INTRODUCTION:

Liposomes are colloidal nanostructured spherical shape vesicles, consisting of one or more bilayers of phospholipids with a particle size ranging from 30 nm to a few micrometres ^1,2. Dr Alec D Bingham FRS, a British haematologist was the first to describe liposomes in 1961³. Liposome is derived from two Greek words 'Lipos' meaning fat and 'Soma' meaning body⁴. Liposomes can be widely used as carriers for various molecules in cosmetic, pharmaceuticals, food and farming industries⁵. Several bioactive elements, antioxidants and antimicrobials can be entrapped in its vesicular structure to protect their functionality and stability. Liposomes can entrap a variety of drugs of varied lipophilicity in its supramolecular geometry.

Lipophilic drugs interact with the bilayer surface while hydrophilic drugs get entrapped into the internal aqueous filled core. Entrapment of drugs with intermediate solubility into the internal core is achieved by modification of pH and chemical composition of the aqueous core. These small spherical vesicles have the flexibility to provide selective passive targeting or active targeting by coupling with site specific ligands. The approach of targeting is associated with improved pharmacokinetics, therapeutic efficacy with reduction in toxicity of the entrapped molecule. Liposomes in general, exhibit a special bio nano interaction in contact with plasma proteins. The presence of serum component opsonin promotes phagocytosis, while dysopsonin suppresses the process. The reason for the phagocytosis of liposomes is the hydrophobicity of the liposomal surface. The plasma protein competes for the surface space of the liposome and forms the protein corona. Formation of the corona depends on the surface charge and length of exposure. This leads to a change in the biological activity and elicits immune response ^6,7. Therefore, a drug in same route of administration thus exhibits a difference in pharmacokinetics and distribution profile when administered in a liposomal formulation and in a non-liposomal formulation. The complexity of the lipid vesicles needs a detailed study on the ADME profile for a new liposome drug product for a safe and efficient dosing regimen. For an innovator product, it is essential to look for comprehensive specifications to achieve the desired quality, with minimum batch to batch variations and optimum therapeutic effect. The FDA regulatory guidelines emphasize the Chemistry, Manufacturing and Control (CMC) with detailed properties and characteristics of a liposome drug product with the human pharmacokinetics, bioavailability, bioequivalence and labelling requirements for the industry ^8,9.

Quality by Design (QbD) is a systematic approach of predefined objectives, based on scientific and risk-based proactive method. Application of QbD in new pharmaceutical product development results in a fast and improved manufacturing and development efficiency¹⁰. Risk assessment helps to identify the potential hazards and parameters which can affect the product quality. The QbD approach for formulations development are described in Figure 1.

Figure 1: QbD approach for formulation development

This review focuses on the critical attributes in process development and regulatory requirement of quality liposomal drug product.

1. QUALITY BY DESIGN METHODOLOGY OF LIPOSOME PREPARATION

A product quality is a feature that meets customer needs and free from deficiency. In a pharmaceutical formulation it is a measure of number of parameters like the drug, excipient, manufacturing factors and packaging system as described in Figure 2.

Figure 2: Determinants of product quality

Identification of quality target product profile (QTPP)

For a liposomal product the performance of drug product and targeting delivery becomes the primary therapeutic aim to ensure the quality, safety and efficacy of the product. Therefore the preliminary step of the product development based on the QbD approach is the description of the target product profile (TPP), which includes the dosage form, route of administration, dosage strength, appearance, pharmacokinetics, bioequivalence, assay, identity, impurities, content uniformity, dissolution etc ⁹.

Identification of critical quality attributes (CQAs)

The second step is to define the quality attributes of the formulation to ensure the desired product quality ^11,12. The CQAs are usually defined properties. These are the physicochemical and biological parameters which affect the performance of the product.

