Photodynamic Therapy Induced Mitochondrial Targeting Strategies for Cancer Treatment: Emerging Trends and Insights.

The perpetuity of cancer prevalence at a global level calls for development of novel therapeutic approaches with improved targetability and reduced adverse effects. Conventional cancer treatments have a multitude of limitations such as nonselectivity, invasive nature, and severe adverse effects. Chemotherapy is also losing its efficacy because of the development of multidrug resistance in the majority of cancers. To address these issues, selective targeting-based approaches are being explored for an effective cancer treatment. Mitochondria, being the moderator of a majority of crucial cellular pathways like metabolism, apoptosis, and reactive oxygen species (ROS) homeostasis, are an effective targeting site. Mitochondria-targeted photodynamic therapy (PDT) has arisen as a potential approach in this endeavor. By designing photosensitizers (PSs) that preferentially accumulate in the mitochondria, PDT offers a localized technique to induce cytotoxicity in cancer cells. In this review, we intend to explore the crucial principles and challenges associated with mitochondria-targeted PDT, including variability in mitochondrial function, mitochondria-specific PSs, targeted nanocarrier-based monotherapy, and combination therapies. The hurdles faced by this emerging strategy with respect to safety, optimization, clinical translation, and scalability are also discussed. Nonetheless, mitochondria-targeted PDT exhibits a significant capacity in cancer treatment, especially in combination with other therapeutic modalities. With perpetual research and technological advancements, this treatment strategy is a great addition to the arsenal of cancer treatment options, providing better tumor targetability while reducing the damage to surrounding healthy tissues. This review emphasizes the current status of mitochondria-targeted PDT, limitations, and future prospects in its pursuit of safe and efficacious cancer therapy.

Dual-Drug-Loaded Topical Delivery of Photodynamically Active Lipid-Based Formulation for Combination Therapy of Cutaneous Melanoma.

Topical administration of anti-cancer drugs along with photodynamically active molecules is a non-invasive approach, which stands to be a promising modality for treating aggressive cutaneous melanomas with the added advantage of high patient compliance. However, the efficiency of delivering drugs topically is limited by several factors, such as penetration of the drug across skin layers at the tumor site and limited light penetrability. In this study, curcumin, an active anti-cancer agent, and chlorin e6, a photoactivable molecule, were encapsulated into lipidic nanoparticles that produced reactive oxygen species (ROS) when activated at 665 nm by near-infrared (NIR) light. The optimized lipidic nanoparticle containing curcumin and chlorin e6 exhibited a particle size of less than 100 nm. The entrapment efficiency for both molecules was found to be 81%. The therapeutic efficacy of the developed formulation was tested on B16F10 and A431 cell lines via cytotoxicity evaluation, combination index, cellular uptake, nuclear staining, DNA fragmentation, ROS generation, apoptosis, and cell cycle assays under NIR irradiation (665 nm). Co-delivering curcumin and chlorin e6 exhibited higher cellular uptake, better cancer growth inhibition, and pronounced apoptotic events compared to the formulation having the free drug alone. The study results depicted that topical application of this ROS-generating dual-drug-loaded lipidic nanoparticles incorporated in SEPINEO gel achieved better permeation (80 ± 2.45%) across the skin, and exhibited the improved skin retention and a synergistic effect as well. The present work introduces photo-triggered ROS-generating dual-drug-based lipidic nanoparticles, which are simple and efficient to develop and exhibit synergistic therapeutic effects against cutaneous melanoma.

Exploring microfluidics and membrane extrusion for the formulation of temozolomide-loaded liposomes: investigating the effect of formulation and process variables.

