailed demonstration of the treatment's usefulness in new biological contexts. The following methods are used to select drug candidates for drug repositioning [10].

#### **2.1 Repositioning based on therapeutic activity**

This method involves testing the therapeutic effectiveness of a drug by performing an in vitro or in vivo study. For the finding of therapeutic entities, comprehensive public library data is used. The therapeutic agent is examined for its protein targets and cellular targets while searching for a suitable drug candidate through activitybased repurposing of the drug (**Figure 3**) [11, 12].

*Drug Repurposing - Advances, Scopes and Opportunities in Drug Discovery*

**Figure 2.** *Strategies to repurpose the drug candidates.*

#### **2.2 Drug repositioning through literature evidence**

This drug repurposing method involves the selection of a drug based on its published therapeutic evidence. The literature study of such drug databases available on PubMed, ClinicalTrail.gov, Drug Quest, MEDLINE, and other available databases is screened, and the required potential molecules are identified by applying such data in a dynamic way (**Figure 4**) [13].

#### **2.3 In silico method: In this method**

Various bioinformatics tools and a public database are used to understand drug protein interactions. For this method, extensive genomic studies and structural evaluations of various proteins are carried out. Most pharmaceutical drug manufacturers adopt the in silico method for drug repurposing. To identify the protein interaction and possible drug candidate, researchers use the science of

*Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting DOI: http://dx.doi.org/10.5772/intechopen.110105*

#### **Figure 3.**

*Diagrammatic representation of Drug Repositioning (Case A).*

artificial intelligence, neural network techniques, and other bioinformation tools (**Figure 5**) [11, 14].

#### **2.4 Advantages of drug repositioning**

#### **Figure 4.**

*Diagrammatic representation of Drug Repositioning (Case B).*

#### **Figure 5.**

*Diagrammatic representation of drug repurposing approaches.*

#### **2.5 Limitations of drug repositioning**

1.More money is spent on licensing requirements for drug repositioning to investigate new therapeutic applications of already approved drugs [16].

*Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting DOI: http://dx.doi.org/10.5772/intechopen.110105*

#### **3. Repurposed drug targeting**

Nanoparticulate drugs (including liposomal drugs) are generally developed for therapeutics developed against cancer to avoid non-specific distribution to healthy cells and tissues that generally causes lethal side effects. Due of the permeable tumour vasculature and decreased lymphatic outflow, the nanomedicines (including liposomes) with prolonged circulation durations preferentially penetrate tumour tissue. This phenomenon is referred to as the "increased permeability and retention (EPR) effect Drug repurposing is a technique for finding new applications for medicines that go beyond their original medicinal indications. On the other hand, drug repurposing is the most effective way to reduce the coast, time and effort required to develop new drug molecules while also lowering treatment costs. The development of nanomedicines (including liposomes) for these repurposed drugs could provide many benefits; increased kinetic, dynamic and biopharmaceutical characteristics, and avoids their primary indications via targeting them to tumour through EPR effect. Furthermore, these nanomedicines could be easily surface modified to passively and actively target tumour cells and cellular components. Therefore, nanomedicines composed of repurposed drug could be preferred over plain drugs or their conventional generic dosage forms currently available in the market.

#### **4. Liposomes (LPs)**

An LPs is a spherical vesicle made up of one or more lipid vesicles that is increasingly being used to deliver therapeutic entities. Liposomes are one of several promising drug delivery systems that represent an efficient approach for delivering active compounds to the target site, and various formulations are currently in clinical use. LPs technology has been developed from typical vesicles to second generation liposomes, which are created by changing the lipid composition, length, and charge of vesicle liposomes and can be employed on a regular basis as what the body does to drugs and what drugs do to the body can be controlled. The LPs provide selective passive targeting to tumour tissues, and the encapsulation method contributes to increased effectiveness, therapeutic index, and stability. Reduced polymer toxicity, site evading effect, helping to enhance the pharmacokinetics of the therapeutic moiety, and suppleness to bind ligands at specific sites to achieve active targeting, to name a few advantages [6]. Liposomes were studied for the first time at the Babraham Institute in Cambridge by two scientists who used an electron microscope to examine phospholipids in dry form with negative staining. These two scientists are Dr. Alec Bengham and R. W. Horne, who identified the liposome assembly in 1961 and

published their study in 1964. Liposome is the name given to a compound made up of lipids (lipo) and body (soma). So that liposome is nothing but a lipid body in which medicine is to be delivered [17]. Many anticancer medications have been designed to terminate tumour cells that are developing uncontrollably because they divide more quickly than normal cells. However, in this instance, ordinary cells grow fast, and a chemotherapeutic agent might harm such cells, resulting in chemotherapy side effects.  Some anticancer medications have the capacity to harm cells in key organs, including the heart, kidney, bladder, lungs, and neurological system. Medication diffusion in solid tumours is hampered by a variety of vascular supply and cellular gravity within tumour cells, particularly in tumour regions. Drug delivery design develops in such a way to ensure that macromolecular medicines are released slowly via the tumour. Advanced technologies are designed to improve tumour tissue permeability. These are triggered by the maladaptive nature of tumorigenesis, which is characterized by structural and physiological abnormalities that lead to hyperpermiability. The medicinal compounds have a larger molecular structure, which leads to the build-up of high-molecular-weight molecules with limited distribution volumes and the ability to circulate for lengthy periods of time through aberrant arteries and concentrate in tumours [18–20].

#### **4.1 Structural features of liposome**

LPs are small cell membrane sacs. Because these LPs can be packed with medications, they are a viable option for treating illnesses and cancer. Liposome membranes are composed of phospholipids with a head and a tail group. Because of the length of the hydrocarbon chain, the head part is hydrophilic and the tail part is hydrophobic. Phospholipids are naturally occurring two-layer stable membranes. Because head groups are hydrophilic, they are fascinated by water and arrange in such a way to form a surface-like assembly away from it when there is water present. In a cell with outside water, while the other is fascinated by water within the cell. They resemble tiny spheres that are smaller than a normal cell's size, whether as bilayers or monolayers. Liposomes are created as bilayers, while micelles are formed as monolayers. Phospholipids form the mainstream of the lipids in the plasma membrane; these phospholipids are phosphatidyl ethanolamine and phosphatidylcholine [19–21]. Liposomes have the capability to penetrate cancer in its natural state. Endothelium cells are contained by tight junctions in the endothelial walls of all healthy human blood vessels. These tight connections prevent large blood particles from spilling out of the vessel. In the event of a tumour vessel, this type of arrangement does not exist, making it symptomatically porous. This capacity is known as the enhanced permeability and retention effect (EPR) (**Figure 6**). Liposomes with a diameter of less than 400 nm can enter tumours quickly from the bloodstream, but they are maintained in the bloodstream by the endothelium wall in healthy tissue [23–25].

#### **4.2 Drug loading mechanism into liposome**

The drug features and the lipids determine how pharmaceuticals are loaded into liposomes. Hydrophilic medications are confined in the inner watery compartment, *Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting DOI: http://dx.doi.org/10.5772/intechopen.110105*

#### **Figure 6.**

*Diagrammatic representation of EPR effect [22].*

#### **Figure 7.**

*Active loading of drug into liposome [26].*

while hydrophobic pharmaceuticals can screen within the lipid hydrocarbon area. In practice, few medicines may segregate into hydrocarbon or aqueous compartments; for example, Amphotericin-B (Amph-B) binds to hydrophobic lipid membranes. The resulting lipid configuration influences Amph-B parcelling and its rate of exchange outside of the liposome envelope (**Figure 7**). Incorporating a negatively charged lipid improves the stability of the membrane's connection [27, 28].

According to the trans-membrane pH gradient, weak bases can concentrate in liposomes. Liposome formation is dependent on two critical steps: the formation of a pH gradient with a lower intra-liposomal pH and the subsequent loading of the drug. Gradient generation of a trans-membrane proton can be done in a variety of ways. Liposomes are made in citrate buffer, and then transferred to a pH 7.5 buffer by an exogenous buffer exchange. Ionophores, on the other hand, can be employed with action gradients. Ultimately, liposomes developed in the presence of significant amounts of ammonium sulphate (**Figure 8**). The withdrawal of salt solution causes the creation of a pH gradient, which is also accountable for the drug entrapment mechanism [22, 30, 31].

**Figure 8.** *Passive loading of drug into liposome [29].*

#### **5. Drug targeting**

Much of the effort in liposome research has been focused on tumour targeting. Liposomes in circulation extravagate through the 'leaky' tumour vasculature; alternatively, attachment of specific antibodies or other proteins to the liposome surface may cause specific targeting. However, the increased clinical efficacy of such targeting in human patients has not been easy to prove. Most of a liposomal drug given intravenously is taken up by phagocytosis into the reticulo-endothelial system, which is extremely efficient at trapping particulate matter circulating intravenously (**Figure 9**). The reticulo-endothelial system may be circumvented by several different methods, such as saturation with large doses of liposome particles or selective macrophage inactivation by pharmacological means However, such a strategy could theoretically further compromise the immune system of cancer patients [33, 34].

Liposomes as a Drug Depot Many drugs are most effective when they are delivered over extended periods of time. For example, agents specific for the division phase of the cell cycle kill cancer cells only when they are dividing. However, even for the most rapidly growing tumours, only a small fraction of the cells are dividing during the drug's residence time. Therefore, depot formulations are needed to maintain therapeutic concentrations for prolonged periods. In contrast to biodegradable polymers or chemical modifications of the standard drug, liposomes and other lipid-based formulations have the advantage of not creating a new chemical entity, and the need for extensive toxicological studies is largely avoided. This is especially the case for the more efficient lipid-based drug delivery systems, where the amount of lipid used is small relative to the amount of drug delivered (**Figure 9**). Even if

*Role of Drug Repurposing in Cancer Treatment and Liposomal Approach of Drug Targeting DOI: http://dx.doi.org/10.5772/intechopen.110105*

**Figure 9.**

*Liposomal drug delivery to treat cancer [32].*

efficacy and toxicity remain unchanged, the convenience and improved patient compliance of fewer painful injections may be sufficient for those drugs that require frequent multiple injections or continuous infusions [35–37].

#### **6. Conclusions**

Nowadays drug repurposing and drug targeting through nanoparticulate drug delivery gain significant attention for delivering various APIs in treatment of cancer through oral and topical route successfully. Loading the repurposing drug in to liposomes escalate therapeutic efficacy and residences toxic effects along with patients compliance. Nanomedicines could be easily surface modified to passively and actively target tumour cells and cellular components. Therefore, nanomedicines composed of repurposed drug could be preferred over plain drugs or their conventional generic dosage forms currently available in the market. The advantages of various methodologies and strategies for drug targeting are outlined in the current chapter, along with information on liposomal drug targeting, liposomal structure, mechanism of liposomal drug loading, and liposomal drug targeting. Drug repurposing and liposomal drug targeting are potent methods for enhancing solubility and bioavailability, minimizing side effects, and developing innovative drug delivery systems to increase the therapeutic effectiveness of drug repurposing to treat cancer.