To identify the CQAS for liposomal products the physicochemical properties of liposomes are very crucial. In a liposomal formulation, the physicochemical parameters like vesicle size, surface charge and number of bilayers affects the circulation half-life and drug encapsulation ¹³. Liposomes in the size range of <0.1 micron are less susceptible to phagocytosis than larger vesicle and enables the slow release of the drug with longer circulation time ¹⁴. Accessibility of liposomes of vesicle size ranging from 190Å to few microns are restricted to some tissues, whereas liposomes of vesicle sizes larger than 600 Å have little access to the capillaries found in skeletal, cardiac, and smooth muscles, lung, skin, subcutaneous tissue. Liposomes larger than 0.5 μm are confined to the intravascular spaces ^15,16. Therefore the residence time of small vesicles is prolonged compared to large vesicles. The surface charge determines the in vivo clearance, tissue distribution, intracellular uptake and stability of the formulations. Charged liposomes are cleared more readily than uncharged liposomes probably due to the formation of protein corona ¹⁷. The stability of bilayers determines the efficient entrapment of the drug without leakage ¹⁸. High drug entrapment permits lower total volume/excipient for administration of formulation. The thermodynamic property are useful indicators of the fluidity and homogeneity of the lipid bilayer membrane. Cholesterol acts as fluidity buffer and can be incorporated into the phospholipid membrane as a high concentration in the range of 1:1 or 2:1 molar ratio of cholesterol and phosphatidylcholine ^19,20. The half-life decreases with increase in vesicle diameter, negative surface charge, density and fluidity of the bilayer membrane. High dispersity index is an indication of tendency to aggregation. Osmolarity determines the tendency of rupture or contraction of the supra molecular structure so that leakage of the drug from the entrapped vesicle can be predicted.

So the critical quality attributes (CQAs) that determines the product specification for liposomal products are mean particle size, polydispersity index, osmolality, zeta-potential and stability of the entrapped drug in the supramolecular structure of liposome^21,22,23.

Critical material attributes

Critical material attribute (CMA) determines the materials whose variability has an impact on critical quality attribute. Therefore, it is needed to be monitored or controlled to ensure desired drug product quality¹⁰.

Lipid Components

The main and the foremost component for liposomes are the lipids. Selection of lipids determines the specificity of the formulations and the stability.

In liposomes the bilayers formation takes place due to the presence of large differences in surface free energy of the aqueous and the hydrophobic environment mediated through the specific molecular geometry of phospholipids ²⁴. The phase transition temperature of the lipids, which in turn affects the bilayers fluidity, influences the behaviour of liposomes. Below the phase transition it makes the membrane less ordered, while above the transition it makes the membrane more ordered ²⁵. The phase transition is directly affected by the hydrocarbon chain length, presence of unsaturation, surface charge, and nature of head group ²⁶. Presence of cholesterol has an important modulatory effect on the bilayer formulation ²⁷. It intercalates into the membrane in a way so that the parallel alignment of the aliphatic chain occurs to the acyl chain, while the hydroxyl group orients towards the aqueous surface. This intercalation alters the free motion of carbon molecule in the acyl chain and restricts the conformational change from trans to gauche form ²⁸. The prevention of aggregation and the improvement of encapsulation efficiency of these composite vesicles of lipids is achieved by using combinations of neutral phospholipids such as phosphatidylcholine or sphingomyelin, mixed with varying amounts of cholesterol as a stabilizing agent. These compositions are referred as conventional liposomes ^29,30. Incorporation of specific glycolipids and phospholipids in the stealth liposome increases the circulation time in the blood and avoid rapid detection and uptake by RES³¹.

The stability of liposome lies in the selection and composition of lipids. Lipids undergo degradation through oxidation, peroxidation and hydrolysis. The stability of lipids is also affected by the methods used in their manufacturing. Drug to lipid ratio is an important concern in the formulation of liposome as it affects the release profile of the drug.

So the selection of lipids plays a vital role in the formulation of liposomes ³². The quality attributes of the lipids are illustrated in Table no. 1.

