formulations: HPLC method
development and stability studies
Antoinette Kiihn
B.Pharm
Dissertation submitted in partial fulfillment of the requirements for
the degree Magister Scientiae in the Department of Pharmaceutics
at the North-West University, Potchefstoom Campus.
Supervisor: Prof. W. Liebenberg
Co-supervisor: Ms. A. Wessels
POTCHEFSTROOM
2008
4 Heel eerste aan my Skepper en Hemelse Vader wat vir my die talent, vermoe en uithouvermoe gegee net om my M.Sc. te voltooi.
4- Prof. Wilna Liebenberg, my studieleier, dankie dat Prof, in my geglo en
my genoeg vertrou net om my eie diskresie met my studies te gebruik. Baie dankie vir die ongelooflike leiding en motivering wanneer ek dit nodig gehad net. Prof, is die heel beste studieleier ooit!
4 Anita Wessels, my mede-studieleier, baie dankie vir die goeie advies en
hulp met die HPLC metode ontwikkeling en validasie en al die HPLC probleemoplossing sessies, ten spyte van hoe besig jy was.
4 Julia Handford vir die goeie taalversorging, baie dankie vir al die hulp en
wenke wat my gehelp het om my verhandeling te vorm.
4 Nicole Stieger vir jou hulp met die taalversorging en baie dankie vir al die
tegnologiese hulp wat jy altyd so vriendelik verleen het.
4- Prof. Banie Boneschans vir die vertaling van my uittreksel.
4- Daleen von Mollendorf and Madelein Geldenhuys, Cenqam personeel
vir die bystand met my studies in metode ontwikkeling. Baie dankie dat julle my tou wys gemaak het om volgens GLP in 'n geakkrediteerde
laboratorium te werk en om my te vorm tot die analis wat ek vandag is. 4- Liezl-marie Nieuwoudt vir die vervaardiging van pro-Pheroid vir my studie.
4- Sharlena dat jy my produkte op stabiliteit geplaas het.
4- Samuel en Martha baie dankie vir die hulp met die glasware en die
vriendelike gesigte.
4 My ouers, Attie en Elna, baie dankie dat julle my die geleentheid gegee
het om te studeer en my die hele pad ondersteun het. Baie dankie dat julle altyd daar was vir my en in my geglo het.
Dankie dat jy in my geglo het, my gehelp het deur die moeilike tye en dat jy my gemotiveer het om dit wat ek begin het, klaar te maak.
4- Nicolene Lubbe, dankie vir jou vriendskap en dat jy altyd daar was
wanneer ek iemand nodig gehad het om mee te gesels.
4- NRF en Inovasie fonds, vir die befondsing, wat my baie gehelp het deur
TABLE OF CONTENT i
ABSTRACT vii UITTREKSEL ix AIM AND OBJECTIVES xi
ABBREVIATIONS xii
CHAPTER 1: HUMAN IMMUNODEFICIENCY VIRUS (HIV) AND ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS)
1.1 Introduction 1 1.2 HIV Statistics 2 1.3 Discovery of HIV 5
1.4 Pathology 8 1.5 Transmission of HIV virus 9
1.6 HIVIifecycle 11
1.7 Prognosis 121.7.1 Paediatric prognosis 12 1.7.2 Adult prognosis 12 1.7.3 Prediction of prognosis 13
1.8 Clinical HIV staging system 13
1.9 Treatment of HIV 15
1.9.1 ARV's classification and mechanism of action 15
1.9.2 Prophylaxis for HIV exposure 16
1.9.3 Adult ARV treatment 17 1.9.4 Children ARV treatment 19 1.9.5 Nutritional support for children 22
CHAPTER 2: PHYSICO-CHEMICAL AND PHARMACOLOGICAL PROPERTIES OF ANTIRETROVIRAL DRUGS
2.1 Introduction 23 2.2 Abacavir sulphate 23 2.2.1 Chemical properties 23 2.2.2 Pharmacology 24 2.2.3 Pharmacokinetics 24 2.2.4 Adverse effects 25 2.2.5 Paediatric and adult dose 25
2.3 Nevirapine 26
2.3.1 Chemical properties 26 2.3.2 Pharmacology 26 2.3.3 Pharmacokinetics 27 2.3.4 Adverse effects 27 2.3.5 Paediatric and adult dose 28
2.4 Efavirenz 28
2.4.1 Chemical properties 28 2.4.2 Pharmacology 29 2.4.3 Pharmacokinetics 29 2.4.4 Adverse effects 29 2.4.5 Paediatric and adult dose 30
2.5 Lamivudine 30
2.5.1 Chemical properties 30 2.5.2 Pharmacology 31 2.5.3 Pharmacokinetics 31
2.5.5 Paediatric and adult dose 32 2.6 Stavudine 33 2.6.1 Chemical properties 33 2.6.2 Pharmacology 34 2.6.3 Pharmacokinetics 34 2.6.4 Adverse effects 34 2.6.5 Paediatric and adult dose 35