Liposomes have gained much attention in drug delivery since the entry of liposomal Doxorubicin (Doxil®) into the market. Liposomes can entrap lipophilic, hydrophilic as well as amphiphilic drug molecules due to their distinctive structural features. Yet the clinical translation of liposomes is limited due to the reproducibility issues owing to a lack of information related to the impact of process parameters and formulation variables on designed liposomes. Recently, preparation techniques like membrane extrusion and microfluidics have been reported to produce liposomes in a reproducible manner. The present research study selected an amphiphilic drug Temozolomide (TMZ). It has a short half-life in the plasma due to its pH-dependent stability. Various critical and non-critical parameters affecting the critical quality attributes were identified and studied using risk-based assessment. The effect of various material attributes and process parameters on the critical quality attributes of the temozolomide-loaded liposomes prepared by microfluidics and membrane extrusion techniques were investigated in detail. Liposomes in the size range of 100-150 nm were targeted. Both techniques were optimized with a minimum number of critical process parameters. The obtained information will be beneficial to formulation scientists for designing liposomes for an amphiphilic drug on a large scale.

Emerging Applications of Hydroxypropyl Methylcellulose Acetate Succinate: Different Aspects in Drug Delivery and Its Commercial Potential.

Hydroxypropyl methylcellulose acetate succinate (HPMCAS) has multi-disciplinary applications spanning across the development of drug delivery systems, in 3D printing, and in tissue engineering, etc. HPMCAS helps in maintaining the drug in a super-saturated condition by inhibiting its precipitation, thereby increasing the rate and extent of dissolution in the aqueous media. HPMCAS has several distinctive characteristics, such as being amphiphilic in nature, having an ionization pH, and a succinyl and acetyl substitution ratio, all of which are beneficial while developing formulations. This review provides insights regarding the various types of formulations being developed using HPMCAS, including amorphous solid dispersion (ASD), amorphous nanoparticles, dry coating, and 3D printing, along with their applicability in drug delivery and biomedical fields. Furthermore, HPMCAS, compared with other carbohydrate polymers, shows several benefits in drug delivery, including proficiency in imparting stable ASD with a high dissolution rate, being easily processable, and enhancing bioavailability. The various commercially available formulations, regulatory considerations, and key patents containing the HPMCAS have been discussed in this review.

An insight into photodynamic therapy towards treating major dermatological conditions.

Photodynamic therapy (PDT), as the name suggests is a light-based, non-invasive therapeutic treatment method that has garnered immense interest in the recent past for its efficacy in treating several pathological conditions. PDT has prominent use in the treatment of several dermatological conditions, which consequently have cosmetic benefits associated with it as PDT improves the overall appearance of the affected area. PDT is commonly used for repairing sun-damaged skin, providing skin rejuvenation, curbing pre-cancerous cells, treating conditions like acne, keratosis, skin-microbial infections, and cutaneous warts, etc. PDT mediates its action by generating oxygen species that are involved in bringing about immunomodulation, suppression of microbial load, wound-healing, lightening of scarring, etc. Although there are several challenges associated with PDT, the prominent ones being pain, erythema, insufficient delivery of the photosensitizing agent, and poor clinical outcomes, still PDT stands to be a promising approach with continuous efforts towards maximizing clinical efficacy while being cautious of the side effects and working towards lessening them. This article discusses the major skin-related conditions which can be treated or managed by employing PDT as a better or comparable alternative to conventional treatment approaches such that it also brings about aesthetic improvements thereof.

Formulating Ternary Inclusion Complex of Sorafenib Tosylate Using ß-Cyclodextrin and Hydrophilic Polymers: Physicochemical Characterization and In Vitro Assessment.

Sorafenib tosylate (SFNT) is the first-line drug for hepatocellular carcinoma. It exhibits poor solubility leading to low oral bioavailability subsequently requiring intake of large quantities of drug to exhibit desired efficacy. The present investigation was aimed at enhancing the solubility and dissolution rate of SFNT using complexation method. The binary inclusion complex was prepared with ß-cyclodextrin (ß-CD). The molecular docking studies confirmed the hosting of SFNT into hydrophobic cavity of ß-CD, while the phase solubility studies revealed the stoichiometry of complexation with a stability constant of 735.8 M-1. The ternary complex was prepared by combining the SFNT-ß-CD complex with PEG-6000 and HPMC polymers. The results from ATR-IR studies revealed no interaction between drug and excipients. The decreased intensities in ATR-IR peaks and changes in chemical shifts from NMR of SFNT in complexes indicate the possibility of SFNT hosting into the hydrophobic cavity of ß-CD. The disappearance of SFNT peak in DSC and XRD studies revealed the amorphization upon complexation. The ternary complexes exhibited improved in vitro solubility (17.54 µg/mL) compared to pure SFNT (0.19 µg/mL) and binary inclusion complex (1.52 µg/mL). The dissolution profile of ternary inclusion complex in 0.1 N HCl was significantly higher compared to binary inclusion complex and pure drug. In cytotoxicity studies, the ternary inclusion complex has shown remarkable effect than the binary inclusion complex and pure drug on HepG2 cell lines.