Stability

The design of stability studies for liposomal products should be carried out by the ICH guidelines - Q1A (R2) stability testing of New Drug Substances and Products⁴¹. Physical and chemical stability of the liposome drug product at varying conditions should be tested as per ICH guidelines. The physical instability of the liposome is because the hydrated bilayers vesicles are thermodynamically unstable, and they exhibit a meta stable state with high energy. The chemical instability is due to the susceptibility of phospholipids that undergo chemical degradation. The consideration of in vivo stability of liposomes in a single dose study depends on the property of liposome. The elements for in vivo stability are the integrity of the encapsulated form of drug in circulation and the constant ratio of unencapsulated to encapsulated drug substance. Liposomes are susceptible to fusion, aggregation, and leakage of the encapsulated drug substance during storage. Depending on the method of formulation and nature of composition, the fluidity, surface charge, permeability and osmolality of the surroundings leads to leakage of the hydrophilic drug from the internal core of the liposome ⁴². So the storage temperature of this dispersion should be strictly defined and controlled.

Regulatory requirement of clinical pharmacokinetics and mass balance study for liposomal drug product

The in vivo availability of the liposome is affected by the route of administration. Following intravenous administration, the administered liposomes undergo phagocytosis or are taken up by the reticuloendothelial system ⁴³. Thus, liposomes get accumulated primarily in the liver, spleen and to a lesser extent in bone marrow, lymph, etc. This alters the tissue distribution and the rate of clearance of the drug. The pharmacokinetics of the drug encapsulated in liposome are mostly determined by its vesicle size, amount of lipids, surface charge and stability of bilayers.

For establishing a dosage regimen and dose concentration response relationship, a pharmacokinetics study should be designed with proper population pharmacokinetics approach^44,45,46. The pharmacokinetics parameters like area under curve (AUC), maximum plasma concentration (Cmax), time to reach the maximum plasma concentration (Tmax), half-life, volume of distribution, renal clearance, total clearance and accumulation should be estimated. Mass balance studies should be conducted on radiolabel moiety (¹⁴C, ³H) of drugs assayed in blood plasma, urine and faecal sample ⁴⁷.

A comparative ADME study on the same drug in same route of administration should be conducted of the liposomal and non-liposomal drug product. A single dose cross over or parallel study design should be employed for the specific diseased population on an appropriate number of subjects ^48,49.

The recommended pharmacokinetic studies are listed in Table 4.

Additional Pharmacokinetics study on effect of food, drug interactions and exposure response study should be provided as an additional pharmacokinetics data for regulatory submission ^50,51.

Table 4: Recommended pharmacokinetics studied for liposomal drug products

Type of Study	Objective
A single-dose pharmacokinetic study	To compare the difference in pharmacokinetics between the liposome and non-liposome drug product
A multiple-dose study	To evaluate the pharmacokinetic parameters of the drug substance of the administered liposome drug product
A dose-proportionality study	To determine the expected therapeutic dose range after administration of the liposome drug product from the pharmacokinetic study.

Bioequivalence

The criteria for regulatory submission of a liposomal drug product for bioequivalence study need to meet the label claim and should be comparable with the non-liposomal product. IVIVC should be established if possible ⁵². The safety considerations of liposomal drug product should be reported with liposome protein interaction ^53,54,55.

Labeling requirements

The CDER guidance web site gives guidance on the current labeling requirements. The nonproprietary name of the liposomal product should include terminology to differentiate the product type as a liposome or a pegylated liposome.

[DRUG] Liposome Type X [DOSAGE FORM]

[DRUG] Pegylated Liposome Type X [DOSAGE FORM]

The innovator liposomal product approved by FDA for a drug and dosage form will be considered type A and subsequent generic products of the same drug and dosage form, should sequentially numbered as type B, C, D, . . . Z with respect to ANDA submission. A caution note should emphasize the different behaviour pattern of liposome drug products. For a reconstituted product, proper reconstitution instructions and appropriate usage period to be provided. Proper storage and reconstitution conditions should also be provided for robustness of the liposomal drug product ⁵⁶.