#### **Acknowledgements**

The authors are thankful to Department of Pharmaceutics Sant Gajanan Maharaj College of Pharmacy Mahagaon and Trustees of Sant Gajanan Maharaj College of Pharmacy Mahagaon for providing required guidance and support for completion of this work.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

Special thanks to **Supriya S. Galatage** and **Shivtej S. Galatage** for their continuous support throughout the work.

#### **Abbreviations**

### **Author details**

Sunil T. Galatage1 \*, Arehalli S. Manjappa2,3 , Raghwendra R. Waghmode4 , Swapnil S. Harale1 , Rushikesh B. Katkar<sup>5</sup> , Sujit A. Desai6 , Swapnil S. Chopade2 , Kranti S. Bille1 , Rubina U. Watangi1 , Shweta N. Kalebere7 , Aditya S. Hebalkar8 , Shradhey V. Dhobale9 , Harshada N. Gunjate1 , Poonam R. Dhenge1 , Purva S. Ikke1 , Saheblal A. Shaikh1 , Rutuja J. Patil1 , Sakshi B. Shinde1 , Rutuja V. Khatavakar1 , Anuja B. Patil1 , Prajakta N. Khatavakar1 , Sourabh S. Hegaje2 and Suresh G. Killedar1

1 Sant Gajanan Maharaj College of Pharmacy Mahagaon, Maharashtra, India

2 Tatyasaheb Kore College of Pharmacy, Kolhapur, Maharashtra, India

3 Vasantidevi Patil Institute of Pharmacy, Kolhapur, Maharashtra, India

4 Krishna Institute of Pharmacy, Krishna Vishwa Vidyapeeth, Karad, Maharashtra, India

5 Vijayrao Naik College of Pharmacy, Shirval Kankavali, Maharashtra, India

6 Annasaheb Dange College of D Pharmacy, Ashta, Walawa, Maharashtra, India

7 Genesis Institute of Pharmacy, Radhanagari, Maharashtra, India

8 IQVIA Biotech, Thane, Maharashtra, India

9 National Institute of Pharmaceutical Education and Research (NIPER), Ahmadabad, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 2**

## A Computational Approach for Identifying Experimental or Approved Drugs That Can Be Repurposed for the Treatment of Type-2 Diabetes

*Gemma Topaz, Dongjun Yoo, Richard Anderson and Kimberly Stieglitz*

#### **Abstract**

Approved and experimental drugs can be utilized for new indications as illustrated in the case study presented herein. In this case study, allopurinol (trade name Zyloprim and Aloprim) which is currently utilized for gout, was retrieved from the Drug Bank and evaluated for a new indication. Utilizing a catechin derivative as a scaffold, a derivative was designed incorporating allopurinol. This novel molecule was predicted to act as an allosteric inhibitor of fructose 1,6-bisphosphatase (FBPase), a control point for entry into the biochemical pathway gluconeogenesis. The predicted inhibition was validated with a colorimetric assay. Potential toxicity was assessed using a HepG2 MTT assay. As an inhibitor of this enzyme, the novel molecule proved to be both potent and non-toxic in cell-based assays. Once optimized and tested *in vivo*, the novel molecule may be potentially used as a therapeutic agent for type-2 diabetes mellitus inhibiting FBPase. This action prevents the *de novo* synthesis of glucose and potentially contributes to lowering blood glucose levels for patient populations that are genetically prone to chronic high blood glucose leading to insulin resistance. The computational approach to the design of the novel potential lead compound is discussed in detail and validation data presented.

**Keywords:** allosteric regulation, enzyme inhibition, structure-based drug discovery, geometric docking, molecular dynamics

#### **1. Introduction**

Type-2 diabetes mellitus is recognized as a global epidemic by the World Health Organization with an estimated 500 million affected worldwide. The Center for Disease Control (CDC) estimates that 30 million in the US are affected. Most of this is caused by lifestyle and improper diet, a direct result of the mismanagement of agricultural

and food distribution systems. Certain populations, such as Native Americans and Polynesians, appear to be predisposed to type-2 diabetes. This might be a result of genetic adaptations brought on by chronic food scarcity [1]. In both cases, type-2 diabetes is characterized by impaired insulin sensitivity or availability and increased endogenous glucose production (EGP). The primary source of EGP is gluconeogenesis in the liver, which is typically three-fold greater in type-2 diabetics. It is widely recognized that when gluconeogenesis is curtailed, this provides a valuable therapy for type-2 diabetes [2]. Analysis of the gluconeogenesis pathway has suggested that the best target for inhibition is the fructose 1,6-bisphosphatase (FBPase) enzyme. Yet after many years of work, there is no FBPase inhibitor that has reached the market. The FBPase molecule is a homotetramer, each monomer with an active site and two allosteric binding sites. The active site contains highly conserved amino acids and so is not a suitable drug target. The AMP binding site is an allosteric site, which has been known and targeted for many years for drug development [1–4]. A novel allosteric site was discovered at the tetramer interface by Pfizer [5]. Although a potent inhibitory site, the volume of the pocket is made up of contributions from the four monomers and is highly mobile, so there is difficulty in predicting the actual binding modes of selected molecules. In addition, a 2:1 molar ratio of drug to protein is often required for effective inhibition, which makes drug studies challenging. This study focuses on the FBPase protein target AMP binding site relying heavily on similarity searches with the natural inhibitor AMP.

Herein is a case study focused on the early stages of the development of an FBPase inhibitor (lead compound) that targets the adenosine monophosphate (AMP) allosteric binding site is presented. This has led to the computational identification and assay verification of several effective compounds in the Drug Bank database. The compounds are currently approved drugs. If any of these drugs can be fully verified to be effective for a new application, this could lead to a breakthrough treatment for type-2 diabetes mellitus.

#### **2. Strategies and methods**

As shown in **Figure 1**, the repurposing workflow is a linear process. The process begins with literature searches then similarity searches of known inhibitors with searches of the Drug Bank database [6]. Once compounds are identified, molecular docking is done using AutoDock Vina for both fixed and flexible (side chain) docking protocols [7]. Molecular dynamics follows for accurate physiological check on the binding score of the ligand/protein complex [8] followed by laboratory validation [9]. In the hit-to-lead optimization stage, there was a reiterative process of modifying the hit repurposed compound (treated as a scaffolding compound) with substituted functional groups. This process refines the molecule eliminating derivatives issues such as low solubility, cellular toxicity, and PAINS (pan assay interference screening) before proceeding to optimize the lead compound for *in vivo* animal studies and finally clinical trials.

Key research performed already published that supports this strategy includes:

*A Computational Approach for Identifying Experimental or Approved Drugs That Can Be... DOI: http://dx.doi.org/10.5772/intechopen.110812*

**Figure 1.** *A virtual drug development platform (VDDP): a schematic representation of repurposing workflow.*

Research conducted by symmetric computing supports this case study includes the development of the virtual drug discovery platform (VDDP), which performs high throughput virtual screening by combining molecular docking with molecular dynamics. Screening of millions of small molecules from the ZINC15 database was done using VDDP, in order to identify high-scoring binders to the AMP binding site on fructose 1,6-biphosphatase. Identification of high-scoring binders that are Phase I approved deployed or experimental drugs that were also included in the Drug Bank database were cross-referenced.

### **3. Flexible docking and MD protocols**

#### **3.1 Preparation of small molecules and protein target for MD and geometric docking**

Once repurposing molecules for the target were identified through literature searches to be the appropriate for alternative medicinal activities, the PubChem and Drug Bank database was searched these compounds and derivatives. Following identification, the total polar surface area (TPSA) was calculated with SwissADME [16]. Small molecules with druggable TPSA of approximately 90–120 A^2 [16–18] were fed into the SwissADME program, followed by the pkCSM program to screen for central ADMET properties to test for predicted toxicity using graph-based patterns [17]. The protein target binding site the FBPase AMP binding site was then selected based on geometric attributes and the docking score of the Drug Bank scaffolds. More specifically, the geometric attributes of the protein binding site coordinates were selected according to the ratio of molecular surface volume of the cavity over the total molecular surface volume of the protein. Ratios below 0.15 A^3 were not considered druggable target binding sites [19]. In addition, the docking score of the ligand to the protein was considered reasonable when less than ~50 micromolar. Once the protein target binding site was correctly identified, docking was performed with all of the selected drugs for repurposing (n = ~525) with both flexible and rigid residues. After initial docking (rigid) followed by flexible docking, a molecular dynamics protocol in NAMD was executed to check contacts and delta G of binding scores. Since convergence with flexible docking scores and MD scores occurred, flexible docking was used for drugs that were then altered with specific functional groups for further protein/ drug docking studies.

The initial workflow for ligand and FBPase protein target preparation is shown in **Figure 2**. Two protein target PDBs that were chosen of FBPase were 1KZ8 [5] and 1FTA [2] to capture the AMP binding site and the dimer and tetramer interfaces in different starting conformations.

A guide of the initial identification of potentially druggable binding sites for a given target and previously approved drugs appropriate for drug repurposing studies.

Initially, the RCSB was searched for human and pig kidney structures of FBPase as these species have >85% identity and ~95% homology of amino acid sequences. Although the location of the AMP binding site of FBPase is known, the conformation of the amino acid side chains that make up the "gateway" and interior of the binding site change position when substrate or product is bound in the active site. The enzyme has two canonical states the T state known as inactive state and the R state the active state. The enzyme rotates around the dimer interface of the tetramer. Since the AMP binding site is on exterior side of the homotetramer on each monomer individual amino acid side chains shift position during this rotation.

#### **Figure 2.** *Ligand/protein target computational platform workflow chart.*

*A Computational Approach for Identifying Experimental or Approved Drugs That Can Be... DOI: http://dx.doi.org/10.5772/intechopen.110812*

The repurposed drugs were selected based on similarity searches to natural allosteric inhibitor in both PubChem and Drug Bank databases. Once the protein target center of position coordinates are selected and the repurposed drugs identified, static docking followed by flexible docking was done with the FBPase-repurposed drug complex. After molecular docking, the coordinates were run in MD protocols to check overall energy of the system and calculate the binding energy of the protein-drug complex. From the output from the VDDP, repurposed drugs from the Drug Bank were chosen for laboratory validation, derivatized as needed to enhance binding and the process of optimization towards lead compounds was done. In addition, the processed target protein can be "recycled" in its altered conformation out of MD to be tested with other repurposed drugs. In a reiterative process tailored to each small molecule, different amino acids can be made flexible prior to computationally expensive MD, simulating an induced fit model of protein-ligand binding using the platform. The reiterative process of flexible docking coupled with MD protocols decreases the computational time to generate new protein conformers significantly.

#### **4. Molecular dynamics**

#### **4.1 MD simulations**

Preparations for MD simulations were done using SwissDock parameter files for the catechin derivatives for MD [20]. The protein structure files were generated with docked ligands in VMD [21]. Molecular dynamics simulations were performed with the NAMD Program [8] using CHARMM37 parameters along with complementary CHARMM General Force Field optimized for ligand parameter files for each protein target/catechin derivative complex [22]. Periodic boundary conditions were imposed and explicit solvent/implicit solvent boundaries defined using a truncated rectangular box, ensuring a solvent shell of at least 10 A around the solute surrounding the protein-ligand complex. The solute was neutralized with potassium ions (K+ /Cl− ion pairs) to a concentration of 150 mM. The ions were initially placed at random, but at least 5 A from ligands and 3.5 A from one another. The resulting systems contained between 10,500 and 15,250 water molecules, corresponding to a total of 30–45,000 atoms.