Potential herbal constituents for psoriasis treatment as protective and effective therapy.

Psoriasis is a multifactorial and chronic skin disorder. It is a recurrent disease that requires incessant therapy. Psoriasis treatment includes topical and systemic routes using synthetic drugs that lead to severe unwanted adverse effects. Herbal therapy is widely used for thousands of years in countries like China and India. The use of herbal therapy in the developed region enhanced to a great extent and showed better efficacy towards psoriasis alone or as adjuvant to synthetic therapy. Herbal medicines have gained great attention in the treatment of psoriasis due to their lesser side effects compared to synthetic drugs. In this review, the various plant sources which have been found effective in psoriasis and can be used to develop novel therapeutics have been discussed. The mechanisms by which the phytoconstituents elicit anti-psoriatic activity and various research studies that have proven the effectiveness of these natural products have also been compiled in this review.

Design of temozolomide-loaded proliposomes and lipid crystal nanoparticles with industrial feasible approaches: comparative assessment of drug loading, entrapment efficiency, and stability at plasma pH.

Temozolomide is a drug approved for treating glioblastomas, which has 100% oral bioavailability but gets degraded at physiological pH thus having very short half-life and only 20-30% brain bioavailability. Due to its amphiphilic nature, reported nanoformulations exhibits poor drug loading. The objective of this work was to formulate lipid-based drug delivery systems to enhance the brain bioavailability by prolonging the drug release and circulation time of the drug to overcome the limitations of the existing therapies and possible reduction of side effects. The size of the nanocarriers obtained was less than 300 nm and the PDI obtained was less than 0.3. The designed formulation showed higher entrapment efficiency as compared to the other reported nanocarriers of temozolomide. The designed formulations showed prolonged drug release from 12 to 20 h compared to 6 h for the pure drug. About 95% of the pure drug was degraded at plasma pH at the end of 12 h, whereas only 68% and 77% was degraded when entrapped inside the lipid crystal nanoparticles and proliposomes respectively. Further, pharmacokinetic and animal studies can confirm the potential of these for improvement of brain bioavailability.

Insightful exploring of microRNAs in psoriasis and its targeted topical delivery.

Psoriasis is a common immune-mediated inflammatory skin disease. It includes multifaceted interaction between the immune system and the keratinocytes. Recent studies depicted the role of microRNAs (miRNAs) in hyperproliferation of keratinocytes and inflammatory cytokine production, which serve as biomarkers for diagnosis, monitoring treatment response, and prognosis. miRNAs are small nucleotide sequenced noncoding RNAs. Deregulation of miRNAs was found to be the most common factor in the studies pertaining to psoriasis. Hence, miRNA-based targeting for psoriasis treatment became the primary field of current research. miRNA due to its spatial and chemical properties offer different challenges in the process of its delivery. The topical delivery of different siRNAs and genes has paved a way to similar delivery of miRNA. The topical delivery of miRNAs to the skin can bring a revolutionary change in the field of psoriasis treatment.

Emerging role of nanocarriers based topical delivery of anti-fungal agents in combating growing fungal infections.

The incidences of fungal infections have greatly increased over the past few years, particularly in humid and industrialized areas. The severity of such infections ranges from being asymptomatic-mild to potentially life-threatening systemic infections. There are limited classes of drugs that are approved for the treatment of such infections like polyenes, azoles, and echinocandins. Some fungi have developed resistance to these drugs. Therefore, to counter drug resistance, intensive large scale studies on novel targeting strategies and formulations are being conducted, which have gained impetus lately. Conventional formulations have limitations such as higher doses, frequent dosing, and several side effects. Such limiting factors have paved the path for the emergence of nanotechnology and its applications. This further gave formulation scientists the possibility of encapsulating the existing potential drug moieties into nanocarriers, which when loaded into gels or creams provided prolonged release and improved permeation, thus giving on-target effect. This review thus discusses the newer targeting strategies and the role of nanocarriers that could be administered topically for the treatment of various fungal infections. Furthermore, this approach opens newer avenues for continued and sustained research in pharmaceuticals with much more effective outcomes.