2.1 REGULATORY VIEW FOR NEW DRUG APPROVAL

In the USA - The first draft guidance on liposomal drug products was issued by FDA in the year 2002. The guidance describes the information required to submit for NDA and ANDA application for a liposomal drug product to be submitted for review to CDER. Since then several liposomal drug products have received market approval in USA. Recent updates of the Food and Drug Administration for industry on Liposome Drug Products, Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labelling documentation has been published in April 2018. This guidance solely focuses on technical aspects of this type of products. Recommendations on clinical efficacy and safety studies, nonclinical pharmacology or toxicology studies or drug-lipid complexes are not provided.

In the European Union - The Common Technical Document (CTD) format is used for submission of liposomal product for marketing approval via Central Procedure in the European Union. The guidelines on the data requirements for the development of intravenous liposomal products with reference to an innovator liposomal product was published on July 2011 by EMA, emphasizes on pharmaceutical quality, quality characterization, establishing pharmaceutical comparability, pharmaceutical developments of the applicant’s product, nonclinical and clinical requirement, dose, design, analytes measurement, assessment of efficacy and safety of the liposomal product as required for CTD format. The agency recommends the generic product should be made like the innovator’s product in qualitative and quantitative terms.

3. CURRENT AND FUTURE DEVELOPMENT OF LIPOSOMES

The potential advantages of liposomal delivery over controlled pharmacokinetics and pharmacodynamics to improve bioavailability with reduction in toxicity have resulted in the exploration of its application in various diseases. The present market scenario of liposomal products reveals that there are fifteen approved formulations available worldwide for the human use. The chronological list of approved liposomal products is described in table 5.

The therapeutic areas covered by liposome-based products are mostly cancer therapy ⁵⁷, fungal diseases ⁵⁸, viral vaccines ⁵⁹, analgesic ⁶⁰ and photodynamic therapy ⁶¹. These marketed products are administered by intravenous, intramuscular, spinal or epidural route. The extensive revolutionized development with the application of quality by design in the field of pharmaceutical research has opened the doors of achieving targeted liposomal products through inhalation and topical therapy. Present data shows two products - Amikacin through aerosol delivery for lung infections and T4 endonuclease V through topical delivery for skin pigmentation have reached the Phase III clinical trial ^62.63,64. Another phase III Clinical trial product ThermoDox®, a temperature sensitive liposomal formulation of doxorubicin for the treatment of Hepatocellular carcinoma and chest wall breast cancer was found to attain the rapid onset of membrane permeability by sharp thermal transition due to local hyperthermia, when administered in combination with radio frequency ablation ⁶⁵. Endotag-I, a cationic liposome of Paclitaxel, under phase II clinical trial has been designed to interact with negatively charged endothelial cells in tumors during angiogenesis, thus damaging the tumor blood supply without affecting healthy tissue ⁶⁶. Several novel liposomal products are under clinical investigation ⁶⁷.

Table 5: List of approved liposomal product.

Sl No	Year of approval	Trade name of liposomal product	Content of Liposomal product
1	1993	Epaxal®	Inactivated hepatitis A virus⁶⁸
2	1995	Doxil®	Doxorubicin
3	1995	Abelcet®	Amphotericin B
4	1996	DaunoXome®	Daunorubicin
5	1996	Amphotec®	Amphotericin B
6	1997	Ambisome®	Amphotericin B
7	1997	Inflexal® V	Inactivated hemaglutinine of Influenza virus strains A and B
8	1999	Depocyt®	Cytarabine/Ara-C
9	2000	Myocet®	Doxorubicin
10	2000	Visudyne®	Verteporphin
11	2004	Mepact®	Mifamurtide
12	2004	DepoDur™	Morphine sulfate
13	2011	Exparel®	Inactivated hepatitis A virus
14	2012	Marqibo®	Vincristine
15	2015	Onivyde™	Irinotecan

CONCLUSION:

The various regulatory guidelines link industry, academia and regulatory bodies for speedy development and marketing approval of new liposomal products. A specific, sensitive assay, with good biopharmaceutical characterization and proper manufacturing control can help to avoid significant differences in expectations in clinical and bioequivalence studies. Liposomoligists can come with novel therapeutically beneficial liposomal products and generic versions of the existing products comparatively faster with the help of the regulatory guidance.