Simulations employed periodic boundary conditions and electrostatic interactions were treated using the particle-mesh Ewald algorithm [23, 24] with a real space cutoff of pair list was built with a buffer region, and a list update was triggered whenever a particle moved by more than 0.5 A with respect to the previous update. Each system was initially subjected to energy minimization with harmonic restraints of 2 kcal mol−1 A ̊−2 on the solute atoms. The system was then heated to 310 K at constant volume during 100 ps. Constraints were then relaxed from 5 to 1 kcal mol−1 A ̊ −2 during a series of 1000 steps of energy minimization (500 steps of steepest descent and 500 steps of conjugate gradient) followed by 50 ps of equilibration with restraints of 0.5 mol−1 A ̊−2 and 50 ps without solute restraints. The 50 ns production simulations were carried out at constant temperature (300 K) and pressure (1 bar) with a 2 fs time step. This was reiterated 10×. During these simulations, pressure and temperature were maintained using the Berendsen algorithm [25] with a coupling constant of 5 ps, and SHAKE constraints were applied to all bonds involving hydrogens [26]. Conformational snapshots were saved for further analysis every 10 ps.

For comparison purposes, the isolated catechin complexes from each complex were also simulated alone using an identical protocol, creating a second set of ten 50 ns trajectories.

#### **4.2 Potential energy of binding and free energy of binding calculations for MD output**

The overall potential energy of protein-ligand system was evaluated by adding the final potential energy of the small molecule alone, and the final potential energy of the protein alone run through the same MD protocol. These values were compared with the protein/ligand complex. An expected, drop in the potential energy of the complex was confirmed. Absolute binding free energy (ABFE) calculation, with total annihilation of the ligand in the binding pocket followed by its reappearance at bulk state where the target protein was absent, was part of this process. As free energy is a state function, the alchemical FEP route to getting binding free energy of these derivates was as follows: (1) 'locking the ligand', restraining conformational, translational, and rotational degrees of freedom at bound state, (2) 'disappearing the locked ligand', turning off the interaction between ligand and its surroundings, (3) 'translocating ligand' of which corresponding free energy is zero, (4) 'reappearance of the locked ligand', turning on the interaction between ligand and its surroundings at bulk state, and (5) 'unlocking the ligand', releasing of the three restraints from [1]. Lenselink et al. tested binding free energies of congeneric ligands to four different using FEP+ from Schrödinger and obtained results in great agreement with our experimental results [27].

#### **5. Laboratory validation**

In order to validate the binding of the Drug Bank compounds, colorimetric kinetic assays were conducted. To perform these tests recombinant FBPase protein was overexpressed and purified. The construct was transformed into competent cells, and the purified construct used to over-express protein, which was harvested and purified prior to use.

#### **5.1 Transformation(s) and purification of FBPase plasmid**

Plasmids containing FBPase sequence underwent transformation with XL Blue super-competent cells [13]. Cells were plated on Luria Bertani (LB) agar plates and colonies selected for 5 mL overnights for plasmid purification using various kits [28]. Following purification, FBPase plasmids were screened for integrity and run on an agarose gel and sequenced. A transformation protocol for over-expression was then performed as previously described [13]. The contents of the tubes were transferred to LB ampicillin plates using a sterile technique. Plates were incubated for 18 h at 37°C and stored for 2.5 weeks at 4°C.

#### **5.2 Protein over-expression, isolation, and purification**

To prepare the recombinant human or closely related pig kidney FBPase enzymes for kinetic and binding assays, the recombinant proteins were over-expressed, isolated, and purified as previously described using ampicillin resistance for selection of cells

#### *A Computational Approach for Identifying Experimental or Approved Drugs That Can Be... DOI: http://dx.doi.org/10.5772/intechopen.110812*

containing the construct [13]. Briefly, the host cell translation was inhibited with 34 mg/ mL chloramphenicol (in isopropanol). The solution was shaken again for 2–3 h at 37°C to ensure optimal growth of the host cells. Cells were isolated by pelleting in 250 mL flasks in a centrifuge at 4000 rpm. After the cells were frozen, each cell pellet was resuspended in 20 mL of 50 mM Tris pH 7.5. The supernatant was lysed *via* sonication to release cell contents. Each protein solution was sonicated as previously described. Sonication settings were at 10% duty cycle for 5 min, pulsing 10 s on, and 10 s off ×3. Each supernatant cell lysate was centrifuged for 30 min at 13,500 rpm at 4°C and transferred to dialysis tubing for dialysis in 50 mM Tris pH 8.0. Protein was then purified *via* NTA nickel affinity column as previously described [13]. In addition, gel filtration was run on a G250 column in Tris buffer pH 7.5 in 0.150 M NaCl as eluent buffer to investigate the oligomeric status of inhibitor-bound protein compared to FBPase enzyme alone.

#### **5.3 Characterization of purified recombinant enzymes and preparation for enzyme kinetic assays**

The purity of the recombinant FBPase was assessed *via* SDS-PAGE, and the oligomeric state was identified with native gel electrophoresis. An SDS-PAGE gel electrophoresis was used to separate and identify proteins with the correct molecular weight [9]. For the kinetic assay, a standard curve was obtained from an ammonium molybdate malachite green inorganic phosphate assay (OD660 nm) [5, 6]. The purified FBPase protein was dialyzed ×3 in 50 mM Tris buffer at pH 7.5 at 4°C. For enzyme concentration, absorbency values were recorded using spectrophotometer readings at OD280 nm. To quantify enzyme concentration, the enzyme concentration was calculated based on the quantity of micrograms per microliter (μg/μL) present. Final concentration FBPase enzyme was between 2.5 and 5.0 μg/μL. Using a colorimetric assay as described below, specific activity (SA) was determined in the absence and presence of Drug Bank inhibitors.

#### **5.4 Kinetic assays on FBPase/ligand complexes**

A colorimetric malachite green kinetic assay was utilized for FBpase activity levels to validate inhibition of the enzyme predicted with selected Drug Bank molecules. FBPase cleaves fructose 1,6-bisphosphate to fructose 6 phosphate and inorganic phosphate. The malachite green colorimetric assay is based on the change in color from brown to blue, observed when a complex is formed between malachite green, ammonium molybdate, and the product inorganic phosphate. For calculating Ki values, data was collected with varying substrate FBP concentrations (100–500 μM at pH 7.5). Malachite green dye was prepared under acidic conditions to activate the color change and quench the FBPase activity at fixed time points [13]. Absorbance readings were recorded at OD660 nm. Data collected was input to the inorganic phosphate standard curve equation to calculate product formed. Micromoles of product inorganic phosphate were calculated to determine the specific activity (SA) of FBPase enzyme +/− inhibitor. The IC50 assay was designed with fixed high concentration of substrate varying the inhibitor concentration to calculate IC50 values. For each Drug Bank ligand, the kinetic assay was performed in duplicates of triplicates. All kinetic data were fit using origin software based on methods previously described [13]. More specifically, for kinetic parameters established to fin Ki's *via* curve fitting the equation y = d + (a – d) / 1 + (x / c)b was used where "x" is represented by apparent Ki when y = Vmax. IC50s were also determined with origin curve-fitting software [29].

To avoid high background due to malachite green dye interaction with some Drug Bank ligands cleavage assays utilizing phosphoglucose isomerase and glucose-6-phosphate dehydrogenase were used as coupling enzymes in validation assays for FBPase [18]. For specific activity measurements, reduction of NADP to NADPH was monitored by absorbance at 340 nm. Other assays used the same coupling enzymes but monitored the formation of NADPH by its fluorescence emission at 470 nm using an excitation wavelength of 340 nm. Assays were performed at 22°C in 50 mM Hepes, pH 7.5. Data for inhibitor (inhibition) were fit to several models using origin or sigma plot software with a Hill equation model [6]. These assays are robust and unbiased as they follow the ASBMB standard for rigor and reproducibility [30]. In addition, these assays have been published in many peer reviewed research articles [1–5, 14].

#### **6. Case study analysis and discussion**

#### **6.1 Case study: allopurinol as a scaffolding molecule for drug design**

The approved Drug Bank compound allopurinol is already on the market to relieve symptoms of gout. Also known as Zyloprim and Aloprim, allopurinol is composed of a dihydroxy substituted pyrimidine ring that is fused to a pyrazole ring. Allopurinol is shown below in green in **Figure 3A**. Allopurinol alone is not large enough to bind to the entire allosteric binding site, and more than one allopurinol molecule may bind at a time resulting in a 1:2 binding ratio. For every target allosteric binding site, 2 allopurinol compounds can fit in the allosteric binding pocket. In this study, allopurinol molecules docked in the FBPase AMP binding site were used as scaffolding molecules upon which functional groups were added to enhance their binding affinity for the FBPase. As shown below in **Figure 3A**, allopurinol top scoring position (Ki ~300 micomolar) overlays with the crystallographic AMP molecule adenine ring. Interestingly, with another allopurinol docked, the predicted Ki dropped to ~150 micromolar. The residues shown here Phe184, Leu173, Glu20, Val160, Arg140, Tyr113, Lys112, Glu 29, and Thr27 were all separately made flexible during reiterative runs of docking followed by full-scale MD runs for scoring predicted Ki's. In **Figure 3B**, the Zinc38643891 (pink) literally straddles the AMP binding site. The predicted Ki is ~50 nM for this catechin derivative discovered in the PubChem database after similarity searches for catechin EGCG as previously described (ref). Note when compared to the crystallographic position of the AMP co-crystallized with FBPase in PDB 1FTA, the predicted position of the phenyl ring aligns in the position where the adenine ring is located. Leu174, Phe184, and Val60 alternate to interact with the zinc molecule and stabilize its position during simulations. During simulations the helix behind these three residues shifts slightly out of position causing a "domino effect" and the zinc molecule moves deeper into the AMP binding site cleft.

**Figure 3C** shows the best docking pose predicted for Zinc38643891 and allopurinol. This figure illustrates how allopurinol may be modified to bind tighter by derivatizing the heterocyclic ring. Details of the synthesis are preserved for a future manuscript.

**Figure 3A** shows the 1FTA protein coordinates with a close-up of the AMP binding site with the crystallographic ligand AMP in dark gray. The best score for the docked allopurinol is in green. **Figure 3B** shows the same coordinates docked with a catechin derivative (pink) from the PubChem database cross-referenced to the zinc database

*A Computational Approach for Identifying Experimental or Approved Drugs That Can Be... DOI: http://dx.doi.org/10.5772/intechopen.110812*

**Figure 3.** *Docking of allopurinol and Zinc38643891 into the AMP allosteric binding site of FBPase.*

as Zinc38643891. **Figure 3C** shows the best docking results of the two compounds Zinc38643891 (pink) and allopurinol (green).