Emerging Trends in Topical Delivery of Curcumin Through Lipid Nanocarriers: Effectiveness in Skin Disorders.

Curcumin is a unique molecule naturally obtained from rhizomes of Curcuma longa. Curcumin has been reported to act on diverse molecular targets like receptors, enzymes, and co-factors; regulate different cellular signaling pathways; and modulate gene expression. It suppresses expression of main inflammatory mediators like interleukins, tumor necrosis factor, and nuclear factor κB which are involved in the regulation of genes causing inflammation in most skin disorders. The topical delivery of curcumin seems to be more advantageous in providing a localized effect in skin diseases. However, its low aqueous solubility, poor skin permeation, and degradation hinder its application for commercial use despite its enormous potential. Lipid-based nanocarrier systems including liposomes, niosomes, solid lipid nanoparticles, nanostructured lipid carriers, lyotropic liquid crystal nanoparticles, lipospheres, and lipid nanocapsules have found potential as carriers to overcome the issues associated with conventional topical dosage forms. Nano-size, lipophilic nature, viscoelastic properties, and occlusive effect of lipid nanocarriers provide high drug loading, hydration of skin, stability, enhanced permeation through the stratum corneum, and slow release of curcumin in the targeted skin layers. This review particularly focuses on the application of lipid nanocarriers for the topical delivery of curcumin in the treatment of various skin diseases. Furthermore, preclinical studies and patents have also indicated the emerging commercialization potential of curcumin-loaded lipid nanocarriers for effective drug delivery in skin disorders. Graphical Abstract.

Recent Expansions on Cellular Models to Uncover the Scientific Barriers Towards Drug Development for Alzheimer's Disease.

Globally, the central nervous system (CNS) disorders appear as the most critical pathological threat with no proper cure. Alzheimer's disease (AD) is one such condition frequently observed with the aged population and sometimes in youth too. Most of the research utilizes different animal models for in vivo study of AD pathophysiology and to investigate the potency of the newly developed therapy. These in vivo models undoubtably provide a powerful investigation tool to study human brain. Although, it sometime fails to mimic the exact environment and responses as the human brain owing to the distinctive genetic and anatomical features of human and rodent brain. In such condition, the in vitro cell model derived from patient specific cell or human cell lines can recapitulate the human brain environment. In addition, the frequent use of animals in research increases the cost of study and creates various ethical issues. Instead, the use of in vitro cellular models along with animal models can enhance the translational values of in vivo models and represent a better and effective mean to investigate the potency of therapeutics. This strategy also limits the excessive use of laboratory animal during the drug development process. Generally, the in vitro cell lines are cultured from AD rat brain endothelial cells, the rodent models, human astrocytes, human brain capillary endothelial cells, patient derived iPSCs (induced pluripotent stem cells) and also from the non-neuronal cells. During the literature review process, we observed that there are very few reviews available which describe the significance and characteristics of in vitro cell lines, for AD investigation. Thus, in the present review article, we have compiled the various in vitro cell lines used in AD investigation including HBMEC, BCECs, SHSY-5Y, hCMEC/D3, PC-2 cell line, bEND3 cells, HEK293, hNPCs, RBE4 cells, SK-N-MC, BMVECs, CALU-3, 7W CHO, iPSCs and cerebral organoids cell lines and different types of culture media such as SCM, EMEM, DMEM/F12, RPMI, EBM and 3D-cell culture.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000 ng/mL (r2  > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Plant-based larvicidal agents: An overview from 2000 to 2018.