ACKNOWLEDGEMENT:

The authors express their sincere gratitude to The Management, Krupanidhi Group of Institutions for supporting the work through Krupanidhi Research Incubator Centre (K-RIC) program under Krupanidhi College of Pharmacy and to Dr Nilanjan Das, Accendere: CL Educate Ltd.

AUTHOR CONTRIBUTIONS:

Both the authors were equally involved in the drafting, gathering information and design of framework of the manuscript.

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

ABBREVIATION USED:

ADME – Absorption, Distribution, Metabolism and Excretion

ANDA- Abbreviated New Drug Application

API- Active Pharmaceutical Ingredient

AUC- Area Under Curve

CDER- Centre for Drug Evaluation and Research

CMC- Chemistry, Manufacturing and Control

CQAs- Critical Quality Attributes

CTD- Common Technical Document

DLS- Dynamic Light Scattering

DNA- Deoxy ribonucleic acid

DSC –Differential Scanning Calorimetry

ELSD- Evaporating Light Scattering Detector

FDA- Food and Drug Administration

FFF- Field Flow Fractionation

HPLC- High Performance Liquid Chromatography

IVIVC- Invitro Invivo Correlation

MS- Mass Spectroscopy

NDA- New Drug Application

RES- Reticulo Endothelial System

SEC- Size Exclusion Chromatography

TBA- Thiobarbituric acid

TEM- Transmission Electron Microscopy

TLC- Thin Layer Chromatography

UV- Ultraviolet

REFERENCES:

1. Cullis PR et al. Generating and loading of liposomal systems for drug-delivery applications. Adv Drug Deliv Rev. 1989; 3(3): 267–82.

2. Tagawa T, Ueda M inventors: Mitshubishi Pharma, Corp. assignee. Liposome. United States patent application US 10/581.169. 2007 Oct 25.

3. Srivastava et al. Development of Liposomal Cosmeceuticals. J Chem Pharm Res. 2016; 8(2): 834-38.

4. Dua JS, Rana AC, Bhandari AK. Liposome: methods of preparation and applications. Inter J Pharm Studies Res. 2012; 3:14-20.

5. Akbarzadeh A et al. Liposome: classification, preparation, and applications. Nanoscale Res Lett. 2013; 8(102): 1–9.

6. Fleischer CC, Payne CK. Secondary structure of corona proteins determines the cell surface receptors used by nanoparticles. J Phys Chem B. 2014; 118(49): 14017–26.

7. Caracciolo G et al. Evolution of the Protein Corona of Lipid Gene Vectors as a Function of Plasma Concentration. Langmuir. 2011; 27 (24):15048-53.

8. US Food and Drug Administration (USFDA). Guidance for industry - Liposome drug products (Draft guidance 2191), August, 2002.

9. FDA, CDER. Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labelling Documentation Guidance for Industry. April, 2015.

10. Jay M J, Girish PK. A Comparative Review of the USFDA Guidelines on Process Validation Focusing on the Importance of Quality by Design (QbD). Research J. Pharm. and Tech. 2017; 10(4): 1257-60.

11. Rajesh BN et al. Formulation and Characterization of Efavirenz Nanosuspension by QbD approach. Research J. Pharm. and Tech 2017; 10(9):2960-72.

12. Hardik BR et al. Development of Sustained Release Pellets of Galantamine HBr by Extrusion Spheronization Technique Incorporating Risk based QbD Approach. Research J. Pharm. and Tech 2018; 11(11): 4899-10.

13. Labiris NR, Dolovich MB. Pulmonary drug delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications. Br J Clin Pharmacol. 2003; 56(6): 600–12.

14. Allen TM. Liposomal drug formulations. Rationale for development and what we can expect for the future. Drugs. 1998; 56: 747–56.

15. Nakanishi M, Noguchi A. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative. Adv Drug Delivery Rev. 2001;52: 197–207.