**Figure 4A** shows allopurinol derivative (orange) and Zinc38643891 (blue) in an FBPase activity assay showing relative activity as a function of compound concentration. The average specific activity of each concentration (triplicates) was normalized against FBPase with no inhibitor. **Figure 4B** shows the relative viability of HepG2 cells as a function of inhibitor concentration. The raw data from the MTT assay was normalized with HepG2 cells with no inhibitor present.

As shown in **Figure 4A**, both the allopurinol derivative and Zinc inhibit the activity of the isolated recombinant FBPase activity with ~50% inhibition in the low nanomolar range. The allopurinol derivative reaches the 50% inhibition mark at exactly 100 nM whereas the Zinc compound reaches 50% inhibition at ~25 nM, nearly 4-fold more potent than the allopurinol derivative in this cell-free activity assay. This is considered a promising hit for moving forward in the drug development pipeline to a lead compound.

However, it was surprising in **Figure 4B** that within the same concentration range, the Zinc38643891 was at 50% viability in the MTT viability assay with the HepG2 cells. Whereas the allopurinol derivative remained 90% viable in this range and beyond. In fact, allopurinol did not show significant drop in viability until the mid-high micromolar range. Initially, the expectation was that a derivative of a natural product catechin, the Zinc compound would have a better toxicological profile. The allopurinol derivative of Zinc38643891 was able to overcome the toxicological barrier of the original zinc molecule by substituting out the fluorophenyl rings for allopurinol in a novel synthesis

**Figure 4.** *Inhibition and toxicity assays comparing Zinc38643891 and sllopurinol derivative.*

protocol (manuscript under preparation). Insights from this case study that have led to the synthesis of the novel allopurinol derivative were discovered by visual analysis of the output frames of the MD runs of the allopurinol and Zinc38643891 protein-ligand complexes. During this visual inspection process (which was laborious), it became apparent that the allopurinol molecule(s) in the allosteric binding site were localizing in the same area as Zinc38643891 fluorophenyl rings. The VDDP was an integral part of the drug discovery process for this project. Currently, the lead compound(s) from this case study are being evaluated for *in vivo* studies.

#### **7. Conclusions**

Using our virtual drug discovery platform, symmetric computing has identified potential therapeutic small molecules used as scaffolding molecules that are approved drugs. The repurposed compounds were selected based on theoretical binding score (Ki) of the FBPase protein-compound complex. Initial validation in a colorimetric enzymatic inhibitory assay and a toxicity assay led the way to derivatives of these repurposed drugs from validated hits to lead optimization. The next steps to validate the predicted activity of each of these compounds would be an animal model (*in vivo* studies targeting the liver) utilizing a rat animal model. In the future, a decision will be made on which Drug Bank derivative will be advanced to human clinical studies.

#### **Acknowledgements**

We thank Jason Gao of Northeastern University for the use of the NMR facility to track synthesis of the Drug Bank derivatives. Also thank you to Jeffrey Turner and the symmetric computing support staff for this project, and Professor Nurit Haspel for valuable insight and discussions on the molecular dynamics protocols. As well as the Biotechnology Division at Roxbury Community College. Thank you to Northeastern Professional Studies College Department of Biotechnology for assistance with HepG2 toxicity assays.

#### **Conflict of interest**

None.

*A Computational Approach for Identifying Experimental or Approved Drugs That Can Be... DOI: http://dx.doi.org/10.5772/intechopen.110812*

#### **Author details**

Gemma Topaz1 , Dongjun Yoo2 , Richard Anderson1 and Kimberly Stieglitz1 \*

1 Venture Development Center, Symmetric Computing Inc., University of Massachusetts Boston, Boston, USA

2 University of Massachusetts Boston Psychology Department, Boston, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Targeted Modification of Physical-Chemical Properties of Drugs as a Universal Way to Transform "Old" Drugs into "New" Drugs

*Aleksandr Urakov, Natalya Urakova, Yulia Sorokina, Aleksandr Samorodov and Evgeny Fisher*

#### **Abstract**

Historically, the bulk of known drugs was created for resorptive action. Therefore, the mechanism of action of drugs was attributed to the specific action of the main ingredients once they were absorbed into the bloodstream. In recent years, it has been found that the mechanism of local action of drugs is determined not only by the specific activity of their main ingredients but also by the nonspecific activity of the excipients and formulation ingredients included in the ready-to-use drug: tablet, injectable solution, aerosol, etc. In this regard, there is an opportunity to repurpose drugs by purposefully changing their quality by changing the physical, chemical, and physical-chemical properties of the finished products. As an example, two new groups of drugs intended for local application to dissolve dense pus and discolor blood stains, namely, piolytics and drugs that bleach bruises, have been created. It is shown that the advantage and at the same time the limitation of the upgraded preparations is their local application, as their peculiarity is the realization of the physicalchemical principle of the drug action in local interaction with the selected part of the patient's organism.

**Keywords:** new drugs, repurposing, physical-chemical features, financial cost, search, development, screening

#### **1. Introduction**

Traditional ways of searching for and developing new drugs require large financial, time, and human costs. It is reported that with traditional research design, it would take more than 12 years and \$800 million to create a new drug and conduct the entire set of preclinical studies [1, 2]. However, even these costs do not guarantee success, since all traditional drugs have toxicity. Therefore, all drug candidates are

traditionally tested for general (resorptive) toxicity. However, no amount of testing can completely eliminate drug toxicity. Therefore, it is no coincidence that most of today's projects aimed at finding and developing new drugs designed for resorptive action are doomed to fail, as they have in the past. In particular, specialists know that under the traditional scheme of search and development of new drugs out of several thousand substances that participated in trials at the beginning of the complex of these trials, only 1–2 substances can survive all trials more or less successfully and obtain the status of a drug [3]. All other substances will be rejected and forgotten.

That is why it is very important to choose the right design for the search and development of new drugs at the very beginning of this journey in order to reduce the risk of a pharmacological project, the financial and human costs, and to win in time. One very profitable decision these days may be the decision to develop a new drug intended not for general (resorptive) but for local application [4, 5]. The essence of this innovation lies in the fact that in order to reduce financial, human, and time costs when searching for and developing new drugs, it is proposed to abandon the idea of discovering absolutely new chemical compounds intended for resorptive action and to aim at modernizing the formulation of "old" (known) drugs by turning them from resorptive drugs into drugs of local action when applied locally. In other words, it is proposed to create new drugs by purposeful changes in the formulation of known drugs providing them with a new mechanism of local action when applied locally. It has been shown that the transformation of drugs known for their general action into drugs with a new mechanism of local action provides a change in their quality indicators and especially in their physicochemical properties [6, 7].

The author's experience in using this design for the search and development of new drugs showed the high promise and competitive benefit of the proposed research design compared to the conventional design. It was also confirmed that artificial imparting of quite certain physicochemical properties to ready-made drugs and/ or artificial provision of certain physicochemical factors of their local interaction during local application can provide the required mechanism of local drug action and become an alternative to traditional performers of this role in pharmacology, namely, original chemical compounds (substances) with specific pharmacological activity inherent in each of them.

#### **2. Methods**

The study is based on Russian research, as there are no similar studies in other countries. The contents of the following materials were included in the analysis:

*Targeted Modification of Physical-Chemical Properties of Drugs as a Universal Way… DOI: http://dx.doi.org/10.5772/intechopen.110480*

#### **3. Results**

The first announcement that new materials can be created from old materials by changing their physical and chemical properties was made at the 3rd International Conference on Competitive Materials and Technology Processes (IC-CMTP3) (6–10 October 2014, Miskolc-Lillafüred, Hungary) [4]. Several examples of creating new drugs created from "old", that is, known drugs by purposeful modification of their physical and chemical properties were shown as evidence of the real possibility of this direction. Thus, there was an example of creating a "floating tablet" due to the fact that the well-known tablet, which is an artificial stone, is made porous, consisting of individual isolated cavities filled with air, and the specific gravity of the tablet is less than 1 g/cm3 (RU 2254121). It has been clearly demonstrated that the specific gravity and strength values of conventional drug tablets are similar to pieces of chalk, natural stone, river pebbles, and even concrete since the tablets are produced from dry powders by pressing them without maintaining isolated cavities filled with air. Therefore, when taken orally, regular pills sink in gastric juice, sink quickly to the very bottom of the stomach and remain at the bottom, no matter how much water the patient drank before, during, or after taking the pills. Moreover, "sinking" pills are very salty and acidic, so they have a local irritating effect on the mucous membrane of the stomach in the area of their contact with it. That is why all the pills cause erosive damage to the mucous membrane of the stomach and gastric ulcers precisely in the pyloric region. At the same time, a floating tablet is lighter and floats on the surface of the gastric juice, not in contact with the pyloric mucosa. Moreover, it has been shown that taking water and raising the liquid level in the stomach cavity causes the floating tablet to move along with the liquid level. Therefore, the floating tablet cannot have a local irritant and ulcerogenic effect on the mucous membrane of the pyloric region of the stomach. The higher the level of liquid in the stomach cavity rises, the higher the floating pill rises.

In addition, it has been convincingly shown that increasing the temperature, alkaline activity, and gas content of conventional drugs can turn them into new drugs, for example—into solvent drugs for thick and dry biological masses (pus, blood clots, blood stains, mucus, sputum, sulfur plugs, etc.). It has been reported that almost any drug can be turned into a solvent for thick pus and blood stains if sodium bicarbonate, hydrogen peroxide, and/or carbon dioxide under increased pressure are additionally introduced into the drug formulation and the drug is heated to a temperature of 37–45°C. In particular, it has been reported that such drug solutions acquire the physicochemical properties of alkaline carbonated drinking beverages.

However, drugs differ from the known carbonated drinking beverages in that they have hydrogen peroxide in their composition. The fact is that the addition of hydrogen peroxide to such solutions further changes their physicochemical properties, especially during local interaction with biological tissues containing the enzyme catalase, which decomposes hydrogen peroxide into oxygen gas and water very quickly. Therefore, warm alkaline solutions of hydrogen peroxide in local interaction with thick pus, mucus, sputum, blood, feces, meconium, sulfur plugs, and other biological objects containing catalase enzyme immediately "explode" them due to cold boiling process. As a result, the above biological objects turn into a soft oxygen foam of white color, because at the same time, oxygen decolorization of hemoglobin and its colored metabolites occurs.

On this basis, it was concluded that by giving drugs physical and chemical properties such as hyperthermia, alkalinity, increased gas content under increased pressure, and the ability to release molecular oxygen under the influence of catalase can provide drugs with the ability to bleach blood spots, bruises, and hematomas, as well as dissolve thick pus and thick sulfur plugs.

Finally, based on the results obtained, it was concluded that a new direction in pharmacology and materials science was discovered, namely, physicochemical pharmacology and physicochemical materials science.