Current review aims to systematically segregate, analyze and arrange the key findings of the scientific reports published on larvicidal plants including larvicidal formulations. The investigation was carried out by analyzing the published literature in various scientific databases, subsequently, the key findings of the selective scientific reports having larvicidal potency (LC50) of extract or isolated oil<100 µg/mL were tabulated to provide the concise and crucial information. Special emphasis was given on reports in which LC50 of extract or isolated oil was reported to be < 10 µg/mL, genus or species documented in multiple independent studies, advancement in larvicidal formulations and activity of isolated phytoconstituents. Extensive analysis of published literature revealed that the larvicidal potency of herbal resources varied from sub-microgram/ml to practically insignificant. Overall, this unprecedented summarized and arranged information can be utilized for design, development and optimization of herbal based formulation having potential larvicidal activity.

Microsponge: An emerging drug delivery strategy.

Microsponges are the spherical particles ranging from 5 to 300 µm in size. These are further made up of clusters of smaller spheres. They are designed for delivering the drug efficiently at a comparatively lesser dose and enhancing the stability, modifying the drug release profile and minimizing the side effects. Microsponge drug delivery system decrease transdermal invasion of the active ingredient into the skin while increasing the time the drug remains on the skin surface or within the epidermis. Preparation of the microsponges includes two techniques: Liquid-liquid suspension polymerization and Quasi-emulsion solvent diffusion method. Their characterization and evaluation can be done in many ways like particle-size measurement and porosity, morphology, true density determination, analyzing the rheological properties, and dissolution studies. Present work focuses on the detailed study of the microsponge drug delivery system. This will help the reader to get all the information regarding the microsponge delivery systems.

The potential of siRNA based drug delivery in respiratory disorders: Recent advances and progress.

Lung diseases are the leading cause of mortality worldwide. The currently available therapies are not sufficient, leading to the urgent need for new therapies with sustained anti-inflammatory effects. Small/short or silencing interfering RNA (siRNA) has potential therapeutic implications through post-transcriptional downregulation of the target gene expression. siRNA is essential in gene regulation, so is more favorable over other gene therapies due to its small size, high specificity, potency, and no or low immune response. In chronic respiratory diseases, local and targeted delivery of siRNA is achieved via inhalation. The effectual delivery can be attained by the generation of aerosols via inhalers and nebulizers, which overcomes anatomical barriers, alveolar macrophage clearance and mucociliary clearance. In this review, we discuss the different siRNA nanocarrier systems for chronic respiratory diseases, for safe and effective delivery. siRNA mediated pro-inflammatory gene or miRNA targeting approach can be a useful approach in combating chronic respiratory inflammatory conditions and thus providing sustained drug delivery, reduced therapeutic dose, and improved patient compliance. This review will be of high relevance to the formulation, biological and translational scientists working in the area of respiratory diseases.

Prediction of in vivo plasma concentration-time profile from in vitro release data of designed formulations of milnacipran using numerical convolution method.

The aim of this study was to predict the in vivo plasma drug level of milnacipran (MIL) from in vitro dissolution data of immediate release (IR 50 mg and IR 100 mg) and matrix based controlled release (CR 100 mg) formulations. Plasma drug concentrations of these formulations were predicted by numerical convolution method. The convolution method uses in vitro dissolution data to derive plasma drug levels using reported pharmacokinetic (PK) parameters of a test product. The bioavailability parameters (Cmax and AUC) predicted from convolution method were found to be 106.90 ng/mL, 1138.96 ng/mL h for IR 50 mg and 209.80 ng/mL, 2280.61 ng/mL h for IR 100 mg which are similar to those reported in the literature. The calculated PK parameters were validated with percentage predication error (% PE). The % PE values for Cmax and AUC were found to be 7.04 and -7.35 for IR 50 mg and 11.10 and -8.21 for IR 100 mg formulations. The Cmax, Tmax, and AUC for CR 100 mg were found to be 120 ng/mL, 10 h and 2112.60 ng/mL h, respectively. Predicted plasma profile of designed CR formulation compared with IR formulations which indicated that CR formulation can prolong the plasma concentration of MIL for 24 h. Thus, this convolution method is very useful for designing and selection of formulation before animal and human studies.

Study the effect of formulation variables on drug release from hydrophilic matrix tablets of milnacipran and prediction of in-vivo plasma profile.