16. Juliano RL, Lin G. The interaction of plasma proteins with liposomes: Protein binding and affects on the clotting and complement systems. In: H.J. Baldwin and H.R. Six (Eds.), Liposomes and Immunology, Elsevier, North-Holland, Amsterdam, 1980.

17. Senior J, Crawley JCW, Gregoriadism G. Tissue distribution of liposomes exhibiting long half-lives in the circulation after intravenous injection, Biochim Biophys Acta. 1985; 839:l-8.

18. Allen TM, Cleland LG. Serum-induced leakage of liposome contents. BiochimBiophys Acta. 1980; 597: 418–26.

19. Collins JJ, Phillips MC. The stability and structure of cholesterol-rich codispersions of cholesterol and phosphatidylcholine. J Lipid Res. 1982; 23(2): 291–98.

20. Kirby C, Clarke J, Gregoriadis G. Cholesterol content of small unilamellar liposomes controls phospholipid loss to high density lipoproteins in the presence of serum. FEBS Lett. 1980; 111 (2):324–28.

21. European Medicine Agency. Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product. EMA/Committee Hum Med Prod 806058/2009/Rev 02. 2013; 44:1–13.

22. Chemin C et al. Supramolecular organization of S12363 liposomes prepared with two different remote loading processes. Biochimica et Biophysica Acta. 2009; 1788: 926-35.

23. Derle DV et al. Development, Characterization and Evaluation of Niosomes and Liposomes of Bacitracin Zinc. Research J. Pharm. and Tech. 2010;3 (4): 1295-1300.

24. Lasic DD. In: Liposomes: From biophysics to applications, Elseiver, New York, 1993.

25. Morata LR, Giannotti MI, Sanz F. Influence of Cholesterol on the Phase Transition of Lipid Bilayers: A Temperature-Controlled Force Spectroscopy Study. Langmuir. 2012;28(35): 12851-60.

26. Jain NK. Controlled and Novel Drug Delivery, CBS Publisher and distributors, New Delhi, 2009.

27. G Gregoriadis. Liposome Technology, 3rd ed.; Vol. 1, CRC Press: Boca Raton, Florida, 1984.

28. New R.R.C. In: liposomes: Apractical approach, IRL/Oxford university press, Oxford, London, 1990.

29. Woodle MC, Papahadjopoulos D. Liposome preparation and size characterization. Methods Enzymol. 1989; 171: 193–217.

30. Jensen GM, Bunch TH. Conventional liposome performance and evaluation: Lessons from the development of Vescan. J Liposome Res. 2007;17(3–4): 121–37.

31. Huang Z et al. Progress involving new techniques for liposome preparation. Asian J Pharm Sci. 2014; 9(4): 176–82.

32. Akbarzadeh A et al. Liposome: Classification, preparation, and applications. Nanoscale Res Lett. 2013; 8(1): 1–8.

33. Zylberberg C, Matosevic S. Pharmaceutical liposomal drug delivery: a review of new delivery systems and a look at the regulatory landscape. Drug Deliv. 2016;23(9):3319–29.

34. Naoki Y, Keiko , Eriko W, Takuya I. Liposome and liposome composition. 16013355 (2018).

35. M Yasmin Begum et al. Ketorolac Tromethamine Loaded Liposomes: Development, Characterization and In Vitro Evaluation. Research J. Pharm. and Tech. 2011;4(11): 1766-1771.

36. Grohganz H. et al. Quantification of various phosphatidylcholines in liposomes by enzymatic assay. AAPS Pharm Sci Tech. 2003;4(4): E63.

37. Hein R, Uzundal CB, Hennig A. Simple and rapid quantification of phospholipids for supramolecular membrane transport assays. Org Biomol Chem. 2016;14(7): 2182–85.

38. Wagner A, Vorauer-Uhl K. Liposome Technology for Industrial Purposes. J Drug Deliv. 2011; 2011:1–9.

39. Porfire A et al. A quality by design approach for the development of lyophilized liposomes with simvastatin. Saudi Pharm J. 2017; 25(7):981–92.