2.7 Conclusion 35
CHAPTER 3: PHEROID™ TECHNOLOGY
3.1 Introduction 38 3.2 Structural characteristics of Pheroids™ 38
3.3 Composition and molecular organisation of Pheroids™ 39
3.4 Design of Pheroids™ 40 3.5 Types of Pheroids™ 41 3.6 Metabolism, targeting and distribution 41
3.7 Advantages of the Pheroid™ system 42
3.8 Formulations 43
3.8.1 Abacavir sulphate 43 3.8.2 Nevirapine and efavirenz 43
3.9 Conclusion 44
CHAPTER 4: METHOD DEVELOPMENT AND VALIDATION
4.1 Introduction 45
4.2.1 Method for abacavir sulphate in pro-Pheroid and
abacavir sulphate with TBHQ in pro-Pheroid 46
4.2.1.1 Chromatographic conditions 46
4.2.1.2 Standard preparation 47
4.2.1.3 Sample preparation 47
4.2.2 Method for nevirapine in pro-Pheroid 48
4.2.2.1 Chromatographic conditions 48
4.2.2.2 Standard preparation 48
4.2.2.3 Sample preparation 49
4.2.3 Method for efavirenz in pro-Pheroid 49
4.2.3.1 Chromatographic conditions 49
4.2.3.2 Standard preparation 50
4.2.3.3 Sample preparation 50
4.2.4 Method for lamivudine in pro-Pheroid 50
4.2.4.1 Method A 50
4.2.4.1.1 Chromatographic conditions 50
4.2.4.1.2 Standard preparation 51
4.2.4.1.3 Sample preparation 51
4.2.4.1.4 Results and recommendations 51
4.2.4.2 Method B 52
4.2.4.2.1 Chromatographic conditions 52
4.2.4.2.2 Standard preparation 52
4.2.4.2.3 Results and recommendations 53
4.3 Validation 53
4.3.3 Range 54 4.3.4 Repeatability 54
4.4 Validation results 55
4.4.1 Method for abacavir in pro-Pheroid and abacavir
with TBHQ in pro-Pheroid 55
4.4.1.1 Accuracy 55 4.4.1.2 Linearity and range 55
4.4.1.3 Repeatability 56 4.4.2 Method for nevirapine in pro-Pheroid 57
4.4.2.1 Accuracy 57 4.4.2.2 Linearity and range 57
4.4.2.3 Repeatability 58 4.4.3 Method for efavirenz in pro-Pheroid 59
4.4.3.1 Accuracy 59 4.4.3.2 Linearity and range 59
4.4.3.3 Repeatability 60
4.5 Conclusion 60
CHAPTER 5: STABILITY TESTING AND RESULTS
5.1 Introduction 61
5.1.1 Reasons for stability testing 61 5.1.2 Problem / adverse effects due to instability 62
5.2 Stability programme 63
5.2.1 Storage conditions 65 5.2.2 Stability tests conducted 65
5.3.1 Abacavir sulphate in pro-Pheroid 65 5.3.1.1 Results 66 5.3.1.2 Discussion 68 5.3.2 Nevirapine in pro-Pheroid 68 5.3.2.1 Results 68 5.3.2.2 Discussion 69 5.3.3 Efavirenz 69 5.3.3.1 Results 69 5.3.3.2 Discussion 70
5.4 Antimicrobial preservation efficacy 71
5.4.1 Introduction 71 5.4.2 Results 72 5.5 Conclusion 74 CHAPTER 6: CONCLUSION 75 BIBLIOGRAPHY 78 ANNEXURE A 83
ANNEXURE B: Conference contributions 105
Pro-Pheroid-based antiretroviral formulations: HPLC
method development and stability studies
H1V/A1DS poses an enormous healthcare challenge worldwide and it has become one of the leading causes of death in children. Globally, the number of people living with HIV has risen from around 8 million in 1990 to more than 33.2 million today (AVERT, 2008). According to UNAIDS (2008) and WHO (2008), 1.7 million adults and 330 000 children died from AIDS related disease in 2007. Due to the continual mutation of the HI virus, causing resistance to existing ARV's, it is essential to either develop new drugs or to design delivery systems that would increase the efficacy of existing ARV's.
Pheroid™ technology is a novel delivery system consisting mainly of modified essential fatty acids. The effectiveness of Pheroid™ technology has been proven by several national and international clinical trials for formulations based on this technology (Grobler et al., 2008). The Pheroid™ is similar to an emulsion, but contains two liquid phases, as well as a dispersed gas phase, which are associated with the fatty acid dispersed phase. The one liquid phases is aqueous, the other is oil-based.
Pro-Pheroid production is identical to that of the Pheroid™, except that no aqueous phase is introduced. Instead, drug substances are introduced to the oil phase.