A few years after the first official communication, the assumption was realized in Russia with the help of the State Fund for Innovation Support. This fund provided a grant for the development of the first drug to bleach the skin in the area of the black eye. The first drug, a skin-bleaching agent in the area of a black eye, was successfully developed by the team of the Institute of Thermology (Izhevsk, Russia) in the period from 2016 to 2018. Analysis of the reports of the Institute of Thermology showed that it took 2 years to find and develop a new drug, a skin-bleaching agent for the bruise area, at a cost of \$27,132. It is important to emphasize that 10 qualified physicians participated in the development. One of them was a doctor of medicine, a professor in the field of pharmacology, and four doctors had Ph.D. degrees. In addition, five students helped the team to carry out the research on their own initiative, that is, free of charge.

At the same time, in Russia, a group of specialists and students conducted laboratory screening of several dozens of known drugs and some physical, chemical, and physicochemical factors of local interaction between 2015 and 2022. The results obtained made it possible to establish that such "old" drugs as hydrogen peroxide and sodium bicarbonate have weak dissolving and bleaching activity, and such physical– chemical factors of local interaction such as hyperthermia, alkalinity and hypergazing, and increase and accelerate their bleaching and pyolytic action several times.

In addition, this research team conducted several series of experiments on the nonspecific pharmacological activity of warm alkaline hydrogen peroxide solutions when injected into the skin in the area of bruises in awake piglets, by cutaneous and intradermal injection in the area of artificial bruises created in isolated pig skin segments and in models of blood spots on dressing materials. In parallel, laboratory studies were conducted on the transformation of thick pus, mucus, sputum, and meconium into fluffy oxygenated foam under laboratory conditions with the corresponding isolated biological objects of patients, as well as in experiments on rabbits and isolated rabbit lungs. The results confirmed the correctness of the assumption that it is possible to create new drugs from old drugs by artificially and purposefully changing their physical and chemical properties such as temperature, acidity (alkalinity), osmotic, explosive, and oxygen-releasing activity. Examples are new drugs-bleaching stains, traces of blood, bruises, and hematomas, as well as new medical technologies of skin whitening in the area of bruises, developed in Russia on the basis of physical–chemical pharmacology (RU 2539380, RU2589682, RU 2573382, RU 2653465, RU 2647371, RU 2639485, RU 2586278, RU 2582215, RU 2577510, RU 2600504, RU 2634268, RU 2631593, RU 2631592, RU 2641386, RU 2639283, RU 2679334) [5, 7].

Another example of the successful use of the basics of physicochemical pharmacology to transform "old" drugs into new drugs is the development of new aerosols for inhalations and new solutions for intrapulmonary injections based on the original *Targeted Modification of Physical-Chemical Properties of Drugs as a Universal Way… DOI: http://dx.doi.org/10.5772/intechopen.110480*

warm alkaline hydrogen peroxide solutions, which provide urgent recanalization of the airways and oxygen saturation of blood through the lungs due to oxygen foaming of thick mucus, sputum, and pus inside the airways during respiratory obstruction caused by COVID-19 (RU Patent No. 2742505, RU Patent No. 2735502, RU Application No.<PHONE_NUMBER>, RU Application No.<PHONE_NUMBER>) [8].

#### **4. Discussion**

In the period from 2012 to 2022, the foundations of physical-chemical pharmacology, pharmacy, and materials science were laid [5, 8]. The essence of the new direction is that new drugs, or rather drugs with a new mechanism of local action, can be created from well-known and proven drugs (that is, old drugs) by purposefully changing their physical-chemical properties. It is reported that a targeted change in the physical-chemical properties of finished drugs (tablets, solutions, aerosols, etc.) allows you to radically change the local mechanism of action of traditional drugs (chemical compounds) with local interaction inside the stomach (with enteral administration), inside blood vessels (with intravenous injections), inside the skin (with intradermal injections), on the surface of the skin and mucous membranes (with applications), as well as inside sulfur plugs, purulent masses, blood clots, thick masses of mucus, sputum, meconium, and other biological masses. It has been convincingly shown that the mechanism of local action of drugs with local application is determined not only by the specific activity of their main ingredients but also by the nonspecific activity of auxiliary and formative ingredients included in the composition of drugs, as well as the physical-chemical properties of finished drugs. That is why for the development of new drugs intended for topical use, "old" drugs can be used, but with artificially altered physical-chemical properties. The new mechanism of local action of "old" drugs can be adjusted by changing the mechanical, physical, chemical, and physical–chemical properties of drugs in specific dosage forms, that is, by changing the quality of tablets, solutions, ointments, creams, aerosols, etc.

It is paradoxical, but the results obtained convince us of the prospects of searching for drugs with new mechanisms of local action among the "old" drugs by purposefully changing their quality (from the standpoint of traditional ideas about the quality of drugs).

Indeed, using the example of ordinary tablets, it was shown that they sink to the bottom of the stomach and move inside its cavity under the influence of gravity in the same way as river sand, pieces of chalk, clay, gravel, and pebbles [9]. It turned out that the specific gravity of all modern tablets exceeds 1 g/cm3 , and, therefore, all tablets sink in gastric juice, water, and milk. It has been shown that in the vertical position of the patient's torso, all tablets fall into the pyloric part of the stomach despite the added liquid. At the same time, it was found that all tablets have an aggressive effect on the gastric mucosa since they corrode the stomach wall and can lead to the formation of ulcers. At the same time, a decrease in the specific gravity of tablets of less than 1 g/cm3 due to the creation of isolated cavities in them by the type of solid or thick foam makes it possible to radically change the intra-stomach pharmacokinetics and pharmacodynamics of tablet drugs.

Further studies have shown that tablets, which are considered high-quality today, cannot be considered such in comparison with natural food lumps that a person swallows with high-quality chewing of food [9]. It is reported that not only the specific gravity of the tablets but also the shape of the tablets distinguish them from natural

food lumps. This approach is recommended for the future adjustment of modern standards of quality control of tablets. It was reported that the manufacturing of tablets by pressing dry powders up to their transformation into artificial stones in the form of a round disk is a pharmaceutical mistake since the disc-shaped shape of tablets with the properties of stones is incompatible with the specifics of the human digestive system. In addition, it was reported that the sizes of modern tablets, which are considered high-quality, do not correspond to the sizes of natural food lumps. It has been shown that high–quality tablets from different manufacturers can differ in height by 3 times, and in volume by 10 times. It was also reported that the natural food lump has the shape of an olive with the largest diameter of up to 1 cm and a maximum length of 2 cm. At the same time, food lumps have an average elasticity, a hardness value of about zero, food lumps are porous and have a specific gravity of less than 1 g/cm3 , and are also devoid of aggressive osmotic action on the mucous membranes of the mouth and stomach. At the same time, modern tablets considered to be of high quality may differ from each other in terms of their hardness by 500 times, since the hardness of the tablets is not controlled [9].

Therefore, it was not by chance that it was stated that the human stomach is not adapted to the introduction of such pills into it (and in fact—artificial stones), since a person is not a bird. Based on these data, it was proposed to modernize the quality standard of tablets, since to increase their safety there is no alternative to their similarity to natural human food lumps in shape, size, and physical-chemical properties. In particular, tablets–analogs of natural food lumps were developed (RU 2533840).

In parallel with the studies of the physical-chemical properties of tablets, the physical-chemical properties of solutions of drugs intended for injection and considered to be of high quality (from the standpoint of drug quality standards) were carried out. It was reported that most of the modern high-quality drug solutions have acidic activity, that is, a pH value of less than 7.0. At the same time, blood and most human tissues have alkaline activity with a pH value of 7.4 [4, 5]. Therefore, some solutions of drugs, which are considered to be of high quality today, can have a local irritating effect and even post-injection necrosis and abscesses at the sites of intramuscular and intravenous injections [10].

In this regard, it was proposed to modernize the pharmacopeia requirements for drug solutions intended for injection. In particular, it was proposed to replace the traditional division of medicinal solutions by the magnitude of their acid (alkaline) activity from pH 7.0 (that is, acidic and alkaline drugs) with a more physiological division of drugs into groups compared to pH 7.4, namely, acidifying and alkalizing drugs (RU 2219958, RU 2221248). The fact is that the acidic activity of drug solutions coagulates proteins and protein-lipid complexes, and also has a local irritant effect on tissues, causing local aseptic inflammation in the injection and/or application sites (for example, when injecting drug solutions into the conjunctival cavity). On the other hand, the alkaline activity of drug solutions liquefies colloidal tissues and can saponify protein and protein-lipid complexes. Therefore, drug's alkaline activity optimizes the rheological properties of blood after local interaction of drug solutions with blood inside veins and vascular catheters, preventing clogging. Additionally, it is reported that alkaline activity of drug solutions promotes liquefaction and dissolution of thick mucus, sputum, pus, and blood clots, especially when local temperature increases, namely, when heating solutions to +37 − +45°C [5, 8].

In addition to this, the great importance of the temperature of drug solutions has been shown. The fact is that temperature according to the Arrhenius and Van Goff law is of great importance for the intensity of all metabolic and vital processes at all levels

#### *Targeted Modification of Physical-Chemical Properties of Drugs as a Universal Way… DOI: http://dx.doi.org/10.5772/intechopen.110480*

of organization of living systems, including protein molecules. At the same time, there are reports that modern standards of treatment do not take into account the temperature of drugs when they are administered to patients. Because of this, drugs are not heated to human body temperature and most often the drugs are at room temperature, that is, about +24°C [5, 8]. Therefore, during local interactions, cold drugs cool the tissues in the area of local interactions. Thus, the drugs cause formation of a zone of local hypothermia. In its turn, local hypothermia compacts biological tissues and slows down the rate of biochemical processes in them. In particular, it was reported that temperature decrease from +37 to +24°C significantly slows down the process of blood clotting inside the vascular catheters and blood vessels, which reduces the risk of vein and vascular catheter thrombosis formation, especially during infusion of infusion fluids. In addition, during intravenous injections, cold drug solutions cool the veins, the localization of which can be detected using a thermal imaging device by appropriate zones of local hypothermia in the area of vein projection. This role of cold solutions during intravenous injections was the basis of a new method of infrared imaging of subcutaneous veins (RU 2389429).

On the other hand, it is reported that another very important physicochemical property of drug solutions is their osmotic activity. For a long time, ready-to-inject drug solutions were thought to be of impeccable quality and to be isotonic. However, studies conducted in recent years have shown that the drug quality standard includes control of the concentration value of drug ingredients, but does not include control of osmotic activity and local irritant effect of drug solutions on tissues at injection sites [11]. In this regard, any drug in a particular series (or batch) with a particular manufacturer may sooner or later have excessive hypertonic and/or acidic activity.

Using the example of nonsteroidal anti-inflammatory drugs (NSAIDs), it was shown that some of these drugs have a local irritant effect due to the high concentration of ingredients and hypertonic activity [11]. In laboratory conditions, using an osmometer, it was found that a solution of 50% sodium metamizole has an osmotic activity of 4638 ± 12.5 mosmol/l of water, that is, it is a hypertonic solution. In experimental and clinical conditions, it was shown that intramuscular and subcutaneous injections of a solution of 50% metamizole sodium caused aseptic inflammation, necrosis, and abscess. However, preliminary dilution of a 50% metamizole sodium solution with water by 10 times completely prevented the development of postinjection necrosis and abscess during subcutaneous and intramuscular injections. At the same time, the effectiveness of the protective action of water was explained by its physicochemical role in reducing the concentration of ingredients and the hypertonic activity of the diluted sodium metamizole solution.