40. Ramandeep S, Ashutosh U, Kale MK. Effect of Liposomes as a carrier on Pharmacokinetics of Cisplatin. Research J. Pharm. and Tech 2018; 11(11): 5073-5077.

41. ICH Expert Working Group. ICH Guideline Q1A(R2) Stability Testing of New Drug Substances and Products. Int Conf Harmon. 2003.

42. Xia Y, Sun J,Liang D. Aggregation, fusion, and leakage of liposomes induced by peptides. Langmuir. 2014; 30(25): 7334-42.

43. Mayer JD, Shabbits JA. The role of liposomal drud delivery in molecular and pharmacological strategies to overcome multidrug resistance. Cancer metastasis Rev. 2001; 20:87.

44. Hope WW et al. Population pharmacokinetics of conventional and intermittent dosing of liposomal amphotericin B in adults: a first critical step for rational design of innovative regimens. Antimicrob Agents Chemother. 2012; 56(10): 5303-08.

45. Briguila ML et al. Influence of cholesterol on liposome stability and on invitro drug release . Drug Delivtransl res. 2015;5(3):231-42.

46. FDA. Guidance for Industry Population Pharmacokinetics. FDA Guid. 1999.

47. Bekersky I et al. Pharmaco- kinetics, excretion, and mass balance of 14C after administration of 14C-Cholesterol-labeled ambisome to healthy volunteers. J Clin Pharmacol. 2001; 41: 963–71.

48. FDA. Guidance for Industry Safety Testing of Drug Metabolites Guidance for Industry. Guidance 2016.

49. FDA. Bioavailability and Bioequivalence Studies submitted in NDAs or INDs - General considerations (Draft). Food Drug AdmGuid Ind [Internet]. 2014.

50. U.S. Food and Drug Administration. Bioequivalence Studies with Pharmacokinetic Endpoints for Drugs Submitted Under an ANDA Guidance for Industry Bioequivalence Studies with. Cder. 2013.

51. FDA. Guidance for Industry: Exposure-Response Relationships - Study Design, Data Analysis and Regulatory Applications. FDA Guid. 2003.

52. Fortuna A. In Vitro In Vivo Correlation (IVIVC): A Strategic Tool in Drug Development. J Bioequiv. 2011;8(4):1–12.

53. Kimelberg HK. Protein-liposome interactions and their relevance to the structure and function of cell membranes. Mol Cell Biochem.1976; 10(3):171-90.

54. Le Maire M, Champeil P, Møller JV. Interaction of membrane proteins and lipids with solubilizing detergents. BiochimBiophys Acta – Biomembr. 2000; 1508 (1–2): 86–111.

55. Verchère A, Broutin I, Picard M. Reconstitution of Membrane Proteins in Liposomes. Methods Mol Biol. 2017; 1635: 259-82.

56. FDA, Cder. Nonproprietary Naming of Biological Products Guidance for Industry. US Dep Heal Hum Serv. 2017.

57. Jaafari MR et al. Liposome composition for cancer treatment. 15/130116 (2016).

58. Walsh T. Liposomal nystatin treatment of fungal infection. 09/950980 (2001).

59. Dwek RA et al. Liposome treatment for viral infections. 11/832891 (2007).

60. Krugner-higby LA, Heath TD, Smith LJ. Liposome-encapsulated opioid analgesics. 10/350207 (2013 ).

61. Albrecht V et al. Liposomal formulation of hydrophobic photosensitizer for photodynamic therapy. 11/298729 (2005).

62. Li Z, et al. Characterization of nebulized liposomal amikacin (Arikace™) as a function of droplet size. J Aerosol Med Pulm Drug Deliv. 2008;21: 245–54.

63. Clancy JP. Clinical trials of lipid-associated aerosolized amikacin: The arikace™ story. In PediatricPulmonology;Wiley-LissDivJohnWileyand Sons Inc,Hoboken, NJ, USA, 2009.

64. Wolf P et al. Topical treatmentwith liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-α. J Investig Dermatol. 2000; 114: 149–56.