This study was conducted to determine the respective stabilities of three ARV's in the pro-Pheroid delivery system. High performance liquid chromatography (HPLC), was used to evaluate the stability of each ARV in the pro-Pheroid system. The analytical methods were developed and validated in-house. The three formulations were stored at 5°C, 25°C+60%RH, 30°C+65%RH and 40°C+75%RH for three months. Accelerated stability trials were performed according to ICH guidelines. It was found that nevirapine is stable in formulation with pro-Pheroid and that its assay results complied with the in-house specifications (lnitial% ± 5%). Due to the positive results obtained, the nevirapine formulation may be further developed to
efavirenz are inconclusive. Assay results of abacavir sulphate and efavirenz in the pro-Pheroid did not conform to in-house specifications. Further study is needed to ascertain the cause(s) of the variable results obtained.
Before these ARVs can be formulated into possibly viable products, the following issues will have to be addressed:
• The Pheroid™/pro-Pheroid manufacturing process will have to be assessed and validated to ensure lot uniformity.
• Analytical methods for evaluating the pro-Pheroid system in particular, need to be developed and validated. Once such methods are available, the stability of the pro-Pheroid system should be evaluated over a minimum of 6-months period. Only once its stability has been confirmed, should active substances be added.
• Separate analytical methods will have to be developed and validated for each formulation containing pro-Pheroid and the respective ARVs.
• The physico-chemical properties of each ARV need to be taken into account in future formulation.
• Parameters, such as viscosity and particle sizes, will have to be optimised to ensure stability of suspensions. This will most likely also increase the uniformity of samples taken for HPLC assay.
Pro-Pheroid gebaseerde antiretrovirale formulerings: HPLC
metode ontwikkeling en stabiliteit studies
MIV/VIGS hou wereldwyd 'n geweldige uitdaging vir gesondheidsorg in en dit het een van die vernaamste oorsake van mortaliteit by kinders geword. Wereldwyd het die aantal mense wat lewe met MIV, van ongeveer 8 miljoen in 1990 na meer as die huidige 33.2 miljoen toegeneem (AVERT, 2008). Volgens UNAIDS (2008) en WHO (2008), het 1.7 miljoen volwassenes en 330 000 kinders in 2007 aan VIGS verwante siektes gesterf. As gevolg van die volgehoue mutasie van die Ml virus en die gevolglike weerstandigheid wat dit teen bestaande antiretrovirale middels tot gevolg het, is dit onontbeerlik dat nuwe geneesmiddels, of nuwe afleweringsisteme wat die effektiwiteit van bestaande antiretrovirale middels sal verhoog, ontwikkel word.
Pheroid™ tegnologie is 'n nuwe afleweringsisteem wat hoofsaaklik uit veranderde essensiele vetsure bestaan. Die effektiwiteit van Pheroid™ tegnologie is deur verskeie nasionale en internasionale kliniese proewe vir formulerings wat op hierdie tegnologie gegrond is (Grobler et al., 2008), bewys.
Die Pheroid™ is soortgelyk aan 'n emulsie, maar bevat twee vloeistoffases sowel as 'n gedispergeerde gasfase wat met die gedispergeerde vetsuurfase geassosieer is. Die vloeistoffase is waterig terwyl die ander 'n oliebasis is.
Pro-Pheroid vervaardiging is identies aan die van die Pheroid™, behalwe dat daar nie 'n waterige fase ingebring word nie, maar in die plek daarvan word 'n geneesmiddelsubstans in die oliefase ingevoer.
Hierdie ondersoek is uitgevoer om die stabiliteit van drie verskillende antiretrovirale middels in die pro-Pheroid-afleweringsiteem te bepaal. Hoedoeltreffendheidvloei-stofchromatografie (HDVC) is aangewend om die stabiliteit van elke antiretrovirale middel in die pro-Pheroid-sisteem te evalueer. Die analitiese metodes is inhuis ontwikkel en gevalideer. Die drie formulerings is by 5°C, 25°C+60%RH, 30°C+65%RH en 40°C+75%RH vir 3 maande bewaar. Versnelde stabiliteitstoetse is volgens die
(aanvanklike waarde ± 5%) en gevolglik as stabiel beskou word. Na aanleiding van die positiewe resultate wat verkry is kan die neverapien-formulering met die oog op kommersialisering verder ontwikkel word.
Die stabiliteitsdata vir abakavirsulfaat en efavirenz is nie beslissend nie. Die geneesmiddels in die pro-Pheroid het nie aan die inhuisspesifikasies voldoen nie. Verdere ondersoek is nodig om die oorsaak van die varigrende resultate wat verkry is, te bepaal. Voordat die antiretrovirale middels in moontlike lewensvatbare produkte geformuleer kan word, behoort die volgende aspekte aandag te kry:
• Die Pheroid™/pro-Pheroid vervaardigingsproses behoort getakseer en gevalideer te word om loteenvormigheid te verseker
• In besonder behoort analitiese metodes vir die evaluering van die pro-Pheroid-sisteem ontwikkel en gevalideer te word. Wanneer sodanige analitiese metodes beskikbaar raak, behoort die stabiliteit van die pro-Pheroid-sisteem vir ten minste 6-maande geevalueer te word. Slegs wanneer die stabiliteit bevestig is kan die geneesmiddels toegevoeg word.