These reports convince us that one of the causes of acute post-injection local complication, which is manifested by aseptic inflammation, necrosis, and abscess of tissues at the injection sites of drugs, may be an excessively strong dehydrating effect of drugs on tissue cells due to their hypertensive activity. The dehydrating effect is exerted by an excessively large hyperosmotic activity (hypertonic activity) of drugs, which is created by a large concentration of dissolved ingredients. It has been shown that medicinal solutions containing ingredients in the range of 1–10% are isotonic, either weak hypotonic or weak hypertonic solutions. Such solutions have moderate postinjection safety. Drug solutions containing ingredients in the range of 10–76% are strong hypertonic solutions and have an excessively strong dehydrating effect on cells with local interaction. In this regard, solutions with a total concentration of ingredients of more than 10% are excessively hypertensive and have excessively low postinjection safety, since their injections cause excessive dehydration of cells, necrosis, and abscess [12].

Based on the results, it was concluded that traditional drug quality control requirements are responsible for the fact that some drug solutions, now considered to be of high quality, may have excessive hypertonic activity. In turn, hypertonic drug activity may cause local irritation, aseptic postinjection necrosis, and injection site abscesses. In this regard, ignoring the actual values of osmotic and acidic activity of drugs in the drug form "solution for injection" during injection reduces postinjection drug safety. Therefore, in order to improve postinjection drug safety, it is proposed to monitor the osmotic and acidic activity and the local irritant effect of drugs on the tissues at the injection sites. At the same time, it is reported that postinjection necrosis and abscess in the place of subcutaneous injection of hypertonic solution of almost any drug can be prevented by immediate injection of water for injection (not later than 5–6 minutes after the injection) under control of the dynamics of local skin temperature in the injection site using a thermal imager (RU 2326662, RU 2304769, RU 2396562).

Consequently, convincing evidence has been obtained that eliminating the hypertonic activity of highly concentrated drug solutions by diluting them 2–10 times with water increases the safety of drug injections. Therefore, there are grounds for including this recommendation in the instructions for medical use of highly concentrated drug solutions.

#### **5. Conclusion**

Thus, in 2014, an affordable way was proposed to transform **"**old**"** quality drugs into new drugs by purposefully changing their quality by artificially changing the physical, chemical, and physicochemical properties of finished drugs: tablets, injectable solutions, aerosols, etc. From the point of view of traditional ideas, the newly created drugs in this way may seem to be spoiled or defective. However, this is not the case. Simply known (old) drugs were traditionally developed mainly for the general (resorptive) mechanism of action. Therefore, the **"**old**"** drugs have outdated requirements for their quality. Pharmaceutical and pharmacological progress is impossible without revising the pharmacopeia and traditional drug quality control standards, because **"**old**"** drug quality standards preclude their qualitative modernization. It is shown that the advantage and at the same time the limitation of modernized drugs is their local application since their peculiarity is the implementation of the physicochemical principle of drug action in local interaction with a selected part of the patient**'**s (or animal**'**s) body.

#### **Acknowledgements**

We express gratitude to Professor László A Gömze for his constant support and assistance in the formation of physicochemical materials science.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Targeted Modification of Physical-Chemical Properties of Drugs as a Universal Way… DOI: http://dx.doi.org/10.5772/intechopen.110480*

#### **Author details**

Aleksandr Urakov1 \*, Natalya Urakova1 , Yulia Sorokina<sup>2</sup> , Aleksandr Samorodov3 and Evgeny Fisher1

1 Izhevsk State Medical University, Izhevsk, Russia

2 Volga Medical Research University, Nizhny Novgorod, Russia

3 Bashkir State Medical University, Ufa, Russia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Wyatt PG, Gilbert IH, Read KD, Fairlamb AH. Target validation: Linking target and chemical properties to desired product profil. Current Topics in Medicinal Chemistry. 2011;**11**:1275-1283. DOI: 10.2174/156802611795429185

[2] Frearson JA, Wyatt PG, Gilbert IH, Fairlamb AH. Target assessment for antiparasitic drug discovery. Trends in Parasitology. 2007;**23**(12):589-595. DOI: 10.1016/j.pt.2007.08.019

[3] Brown M, Bennett P, Sharma P. Discovery and Development of Drugs. 11th ed. Edinburgh: Churchill Livingstone; 2012. p. 680

[4] Urakov AL. Development of new materials and structures based on managed physical-chemical factors of local interaction. IOP Conference Series Materials Science and Engineering. 2016;**123**:012008. DOI: 10.1088/ 1757-899X/123/1/012008

[5] Urakov AL, Urakova NA, Stolyarenko AP. How to turn an old medicine into a new medicine. Journal of Bio Innovation. 2020;**9**(5):774-777. DOI: 10.46344/JBINO

[6] Urakov AL. The change of physicalchemical factors of the local interaction with the human body as the basis for the creation of materials with new properties. Epitőanyag – Journal of Silicate Based and Composite Materials. 2015;**67**(1):2-6. DOI: 10.14382/ epitoanyag-jsbcm.2015.1

[7] Urakov A, Urakova N, Nikolenko V, Belkharoeva R, Achkasov E, et al. Current and emerging methods for treatment of hemoglobin related cutaneous discoloration: A literature

review. Heliyon. 2021;**7**(1):e059542. DOI: 10.1016/j.heliyon.2021.e05954

[8] Fisher E, Urakov A, Svetova M, Suntsova D, Yagudin I. COVID-19: Intrapulmonary alkaline hydrogen peroxide can immediately increase blood oxygenation. Medicinski Casopis. 2021;**55**(4):135-138. DOI: 10.5937/ mskg55-3524

[9] Urakov A, Urakova N, Reshetnikov A, Kasatkin A, Kopylov M, Baimurzin D. About what is happening in the stomach after swallowing human river pebbles, gravel, chalk, clay and tablets drugs. Epitőanyag – Journal of Silicate Based and Composite Materials. 2016;**68**(4):110-113. DOI: 10.14382/ epitoanyag-jsbcm.2016.19

[10] Urakov AL. Nikolau syndrome: Necrotic activity of drugs and ways to prevent post-injection abscesses (In memory of Professor László A Gömze). Creative Surgery and Oncology. 2022;**12**(2):159-163. (In Russ.). DOI: 10.24060/2076-3093-2022- 12-2-159-163

[11] Kasatkin AA, Urakov AL, Lukoyanov IA. Nonsteroidal antiinflammatory drugs causing local inflammation of tissue at the site of injection. Journal of Pharmacology and Pharmacotherapeutics. 2016;**7**(1):26-28. DOI: 10.4103/0976-500x.179359

[12] Urakov A, Urakova N. Osmotic activity of drugs is an important factor of their local action at their injection site: What we don't use to prevent post-injection abscesses. Journal of Pharmaceutical Research International. 2021;**33**(59B):647-650. DOI: 10.9734/ jpri/2021/v33i59B34428

#### **Chapter 4**

## Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain Disorders

*Bhabani Sankar Satapathy, Snigdha Pattnaik, Sangram Keshari Biswal, Biswabhusan Biswal, Pralaya Kumar Sahoo, Himansu Bhusan Samal and Binapani Barik*

#### **Abstract**

Effective treatment of brain disorders remains a tough task in medical science. Age-old brain disorders like Parkinson's (PD) and Alzheimer's (AD) are yet to be managed effectively in spite of fabulous scientific progress over the last decades. Presently available treatment strategies have been found insufficient to tackle the out bursting cases of AD and PD. Indeed, presence of blood-brain barrier (BBB) highly hijacks success of conventional drug therapy. In this regard, phyto bioactive components delivered through nanocarrier (NCs) systems hold ray of hope in improving treatment benefits in brain disorders. Several NCs including polymeric nanoparticles, nanoliposomes, micelles, dendrimers have now being heavily researched to effectively deliver the phyto active components to brain tissue. NCs owing to their structural and physiological uniqueness have now been evolved with great potential for the treatment of brain disorders. Functionalization of brain specific ligands on the surface of NCs further makes them target specific, which might significantly improve bioavailability or reduce the off-target adverse effects. This chapter primarily focuses on recent advancements in phyto component loaded NCs employed for the treatment of brain disorders. The chapter especially covers existing impediments of phyto component based NCs for Parkinson and Alzheimer's disease.

**Keywords:** phyto component, nanocarriers, blood-brain barrier, brain disorder, treatment strategies, recent advances

#### **1. Introduction**

Brain disorders refer to non-transmittable but widely inherited disease problems caused by disruptions in normal body structure and functioning caused by birth

abnormalities or genetic malfunctions [1]. There are many different types of brain problems and diseases that impact the brain, such as infections, tumors, traumas, and neurological abnormalities [2]. Present time demands rapid development of diagnostic, therapeutic, and effective preventive agents to tackle the rising prevalence of cerebral illnesses brought on by the aging global population. The BBB, a unique brain capillary control mechanism that prevents the majority of blood molecules from accessing the central nervous system, has severely hampered brain drug development [3]. As a result, unlike other organs, over than 98% of micro therapeutic compounds and about 100% of bigger therapeutic molecules are unable to reach the brain via the circulatory system. Researchers have found that because of their small size, nanocarriers (NCs) can enter most cells through endocytosis and transcytosis. NCs are sub micrometer objects that act as a unified entity in terms of transport and characteristics. NCs have been extensively researched in recent times for brain medication delivery [4]. Previously, various cosolvents/surfactants like dimethyl sulfoxide, polysorbate 80, ethyl alcohol etc. were used with medications to promote BBB penetration. However, such substances pose high risk for integrity of BBB and might disrupt its protective features. NCs-based therapeutics have recently emerged as a prospective therapeutic for brain diseases and disorders due to their easy transportability across the BBB and distinctive qualities like low toxicity, specificity, biocompatible, tiny size, and solubility [5]. However, conventional drugs used for brain disorders possess lots of severe healthy tissue toxic effects along with low bioavailability at brain tissue, which limits their effective clinical application. In view of this, the secondary metabolites of plants known as phytochemicals or phytonutrients such as alkaloids, saponins, indoles, phytosterols, phenolic acids, isothiocyanates, and phytoprostanes/furanes etc. are being largely investigated in recent days [6]. Phyto bioactive constituents have a long history of use in the management of illnesses and brain disorders in people. Nanocarriers are intended to more effectively carry phytochemicals to the target region (brain). Depending on the carrier material, both hydrophilic and hydrophobic molecules can be loaded inside them. For instance, polar and non-polar molecules are both transported by lipid-based nanocarriers (in the aqueous core and the membrane, respectively) [7]. Owing to their nanosize, tunable surface nature, several nanocarriers like polymeric nanoparticles, nanoliposomes, niosomes, magnetic nanocarriers, dendrimers etc. have been investigated largely over the past years to improve treatment outcomes in brain disorsders. Many phytochemicals delivered through polymeric/lipid based nanocarriers have already shown effective potential in increasing the therapeutic efficacy of phytochemicals. In the present chapter, we want to highlight the major phytocomponents those have been delivered through nanocarriers for improved therapeutic effect in brain disorders. We have mostly covered two important brain disorders, *Viz*. Parkinson's (PD) and Alzheimer's disease (AD) with their recent reports on the use of phtonanocarriers. We summarized potential targets, phytoconstituents, brain medication delivery methods, and nanocarrier systems used in the disease management and therapy in this chapter.