65. Wood B et al. Phase I study of thermally sensitive liposomes containing doxorubicin (ThermoDox) given during radiofrequency ablation (RFA) in patients with unresectable hepatic malignancies. In Proceedings of the Gastrointestinal Cancers Symposium, Orlando, FL, USA, The American Society of Clinical Oncology: Alexandria, VA, USA, 2007.

66. Eichhorn ME et al. Vascular targeting by EndoTAG™-1 enhances therapeutic efficacy of conventional chemotherapy in lung and pancreatic cancer. Int J Cancer. 2010; 126: 1235–45.

67. Bulbake U et al. Liposomal formulations in clinical use: An updated review. Pharmaceutics. 2017;9(2):1–33.

68. 68. Bovier, P.A. Epaxal®: a virosomal vaccine to prevent hepatitis A infection. J Expert Rev of Vaccines. 2008;7: 1141-50.

Received on 03.05.2019 Modified on 22.05.2019

Research J. Pharm. and Tech 2019; 12(8):4057-4065.

DOI: 10.5958/0974-360X.2019.00699.1

Quality Attributes	Regulatory requirement	Justification
Quantity of lipid in the formulation	Expressed in molar ratio or by %w/w of the lipid to the drug substance or as milligram (mg) per millilitre (ml) of the formulation³³	Chain length and presence of saturation and unsaturation affects formation of bilayers and drug loading capacity, drug release and circulation time
Selection of Lipids 1. Natural lipids 2. Synthetic and semi synthetic lipids	1. Percentage of each lipid, percentage of each fatty acid, country of origin of the source material, storage conditions, stability profile, content of impurity and specifications ^34,34,36,37 2. Specifications of the starting materials with a proof of structure based upon standard spectroscopic techniques ⁹	The pharmacological and toxicological property can vary significantly with the compositions of the lipids
Stability and stress testing	Exposure to temperature, pH, light and oxygen at high and low levels	It determines the stability of the liposomal formulation,

Type of test	Methods used	Objective
Lipid Content and purity	HPLC (UV, MS, ELSD detection)	Efficacy and stability of liposomal product
Lipid Compositions	Spectroscopic techniques, other analytical techniques	Stability of liposomal product
Degradation products of the lipid components	HPLC, TLC, UV absorbance, TBA reagent, and iodometry	Efficacy and stability of liposomal product

Type of test	Methods used	Objective
Size distribution	DLS, Nanosight, SEC, FFF or TEM	Represents the idea for bio distribution and the size should be within 50- 200nm.
Polydispersity index	DLS	Detection of presence of aggregates
Surface charge	Zeta potential	Detection of in vivo clearance, distribution characteristics to tissue, and intracellular uptake of the liposome.
Lamellarity and thickness of the membrane	TEM	Evaluate the extent of release of active substances from liposomal product.
Phase transition temperature	DSC	Evaluate permeability of the membrane and predicts premature release through leakage.
Internal volume	FFF, Nanosight, DLS, SEC or TEM	Determination of drug content.
Immunochemical properties	Validated assay techniques	Evaluation of hypersensitivity reactions.
Estimation of residual solvents	Validated analytical techniques	Evaluation of safety.
Osmolarity	Validated analytical techniques	Evaluation of tendency to rupture or contraction of the liposome structure.
pH	Validated analytical techniques	Determine the property and function of the product.
Loading efficiency	Chromatography, ultracentrifugation, gel filtration, or dialysis	Evaluation of amount of the active substance in each fraction, determination of leakage.
Estimation of antimicrobial, anti-oxidant and preservative content	Validated analytical techniques	Evaluation of toxicity.
In Vitro release study	Suitable release testing method	Study of release profile.
Biological activity	In vivo test	In vivo release study.
Sterility	Dry-heat sterilization and autoclaving, sterilizing filtration	Suitability for administration.
Bacterial endotoxins	Lysate reagent and other validated method as applicable or by pyrogen test	Presence of pyrogens.
Long term stability study	ICH Q1A(R2) guideline	Evaluation of stability and integrity of liposomal product.