• Analitiese metodes vir die onderskeie formulerings van die pro-Pheroid en antiretrovirale middels behoort ontwikkel en gevalideer te word.
• Die fisies-chemiese eienskappe van elk van die onderskeie antiretrovirale middels behoort in toekomstige formulerings in aanmerking geneem te word.
• Parameters soos viskositeit en deeltjiegrootte behoort geoptimaliseer te word om die stabiliteit van suspensies te verseker. Hierdeur behoort die uniformiteit van die monsters wat vir die HDVC analise geneem word, te verbeter.
Pro-Pheroid-based antiretroviral formulations: HPLC
method development and stability studies
Acquired immune deficiency syndrome (AIDS) is a deficiency of the immune system, caused by the human immunodeficiency virus (HIV). In South Africa alone, 5.5 million people of which 240 000 children, are currently infected with AIDS. During 2000-2004 a total of 2.49 million in South Africa died of AIDS of which 257 224 were children between the ages 0 and 9. AIDS related deaths in South Africa raised with 79% between 1997 and 2004. Research state that by 2015 over 6 million South African people will live with AIDS and 5.4 million South Africans will have died of AIDS.
Although effective in adults to some extent, many obstacles impede the treatment of infected elderly, infants and children. There is a major need for alternative dosage forms to optimise AIDS therapy in babies and children and those who find it difficult to swallow a number of tablets or capsules.
Therefore, this project aims to optimise the delivery of drugs currently in use for the treatment of HIV/AIDS by entrapment of the drugs in the novel Pheroid™ carrier system. The Pheroid™ system increases the absorption and enhances the bioavailability of certain drugs as reported in literature, with fewer side effects (Grobler, 2004).
The above issues formed the basis of the aims and objectives of this study, which are:
• To develop and validate a HPLC method for the different ARV's in the pro-Pheroid system according to ICH guidelines.
• To analyse the active content of the products by means of a stability indicating HPLC method during and after the stability period of three months.
• To determine the preservative efficacy of propylparaben within this system. • To determine the suitability of the pro-Pheroid system for the ARV's used in
AIDS ART ARV BD BP CDC D HAART HIV HPLC ICH NNRTI NRTI QID TDS UNAIDS UNICEF USP WHO
Acquired Immunodeficiency Syndrome Antiretroviral Therapy
Antiretroviral Twice a day
British Pharmacopoeia
Centres for Disease Control and Prevention Daily
Highly Active Antiretroviral Therapy Human Immunodeficiency Virus
High Performance Liquid Chromatography International Conference on Harmonisation
Non-Nucleoside Reverse Transcriptase Inhibitors Nucleoside Reverse Transcriptase Inhibitors Four times a day
Three times a day
Joint United Nations Programme on HIV/AIDS United Nations Children' Fund
United States Pharmacopeial Convention World Health Organisation
HUMAN IMMUNODEFICIENCY VIRUS AND ACQUIRED
IMMUNODEFICIENCY SYNDROME
"You must be tfie change you wish to see in the world."
- Mahatma
Gandhi-1.1 INTRODUCTION
The Human Immunodeficiency Virus (HIV) and the Acquired Immunodeficiency Syndrome (AIDS) are of the greatest challenges in developing countries, and hugely impact on social-economics and resources. HIV belongs to a subset of retroviruses, called lentiviruses (or slow viruses), which means that there is an interval, of years even, between the initial infection and the onset of symptoms when HIV leads to AIDS (HIV.com, 2008).
Figure 1.1 HIV {Anon., 2008).
HIV destroys CD4+ T cells, a certain white blood cell, when entering the bloodstream, through mucous membranes or with blood-to-blood contact, where the HIV then begins to replicate. The CD4+ T cells are critical to the
normal functioning of the human immune system, the body's defending
Figure 1.2 HIV binds onto a CD4+ T cell (FrontierAids, 2008).
Immunosuppression that leads to AIDS, is triggered by gradual deterioration of the immune function and the eventual destruction of lymphoid and immunologic organs (HIV.com, 2008).
Acquired Immune Deficiency Syndrome or AIDS means: • Acquired: One can become infected;
• Immune Deficiency: A weakness in the immune system of the body that fights diseases; and
• Syndrome: A group of health problems that constitute a disease (AIDS.org, 2008b).