The objective of this study was to design oral controlled release (CR) matrix tablets of Milnacipran using hydroxypropyl methylcellulose (HPMC) as the retardant polymer and to study the effect of various formulation factors such as polymer proportion, polymer viscosity, compression force and also the pH of dissolution medium on the in-vitro release of drug. Two viscosity grade of HPMC (15 K and 100 K) were used in the proportion of 50, 100, 150 and 200 mg per CR tablet. In-vitro release rate was characterized using various model dependent approaches and model independent dissolution parameters [T50% and T80% dissolution time, mean dissolution time (MDT), mean residence time (MRT), dissolution efficiency (DE)]. The statistical analysis was performed on all the model independent approaches using student t test and ANOVA. Results were found that as polymer concentration (50 mg to 200 mg) and viscosity (15 K to 100 K) increases, the MDT, MRT, T50% and T80% extended significantly. Drug release rate was found to be significantly different at different hardness. In-vivo human plasma concentration--time profile was predicted from in-vitro release data using convolution method. Predicted human pharmacokinetic parameters shows that the design CR formulation has capability to sustained the plasma drug level of milnacipran.

Determination of 3-acetyl-11-keto-ß-boswellic acid in analytical and biological samples using streamlined and robust RP-HPLC method.

AKBA (3-acetyl-11-keto-ß-boswellic acid) is a phytoconstituent derived from Boswellia serrata extract and utilized in the management of rheumatoid arthritis. Drug delivery approaches showed interest in delivering AKBA with advanced nanotechnology. There is a need for a simple, sensitive, and robust HPLC method that can determine AKBA in complex nanoformulation and in vitro and ex vivo samples. In the proposed work, the RP-HPLC method was developed using a mobile phase comprising a mixture of acetonitrile : milli Q (90 : 10) at detection λmax 250 nm. The method exhibited a linearity of 250 to 20 000 ng mL-1 with a high correlation coefficient of 1. The limit of detection and limit of quantification for the analytes were found to be 41.32 ng mL-1 and 125.21 ng mL-1, respectively. In the accuracy study, the % recovery of AKBA was found to be 98% to 102%, and the precision study showed less than 2% relative standard deviation. The developed method was found to be robust under chromatographic conditions with changes in pH and mobile phase mixture ratio. The method was also explored for forced degradation study, and the results showed the successful separation of degradation products from the AKBA. Further, the RP-HPLC method was applied for the quantification of AKBA in topical nanoformulations and different matrices, such as skin matrices and adhesive tapes. The method was able to measure entrapment efficiency (93.13 ± 1.94%), drug loading (25.83 ± 0.54%), drug assay in a gel matrix (96.99 ± 3.89%), drug amount in stratum corneum (7.90 ± 0.62 µg cm-2), and drug amount in viable skin layers (33.94 ± 0.21 µg cm-2) with high-speed reproducibility. The developed method can be utilized for the routine analysis of AKBA in conventional and complex formulations in academia and industry.

Bridging the gap in rheumatoid arthritis treatment with hyaluronic acid-based drug delivery approaches.

Rheumatoid Arthritis (RA) is a chronic, inflammatory, auto-immune disease that is majorly associated with the degradation of the synovial linings of the joints. It is a progressive disease that reduces the life span in affected individuals. Nanoparticles involving hyaluronic acid (HA) have gained the limelight for designing target-specific and more effective drug delivery options for RA. HA is found abundantly in the synovial fluid and acts as a natural ligand for the CD44 receptors. The targeted delivery approach using CD44 as the target can help in minimizing off-target drug distribution. These HA-based surface-decorated nanocarriers, hydrogels, and MNs are cutting-edge strategies that promise tailored delivery, fewer side effects, and more patient adherence to address the common issues associated with RA therapy. Considering the above facts, this review attempts to discuss the role of HA in making more effective formulations for therapeutic delivery in treating RA. Additionally, it provides a comprehensive overview of the potential advancements, mainly in treating RA by HA-based topical, transdermal, and parenteral drug delivery systems, with relevant case studies. The existing difficulties and potential paths for future research on HA-based non-conventional formulations for the management of RA are also discussed.