#### **1.1 Blood brain barrier (BBB)**

BBB is essential for allowing biomolecules to enter and exit the brain's neuronal system. Therefore, comprehension of the functional structure and characteristics of BBB is required to increase medication transport to the brain. This protective unit component helps to stop the shuttling of chemicals between the blood and the brain [8]. It consists of layers of vascular endothelial cells that are restrained by tight junctions and other supporting structures. The astrocyte end-feet of the basement

*Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*

membrane surround the endothelial cells discontinuously scanned by microglial cells. Endothelial cells cohesive regions give persistence for the deliberate movement of tiny substances across the BBB. Transcytosis is a type of regulated intracellular transport that occurs to meet the protein and peptide needs of the brain [9]. Depending on the type of molecules, endothelial cells may be able to promote transfer with the help of numerous unique transporting proteins (hydrophilic or hydrophobic) [10]. In recent times, many NCs based novel formulations have been investigated at pre-clinical stage to treat brain disorders like AD and PD. The NCs can encapsulate anti-AD/anti-PD drugs and can efficiently transport them across the BBB.

#### **1.2 Role of P-gp in brain disorders**

The most difficult challenges have been passing the BBB and the bloodcerebrospinal fluid (CSF) barrier in treating brain diseases and disorders. As P-gp carefully mediates material efflux across the BBB, its down-regulation has been linked to the advancement of neurodegenerative disorders and tumors [11]. P-gp inhibition improves medication penetration and subsequent effects across the BBB. Poly(butylcyanoacrylate) NCs have been shown to reduce with P-gp-mediated phenytoin tolerance in rats [12]. Furthermore, when compared to free drug, the incorporation of andrographolide (a neuroprotective agent) into solid lipid NCs increases their permeation to the BBB. In summary, the data suggest that by controlling p-gp, NCs can improve predicted drug penetration and targetability [13].

#### **1.3 Types of nanodrug carriers for brain targeting**

#### *1.3.1 Solid lipid nanoparticles (SLNs)*

SLNs are nanoscale dispersal made of fatty acids, biocompatible triglycerides, or waxes that have been steady by emulsifiers with HLB values less than 12 [14]. SLNs are unique lipid-based biocompatible nanocarrier systems mainly constituting lipid or modified lipid (triglycerides, fatty acids, or waxes) nanostructures (10–1000 nm diameter size range). SLNs have a solid hydrophobic lipid core, in which both hydrophilic and lipophilic drugs can be dispersed.  In vivo testing on rats revealed that Riluzoleloaded SLNs were more effective at transporting the medication into the brain [15]. Docetaxel-loaded SLNs composed of soya lecithin, monostearin, vitamin E, and stearyl amine-betreliesoxybutyric acid (HBA, a ketone body and substrate for the monocarboxylic acid carrier conveyed on the BBB) conjugation showed effective permeation across the intact BBB. Despite the fact that SLNs are easily cleared by the reticuloendothelial system due to their hydrophobicity, they have demonstrated the benefits, biocompatibility, good degradability, and the ability to be surface-covalently for brain targeting [16].

#### *1.3.2 Nanoemulsions*

Nanoemulsions are biphasic dispersion of two immiscible liquids: either water in oil (W/O) or oil in water (O/W) droplets stabilized by an amphiphilic surfactant. These are the thermodynamically stable isotropic system in which two immiscible liquids are mixed to form a single phase by means of an emulsifying agent, i.e., surfactant and co-surfactant [17]. Nano emulsions are promising drug delivery vehicles across the BBB, because they

can solubilize either hydrophilic (W/O nanoemulsions) or hydrophobic (O/W nanoemulsions) drugs. The typical mean droplet diameter of nanoemulsions obtained is around 500 nm or smaller [18]. They have a transparent or hazy look due to their small droplet size, as opposed to the milky white due to attachment with coarse emulsion (whose micron sized droplets partake in multiple light scattering). Additionally, surface functionalization for targeting may make it easier for cells to take up nanoemulsions and the drugs they contain through receptor-mediated endocytosis [19].

#### *1.3.3 Gold NPs*

Gold nanoparticles (AuNPs) provide non-toxic carriers for drug and gene delivery applications**.** AuNPs have been widely studied for their neuroprotective and BBB penetration characteristics in the diagnosis of AD [20]. AuNPs use their special chemical and physical characteristics to load and unload medicines. The gold core is basically inert and non-toxic, which is the first advantage. Another benefit is their simple steps of production. A study reported the production of Au-NPs in the range of 150 nm and the experimental Au-NPs conjugated with glutathione were examined for potential anti-AD effect and it was found that these nanoparticles prevented amyloid aggregation and had a strong anti-AD effect [21].

#### *1.3.4 Nanoliposomes (NLs)*

NLs are lipid based nanoparticles with one or more layers of phospholipid bilayers [22]. Natural sphingomyelin, phosphatidylcholine, and glycerophospholipids are common elements of the phospholipid bilayer. NLs have received a lot of attention because of their good biocompatibility and potential for uploading pharmaceuticals in the aqueous core for systemic therapeutic medication delivery [23]. Another strategy to increase the penetration rate of NLs crossing the BBB is cationization of the conjugated ligands. Other challenges affecting NLs brain delivery include poor stability and low drug loading capacity. Due to the limited number of accessible surface groups, binding ligands to the surface is problematic, and steric hindrance exists [16].

#### *1.3.5 Polymeric nanoparticles (PNPs)*

PNPs have attracted considerable interest over the last few years due to their unique properties and behaviors resulting from their small size. PNPs have the potential for a variety of uses including medication delivery and diagnostics. PNPs are formed of natural or synthetic polymers and have a diameter of 1 to 1000 nm [24]. Depending on the drug-loading techniques, PNPs can either form thermodynamically stable nanocapsules (drugs are encircled by a polymeric shell) or nanospheres (drugs are embedded into polymeric matrix) [25]. PNPs possess certain qualities such as controlled and/or sustained medication release profiles, better half-life, along with easy surface manipulation for site-specific targeting [26].

One of the best-studied biodegradable copolymers is poly (lactic-co-glycolic acid) (PLGA), which breaks down into non-toxic chemicals that the body excretes (H2O and CO2). Through hydrolysis of the ester linkages to its monomeric anions, the polymer degrades in vivo (lactate and glycolate). PEG-PLGA/PLA-PEG NPs were only used in one Phase II clinical trial for metastatic castration-resistant cancer (BIND-014) [27]. There is no PLGA NPs available for the clinical trial in the market for treatment of brain disorders right now. In reality, a variety of pre-clinical studies

#### *Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*

based on drug-loaded NCs are being done. Drugs like curcumin, levodopa, cholesterol, rapamycin etc. have already been investigated for the treatment of AD, PD, high blood pressure, and multiple sclerosis [28]. As a result of the lack of drug-specific transport systems at BBB, many neuroprotective medications are unable to reach the brain. PLGA NPs in such cases have the capacity to carry the loaded drugs across BBB to elicit improved brain delivery [29].

#### *1.3.6 Dendrimers*

The symmetric monodispersed macromolecules known as dendrimers are built from a collection of branching units that are clustered around in an inner core [30]. When there are numerous responsive groups on the surface, the number of arms and

#### **Figure 1.**

*Selected phyto chemicals and their delivery mechanism through various nanocarrier platforms for improved brain permeation.*

surface groups increases dramatically with each generation [31]. They can be used to determine receptor-ligand affinities, administer targeted drugs, or to conduct imaging studies. In case of insoluble and hydrophobic drugs, it is considered as a useful nanocarrier. The interaction of dendrimers with cells is dependent on the hydrophobic contact between the dendrimer arms and the lipid chains of the cell membrane's phospholipid bilayer. Drug distribution to the target site is enabled by the functional groups on the dendrimer's exterior because they act as attachment sites for ligands [32]. Drug is released from dendrimers either through enzymatic dendrimer-drug bond degradation or as a result of environmental changes like temperature and pH [33].

#### *1.3.7 Carbon dots (CDs)*

The performance of CDs, a new type of zero-dimensional carbonaceous nanomaterials, is superior to that of novel metal nanoclusters or inorganic nanocrystals, making them potentially effective information-carrying nanomaterials [34]. The ability to easily modify the surface of CDs for targeted distribution and their tunable fluorescence, which is completely color-tunable from blue to close, regions are two of its most notable things [35]. These novel carriers could be used for chemo- or bio sensing due to their customizable luminous capabilities.

#### *1.3.8 Micelles*

These are single-layered amphiphilic NCs that enable for regulated drug release. These NCs boost the BBB's ability to absorb hydrophobic medicines for the purpose of treating the brain disorders [36]. Micelles have also demonstrated encouraging outcomes to carry different potent compounds, including peptides and small molecules [37]. It has been observed that micelles deliver magnetic resonance imaging for strokes and brain swelling, as well as possesses effective potential for treating AD [38].

Basic mechanism of action of phyto components delivered through NCs has been presented in **Figure 1**.

#### **2. Widely used diagnostic tools in brain disorders**

#### **2.1 Molecular imaging (MI)**

An important area of biomedical science called MI examines pathogenesis or bodily processes at the molecular level. With great sensitivity and specificity, imaging techniques make it simple to visualize, characterize, and quantify activities of interest in the body [39]. It uses cutting-edge methods with a variety of capabilities, including as bioluminescence imaging, microscopy, magnetic resonance imaging, ultrasound, single-photon, x-ray radiography emission computed tomography, and positron emission tomography etc. Infections, brain tumors, and neurological illnesses are just a few of the several brain diseases that have been analyzed and characterized using MI techniques [40].

#### **2.2 Biomarker detection**

Basically, a biomarker is a distinguishable molecule that is linked to a specific disease or protein. A biomarker's ability to distinguish between healthy and ill

*Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*

persons, as well as its precision in determining illnesses stage is the critical component in disease management [41]. Many biomarkers for illnesses and abnormalities of the brain have been identified. However, the lack of suitable methodologies makes its clinical application difficult. Ubiquitin-C-terminal hydrolase-L1 (UCH-L1) plasma limits have been found to be significantly higher in TBI patients than in healthy people, implying that UCH-L1 could be a viable diagnostic marker for the situation [42]. An innovative technique based on the surface plasmon resonance of Au NPs has recently been shown to successfully and swiftly identify the biomarker UCH-L1 in TBI patients with 100% sensitivity and specificity [43].