AIDS was initially reported in adults with Pneumocystis carinii pneumonia in 1981, followed by paediatric reports in 1982 (Pizzo & Wilfert, 1998). These paediatric reports were initially difficult to validate, especially because linkage of the transmission of AIDS with intravenous drug use, or to heterosexual
mechanisms, had not at the time been firmly established or recognized (Pizzo & Wilfert, 1998).
HIV infections progress more rapidly in adults, whilst some untreated children die within the first 2 years after infection (Pizzo & Wilfert, 1998).
1.2 HIV STATISTICS
Approximately 68.8% of people having HIV, live in sub-Saharan Africa, constituting just over 10% of the world's population. 5.5 million of these HIV infected people at the end of 2005, were South Africans, including 240 000 children (AVERT, 2008a).
African women are considerably more likely, at least 1.4 times more, to be infected with HIV than men. Reasons for this include the younger age at which initial infection in women occur and the greater efficiency of male-to-female HIV transmission through sex (AVERT, 2008b).
IS
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:990 ; w ; '.9)7 :99s - . w : « 5 :99t : « / :99B "-W HXM TOO: KOI JOOJ zoc« 2005 ixt, 200;Figure 1,3 The total number of people living with HIV worldwide rising from 8 million in 1990 to 33.2 million in 2007 (AVERT, 2008a). The global and sub-Saharan Africa HIV prevalence, i.e. the proportion of people
living with HIV, is shown in figure 1.4 below. Although this percentage has stabilised, continued new infections contribute to the estimated number of 33.2 million of persons with HIV (UNAIDS, 2007).
The HIV incidence appears to have fallen slightly in sub-Saharan Africa over recent years, since the number of new infections each year is exceeded by the matching number of deaths. However, because of overall population growth here, the total number of people with HIV is still rising (AVERT, 2008b).
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z.o — A — Global z.o — A — Global z.o — A — Global C j l l — A — Global\•7^0 ' > I 92 * 3 W ■?f. S t 07 'Ofl ^ ? 2O0G Ol ■ 02 Qi 04 'OS "Gfc ' 0 7
Figure 1.4 Estimated adult HIV prevalence in sub-Saharan Africa 1990-2007 (UNAIDS, 1990-2007).
The following data regarding worldwide AIDS for 2007 combines statistics from UNAIDS (2007) and AVERT (2007).
Table 1.1 Worldwide AIDS statistics (UNAIDS, 2007; AVERT, 2007)
People living with Newly infected Deaths due to AIDS in 2007 j with AIDS in 2007 AIDS in 2007 Total 33.2 million 2.5 million 2.1 million
Adults 30.8 million 2.1 million 1.7 million
Children under 15 2.5 million 420 000 330 000
The distribution of the people living with HIV worldwide during 2007 is illustrated in figure 1.5.
5.5% 4.6%
oy.s-t
Region
Asia
r-igl" Income Countnes Latin America St iho Cat bbean Eastern Europe & Central Asia
Africa
Figure 1.5 Chart showing the 2007 distribution of people living with HIV worldwide (AVERT, 2008b).
68.8% of all people having HIV live in sub-Saharan Africa, constituting just over 10% of the world's population. African women are considerably more likely, at least 1.4 times more, to be infected with HIV than men. Reasons for this include the younger age at which initial infection in women occur and the greater efficiency of male-to-female HIV transmission through sex (AVERT, 2008b).
1.3 DISCOVERY OF HIV
The earliest known case of HIV-1 in a human was in 1959, in a blood sample collected from a man in Kinshasa in the Democratic Republic of Congo. It is unknown how he became infected. Genetic analysis of this blood sample suggested that HIV-1 may have stemmed from a single virus in either the late
1940's or early 1950's (CDC, 2008a).
It is known that this HIV has existed since the mid to late 1970's in the United States. From 1979 to 1981 doctors in New York and Los Angeles reported rare types of cancer, pneumonia and other illnesses in a number of male patients who had sex with other men. Conditions like these were not usually found in people with healthy immune systems (CDC, 2008a).
In 1982 public health officials began to use the term AIDS or "Acquired Immunodeficiency Syndrome", to describe the occurrences of opportunistic infections like Kaposi's sarcoma, a kind of cancer, and Pneumocystis carinii pneumonia, in previously healthy people. In that year, formal tracking of AIDS cases began in the United States (CDC, 2008a).
In 1983, scientists discovered the virus that causes AIDS, and an international scientific committee first named it HTLV-III / LAV (human T-cell lymphotropic virus-type III / lymphadenopathy-associated virus), which was later changed to HIV (Human Immunodeficiency Virus) (CDC, 2008a).
For many years scientists theorised as to the origins of HIV and how it appeared in the human population, most believing that HIV originated in other primates. An international team of researchers in 1999 reported that they had discovered the origins of HIV-1, the predominant strain of HIV in the developed
world. In West Equatorial Africa, a subspecies of chimpanzees had been identified as the original source of the virus. These researchers believed that when hunters became exposed to infected blood, HIV-1 was introduced into the
human population (CDC, 2008a).
HIV, a lentivirus, and part of a larger group of viruses, known as retroviruses, which attacks the immune system, has been found in a number of different animals, including sheep, cats, cattle and horses. The Simian Immunodeficiency Virus (SIV) that affects monkeys, is the most significant lentivirus in terms of the investigation into the origins of HIV (CDC, 2008b).
Now it is generally accepted that HIV is a descendant of the SIV, because certain strains of SIVs bear a very close resemblance to the two types of HIVs, i.e. HIV-1 and HIV-2 (AVERT, 2008c).
HIV-2 for example corresponds to SIVsm, a strain of the SIV that is found in the indigenous, white-collared monkey in Western Africa, the mangabey (AVERT, 2008c).
HIV-1, the more virulent, pandemic strain of HIV, was more difficult to place, until 1999, when the SIV strain, SIVcpz, that is found in chimpanzees, was identified as being its closest counterpart. This virus, however, still showed certain significant differences from HIV (AVERT, 2008c).
A group of researchers from the University of Alabama announced in February 1999 that they had found a type of SIVcpz that was almost identical to HIV-1. This particular strain was identified as originating from a captive member of the sub-group of chimpanzees, known as Pan troglodytes troglodytes (P. t.
troglodytes), which were once common in West-Central Africa (AVERT, 2008c).
The discovery was made by researchers (led by Paul Sharp from Nottingham University and Beatrice Hahn from the University of Alabama) during the course of a 10-year study into the origins of the virus. They claimed that this sample proved that chimpanzees were the source of HIV-1 and that the virus had at some point crossed species from chimpanzees to humans (AVERT, 2008c).
Two years later, their final findings were published in Nature magazine, where they concluded that wild chimpanzees had been infected simultaneously with two different SIV's to form a third virus that could be passed on to other chimpanzees and, more significantly, was capable of infecting humans and causing AIDS (AVERT, 2008c).
The research group traced these two different viruses back to the SIV that infected red-capped mangabeys and to the SIV that is found in spot-nosed monkeys, two smaller species of monkeys. They believed that the hybridisation took place inside of the chimpanzees, after hunting and killing the two smaller species of monkey, and becoming infected with both strains of SIV (AVERT, 2008c). They also concluded that all three 'groups' of H1V-1, namely Groups M, N and O, represented separate crossover 'events' from chimpanzees to humans, where the three groups came from the SIV found in P. t. troglodytes (AVERT, 2008c).
It is known that certain viruses can pass between species, and humans are just as susceptible. The very fact that chimpanzees obtained SIV from two other species of primate shows just how easily this crossover can occur.
Zoonosis occurs when a viral transfer between animals and humans takes place (AVERT, 2008c). The most common theories about how 'zoonosis' took place, and how SIV became HIV in humans, is shown below (AVERT, 2008c):
■ The Hunter: Normally the hunter's body would have defended itself against SIV, but in this scenario SIVcpz was transferred to humans as a result of chimpanzees being killed and eaten, or their blood entering the hunter's cuts or wounds. The SIV then re-adapted itself a few times within its new human host to become HIV-1.
■ Oral polio vaccines, called Chat: Edward Hooper believes that Chat was grown in kidney cells that were taken from local chimpanzees and infected with SIVcmz. He claims that this would have resulted in the contamination of the vaccine with chimpanzee SIV, and a large number of people subsequently becoming infected with HIV-1.
■ Contaminated needle theory: Health workers used needles repeatedly to inject patients with, due to costly needles back in the 1950's.
■ Colonialism Theory: People were forced into labour camps with poor sanitation and insufficient food. It is believed that because of their weakened immune systems, SIV could easily have infiltrated the labour force to become HIV infected and sick, as stray chimpanzees with SIV would have made a welcome extra source of food for the workers.
■ Conspiracy Theory: Many people believe that HIV was manufactured as part of a biological warfare programme, designed to wipe out large numbers of homosexual and black people (AVERT, 2008c).
Until recently, the origins of the HIV-2 virus had remained relatively unexplored. HIV-2 is far rarer, significantly less infectious and progresses more slowly into AIDS than HIV-1. As a result, it infects far fewer people and is mainly confined to a few countries in West Africa. It is thought that HIV-2 came from the SIV in sooty mangabeys, rather than from chimpanzees, but the crossover to humans is believed to have happened in a similar way as for HIV-1, i.e. through the butchering and consumption of monkey meat (AVERT, 2008c).
1.4 PATHOLOGY
The HIV outer envelope protein has a strong affinity for cells that express the CD4 molecule, a protein that coats the surface of various normal cells, including specific T lymphocytes and macrophages. The virus then attaches to and penetrates these cells, followed by transcription, using viral reverse transcriptase, of viral RNA into double-stranded DNA. This viral DNA is then integrated into the host cell genome and will remain there for the lifetime of the cell.