#### **3. Some important brain disorders, their etiology and treatment strategies**

#### **3.1 Alzheimer's disease (AD)**

AD is caused by a mix of genetically determined and environmental risk factors. Age is the most major risk factor [44]. At the age 65, the chance of developing this disease is about 3 percentages and increasing in to more than 30 percentages by the age of 85. Although the frequency of AD among persons under the age of 65 is unclear, it is estimated that this age group accounts for around 3 percentages of all AD patients [45]. While overall numbers of AD continue to rise with the aging population, age-specific incidence too appears to be declining in a number of countries [46]. The accumulation of amyloid-β protein on neuronal cells after exposure to iron, copper, aluminum, cadmium, and zinc chloride salts suggests that heavy metals might be associated with the development or progression of AD [47]. Researchers have also interlinked the AD pathogenesis with presence of toxic metals such as vanadium, nickel, lead etc. Along with that, certain gases like SO2, NOx, and CO found in polluted air may cause chronic neuroinflammation, cerebrovascular damage, oxidative stress reactive oxygen species (ROS) production, neuron damage/loss and peptide accumulation [48]. Also exposure to high amount of pesticides and insecticides in the environment has also been assigned as other major contributing factors for the progression of AD [49]. The etiology of AD has been summarized in **Figure 2**.

Current therapeutic approaches give symptomatic relief of AD, but do not ensure complete recovery. Phytochemical components in this regard are getting more importance owing to their neuroprotective properties, less toxicity and potential to target various pathogenic pathways implicated in AD. Given the limits of currently available AD medications, the different types of phytocompounds have been suggested as therapeutic agents for the disease management. However, phyto components in their native form show poor bioavailability, low solubility and insufficient BBB permeability, which restrict their effective application. Nanotechnology in this regard has been accepted as an innovative technique to overcome these brain medication delivery restrictions [50]. Phyto constituents loaded NCs have the potential to overcome these challenges while also improving neuroprotective effects in BBB. A numbers of pre-clinical studies on phyto component loaded NCs nanocarriers to treat AD have been reported [28, 50]. However, development of NCs for delivery of phyto componets in AD is still limited due to challenging formulation procedures at industrial prospects [51]. Though, several in-vitro and in-vivo studies have been carried out; however, detailed studies by using small laboratory animals to human testing is still very necessary. When administering medicines for the treatment of AD, precise analysis of important parameters like physicochemical

**Figure 2.** *Probable causes/risk factors involved in the progression of Alzheimer's disease.*

properties, particle size, biodistribution, and bioavailability of loaded drug(s) is extremely necessary [52]. Furthermore, the stability of NCs is another striking factor in decreasing the toxic metabolites agglomeration in the BBB. While the conventional formulation of compounds remains difficult to show their action at brain tissue, NCs could be alternative strategy for effective permeation across the BBB with utmost specificity. Problems of instability, limited solubility, low bioavailability issue associated with phytocomponents could be sufficiently addressed by suitable NCs. Clinical studies have also demonstrated efficacy of phyto component loaded NCs in various brain disorders [53].

Further, concerns of safety and brain absorption of NCs must be addressed in AD patients. At present, creating nano phytopharmaceuticals for large-scale production in line with good laboratory practice standards is required to maintain the quality of end product, while retaining patient compliance.

#### *3.1.1 Recent research findings on phyto component based NCs for AD*

Over the past years, many active phyto components have been delivered through NCs and have been shown to elicit better pharmacological effect than the free phyto components. Multi-functional liposomal NPs were prepared and coated with an amyloid-binding curcumin-lipid ligand to target amyloid. Along with that two other ligands were also conjugated with the liposomal NPs to target transferrin and LDL receptors present over BBB. The study indicated excellent brain targeting and amyloid peptide aggregation of experimental NCs [54].

The functionalization of NPs with plant phytochemicals has been investigated by Zhang J. et al., 2014. Polyphenols were used to functionalize selenium NPs. The nanosized selenium in the study was coated with epigallocatechin gallate (EGCG), a polyphenol found in green tea. EGCG is well-known for its neuroprotective

*Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*

properties, specifically its ability to limit the formation of many amyloid-forming proteins engaged in the course of Alzheimer's disease. The EGCG-stabilized selenium prevented atrial fibrillation while also dissolved the developed fibrils. Furthermore, at very low concentrations, the specified NCs was observed to decrease DNA fragmentation and ROS generation [55].

In another research, A. Mathew, et al., 2012 investigated the potential of curcumin loaded NCs in in neuronal targeting in vitro. Curcumin is one of the widely investigated phytocomponent over the past decade for the treatment of several brain disorders. In the study, Tet-1 targeted PLGA loaded curcumin nanoparticles with antiamyloid and anti-oxidant capabilities was found effective in the diagnosis of AD. The incorporation of Tet-1 neuropeptide into the PLGA-curcumin nanoparticle increased its neuronal targeting efficacy in vitro. Although additional conclusions can only be formed after extensive in vivo research, the findings of this exploratory investigation suggested that curcumin could be a promising drug in the treatment of AD [56]. Resveratrol is another important phytocomponent, which is now heavily investigated for its neuroprotective properties. Grape seed and grape skin extracts containing resveratrol were shown to be more efficient at suppressing aggregation. However, after the intravenous injection, resveratrol is quickly metabolized (in less than 2 hours) in the liver and intestinal epithelial cells into glucuronic acid and sulphate conjugations of phenolic groups, which are subsequently excreted. According to a recent study, anti-transferrin receptor monoclonal antibody (OX26 mAb) functionalized SLNs was found as an efficient carrier system for transporting the extract into the target encephalon [57]. In vitro studies on human brain like endothelial cells showed that OX26 SLNs were significantly more effective at cellular absorption than conventional SLNs and SLNs functionalized with an unspecific antibody. Experimental SLNs functionalized with OX-26 showed higher transcytosis capacity [57].

#### **3.2 Parkinson's disease (PD)**

PD is a complicated condition with biological as well as environmental origins. The common major risk factor for PD is age, with a median onset age of 60 years. The prevalence of the condition rises with age, peaking at 93.1 (per 100,000 person-years) in people aged 70 to 79 [58]. Though, the exact etiology of PD is yet to be understood. But several risk factors like accumulation of heavy metals, cigarette smoking, pesticides, herbicides, genetic factors, high amount caffeine consumption etc. have been identified for the development and progression of PD. The basic etiology of PD has been summarized in **Figure 3**.

Nanosizing the formulation is an option for increased PD protection to improve the efficiency and bioavailability of crude extracts. Furthermore, adding one or two phytonanocarrier of nano-sized range bioactive chemicals delivers considerable health advantages for specific conditions, thus reducing the need for several drugs. Example like Curcumin and resveratrol NLs exhibited anticancer activity against prostate cancer. According to research curcumin loaded NCs improved the therapeutic and bioavailability efficacy during PD [59]. Experimental NCs significantly lowered oxidative stress and apoptotic cell death in fly model of PD. Similarly, an alginate curcumin nanocomposite also showed a lowering in brain oxidative stress and cell death in a transgenic Drosophila PD model [60]. Curcumin loaded NCs improved bioavailability of curcumin in the blood circulation and also the brain pharmacokinetic [61, 62]. The methods of administration utilized were more significant in increasing nanocurcumin bioavailability in the circulatory system and penetrating to the BBB. Ginsenoside also

**Figure 3.** *Probable causes/risk factors involved in the progression of Parkinson's disease.*

protects neurons against 6-hydroxydopamine- and iron-induced neurotoxicity. These compounds have an important role in lowering oxidative stress as reported in the recent experiment [63]. Various studies have also indicated that nanoresveratrol can aid in the prevention of PD and enhance neuronal survival in the face of oxidative stress [64].

#### *3.2.1 Recent research findings on phyto component based NCs for PD*

Aggregation of amyloid protein in the brain as a result of oxidative stress causes the formation of Lewy bodies and the selective death of dopamine neurons [65]. Polyphenolic substances have poor oral bioavailability, higher metabolic turnover, and decreased BBB permeability. The effect of a produced alginate-curcumin nanocomposite on the climbing capacity of PD model flies, apoptosis, and lipid peroxidation in the brain of PD model flies was investigated in a recent work [65]. The chow was supplied at the known concentrations of 105, 103, and 101 g/mL of alginatecurcumin nanocomposite, and the flies were allowed to consume for 24 days. In the Drosophila model of PD, there was a substantial dose-dependent delay in the reduction in climbing ability, as well as a lowering in apoptosis and oxidative stress.

Another recent work highlighted the neuro protective effect of *Bacopa monnieri* loaded SLNs in vivo. The work involved incorporation of *Bacopa monnieri* extract into SLNs to develop dissolvable microneedle arrays and evaluate its neuroprotective activity. Quality by design approach was employed for optimizing the SLNs formulations based on several in vivo and in vitro properties. Mechanical strength, in vitro release studies, permeation studies, skin irritation test, histopathological analysis, biochemical studies, and behavioral tests of SLNs loaded microneedle arrays were performed. The experimental phyto constituent loaded microneedle patches were shown to be mechanically robust, nonirritant. The optimized formulation was also found to produce decreased degree of bradykinesia along with high motor coordination, and balance ability [66].

*Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*

In another study, neuronal protective effect of photoactive component of Ginsen delivered in nanoparticle formulation was investigated. The principal active component of the plant, ginsenoside (Rg1) has been reported to protect the neurons against 6-hydroxydopamine-induced death and neuronal toxicity [67]. Although the photoactive components of Ginsen play a critical role in reducing oxidative stress, but the poor bioavailability problem has always been an issue In the work, Rg1 loaded nanoparticles were found to enhance the in vivo activity and bioavailability of these compound than the crude extracts. Nanosizing the formulation showed an enhanced protective effect against PD. The nano ginsenosides were developed using a nanoemulsion technique with average particle diameter of 19.9 nm. Bioavailability of the selected Rg1 loaded nanoparticles showed significantly higher bioavailability than the crude extract in the rat brain tissue [63].

Numerous studies have reported that different nanoparticle-loaded phytochemicals (e.g., vitamin E, resveratrol, curcumin, and hyaluronic acid) with an average particle size of 100nm resulted in higher ROS scavenging efficiency and lower lipid peroxidation in patients with PD. A work by Pangeni R et al., evidenced antioxidant and neuroprotective effect of nanoencapsulated thymoquinone (TQ ) in PD induced rat model. The experimental TQ-loaded mesoporous silica NPs (90nm in size) were able to cross the BBB. Results showed that the experimental silica NPs enhanced drug delivery to all major brain areas including cortex, thalamus, midbrain, and hypothalamus and significantly reduced oxidative stress biomarkers [64]. Few significant recent research outcomes on phyto component based NCs on AD and PD has been summarized in **Table 1**.

#### **Table 1.**

*List of some important phyto components delivered through nanocarrier systems for the treatment of Parkinson (PD) and Alzheimer's disease (AD) [63].*

#### **4. Conclusion**

Technological advancements in the scientific tools along with exploration of novel formulation strategies have made a substantial impact on diagnosis and treatment of major brain disorders like AD/PD. Undoubtedly, delivery of phyto components through nanocarrier based platforms will bring significant impact on the

*Recent Advancements in Phyto Component Based Nanocarriers for Improved Treatment of Brain… DOI: http://dx.doi.org/10.5772/intechopen.110585*