Active viral replication continues after the primary infection, even during the clinically latent period, where more than 106 viral particles are produced daily. Over time, the host immune system loses the ability to suppress viral replication and the replacing of lost CD4 cells (Schwartz, 2000).
1.5 TRANSMISSION OF HIV
Worldwide, the most common transmission of HIV is through sexual contact, whether anal or vaginal. Male to female transmission is more efficient than female to male; where anal receptive sex is more likely to transmit than vaginal sex (Schwartz, 2000).
Other ways of HIV transmission:
• Exposure to infected blood: It almost always involves parenteral exposure to infected blood via transfusions or needle sharing. In occupational exposure, the risk of transmission from percutaneous exposure to needles being contaminated with HIV infected blood, is 1/300 (Schwartz, 2000).
• Mother to child: Breast milk, perinatally, either in utero (25% to 50%), or during labour and delivery, with increased rate of transmission for women who were previously diagnosed with AIDS, or with low CD4 counts, or with higher viral titers (the concentration of infectious viral particles per millilitre of suspension fluid) (Schwartz, 2000; Biology Van, 2000).
• Vaginal sex, anal sex, oral sex, injecting drugs, blood products, blood transfusions, mother to child transmission, infection in the health-care settings, tattooing / piercing.
HIV is not believed to be transmitted by:
• Sharing of utensils, glasses / cups / similar, bathrooms, or bathtubs. • Kissing, coughing, sneezing, or through bites or insects.
• Drugs injected with sterile needles (CDC, 2008b).
• Exposure to urine, faeces, vomitus, except where these fluids may be grossly contaminated with blood, in which case transmission is rare, if at all.
• Saliva, tears and sweat. HIV has been isolated from saliva in the past, but only in a few people, and in such minute quantities that the risk of infection is virtually zero. Saliva is very good at destroying HIV, so that
the HIV found is often only particles of HIV, rather than the whole virus, meaning they can't infect (De Boer, 2008).
• Screened blood in blood transfusions. Blood that is screened with heat-treating techniques destroy the HIV in blood products, such as albumin
and factor 8 (Lab Tests Online, 2008).
• Protected sex: It is said that if used correctly and consistently, condoms are highly effective at preventing HIV transmission. A small minority of people believe condoms are inadequate protection and that some very small viruses can pass through latex. Scientific tests have, however, proven this theory to be unfounded (CDC, 2008b).
1.6 HIV LIFE CYCLE
The life cycle of HIV is illustrated in figure 1.6.
HIV LIFE CYCLE
Free Virus
O Infection: Virus
penetrates cell. Contents emptied into cell.
4 Reverse Transcription: Single strands of viral RNA are converted into double-stranded DNA by the reverse transcriptase enzyme.
5 Integration: Viral DNA
is combined with the cell's own DNA by the Integrase enzyme.
6 Transcription: Vvhen the infected cell divides, the viral DNA is "read" and long chains of proteins are made.
7 Assembly: Sets of viral protein
chains come tog ether.
2. Binding and Fusion: Virus binds to
a CD4 molecule and one of two "coreceptors" (either CCR5 or CXCR4). Receptor molecules are common on the cell surface. Then the virus luses with the cell.
CCR5 Receptor CXCR4 Receptor
O Budding:
Immature virus pushes out of the ceil, taking some I cell membrane ) with it. The
protease enzyme starts processing the proteins in the newly forming virus. 9 Immature virus breaks free of the infected cell.
10 Maturation: The protease enzyme finishes cutting HIV protein chains into individual proteins that combine to make a new working virus.
1.7 PROGNOSIS 1.7.1 Paediatric prognosis
Prior to the advent of aggressive combination therapies, data suggested a bimodal survival curve, with 25% of perinatally infected infants developing early symptomatic disease, an AIDS diagnosis at age 1 to 2 years, and frequently dying by 3 years of age. The remaining 75% had a delayed onset of symptoms, usually after 5 years of age, with the median survival of this group being 8 to 12 years old.
Since the use of three drug combinations has become standard, morbidity and mortality have both largely decreased and the median survival is now clearly into adolescence. In addition, the incidence of new opportunistic infections has significantly decreased with treatment (Schwartz, 2000).
1.7.2 Adult prognosis
The prognosis for people with HIV has changed dramatically since the early 1980's, when the first cases of AIDS were diagnosed and it was thought that most people would die within a few months of first being diagnosed with this condition. This improved, partly because it was recognised that HIV was the cause of AIDS, taking many years to gradually destroy the immune system, and because doctors gradually learnt more about recognising and treating HIV people with infections and cancers.
Before the introduction of Highly Active Antiretroviral Therapy (HAART) in the mid 1990's, it was thought that in rich countries, such as the UK, it would on average take between eight and fifteen years after infection, for HIV to cause life-threatening illness or death (NAM, 2008).
Many doctors who now treat HIV patients believe that, provided a person who has HIV receives effective and proper anti-HIV treatment before the immune system is severely damaged by the virus, could live a more or less normal life span (NAM, 2008).
1.7.3 Prediction of prognosis
CD4 T-cell counts are key tests used to assess prognosis. These counts give an indication of the health of the immune system, whilst viral load tests determine the amount of HIV in the blood. As the CD4 count falls, the viral load rises, and the risk of becoming ill or dying as a consequence of HIV becomes higher over the short-term (NAM, 2008).
Research into the prognosis of people starting HAART indicates that the risk of becoming very ill or dying because of HIV within the next three years, is linked to five key factors, i.e. (1) having a viral load above 100 000, or (2) a CD4 count below 200 at the time of starting treatment, (3) being over the age of 50, (4) being an injecting drug user, or (4) having had a prior AIDS-defining illness (NAM, 2008).
Doctors often refer to research involving the Multicenter AIDS Cohort Study when discussing HIV prognosis. This established the relationship between viral load, CD4 count and the risk of developing AIDS, or dying within the next few years. This information is often used to help with decisions about when to start HIV treatment (NAM, 2008).
1.8 CLINICAL HIV STAGING SYSTEM
Specific combination antiretroviral therapy delays progression of illness, promotes improved growth and (possibly) neurological outcome. The standard of care now involves the administration of combination (usually three or more drugs) therapy and the most potent agents are those that inhibit viral protease (termed protease inhibitors). Drug regimens are complex, with as many as 9 doses of medicine a day, with adherence to prescribed schedules being critical. The durability of response is short, even when patients miss only 20% to 25% of doses (Schwartz, 2000).
There are four different stages of HIV / AIDS for infants and children, according to the World Health Organisation (WHO). The symptoms for adults (blue) and children (purple) are indicated in table 1.2, whilst the symptoms for both adults and children are illustrated in black.
Table 1.2 WHO staging system for HIV infection and disease in adults and children (Department of Health: Republic of South Africa, 2003)
STAGE 1 STAGE 2
• Asymptomatic. • Weight toss of failure to thrive. • Generalised
lymphadenopathy.
• Primary HIV infection.
• Herpes zoster.
• Minor mucocutaneous manifestations. • Recurrent upper respiratory tract infections.
Performance Scale 1: asymptomatic, normal activity.
• Unexplained chronic diarrhoea. • Persistent or recurrent candidiasis. • Persistent fever.
• Recurrent severe bacterial infections.
Performance scale 2: symptomatic, normal activity.
STAGE 3 STAGE 4
• Weight loss > 10% of body weight.
• HIV wasting syndrome: weight loss > 10% of body weight, plus either of the following persisting for > 1 month:
• Unexplained chronic diarrhoea of > 1 month.
• Unexplained chronic diarrhoea; • Chronic weakness; • Unexplained prolonged fever > 1 month. • Oral candidiasis. • Oral hairy Ieukopiakia.
• Unexplained prolonged fever. • Toxoplasmosis of the brain.
• Pneumocystis carinii pneumonia.
• Recurrent bacterial pneumonia infections.
• Cryptosporidiosis with diarrhoea for more than 1 month.
• Pulmonary tuberculosis.
• Cryptococcosis, extra pulmonary. • Herpes simplex virus infection.
• Severe bacterial infections.
• AIDS defining opportunistic infections.
• Candidiasis of the oesophagus, trachea, bronchi or lungs. • Kaposi's sarcoma.
• HIV encephalopathy, clinical findings of disabling cognitive and / or motor dysfunction over weeks to months.
• Severe failure to • Cytomegalovirus disease of an organ, spleen or lymph nodes.
thrive.
• Progressive encephalopathy. • Malignancy. • Recurrent
• Progressive multifocal leukoencephalopathy. • Any disseminated endemic mycosis.
• Cancer of the cervix.
• Atypical mycobacteriosis, disseminated.
septicaemia. ♦ Non-typhoid salmonella bacteraemia.
Performance scale 3: bedridden < 50% of the day during the last month.
• Extrapulmonary tuberculosis.
• Lymphoma.
Performance scale 4: bedridden > 50% of the day during the last month.
1.9 TREATMENT OF HIV
The very rapid discovery of effective antiretroviral agents was made possible by drug development programs already established for other diseases (Hardman & Limbird, 2001).
1.9.1 ARV's classification and mechanism of action
• Nucleoside reverse transcriptase inhibitors (NRTI's): Act as false substrates for reverse transcriptase and terminate the DNA chain (Hardman & Limbird, 2001).
• Non-nucleoside reverse transcriptase inhibitors (NNRTI's): Inhibit reverse transcriptase activity directly, by potently suppressing HIV replication (Hardman & Limbird, 2001).
• Protease inhibitors (Pi's): Inhibit the HIV protease enzyme, which prevent cleavage of viral polyproteins and result in immature, non-infectious HIV viral particles (Hardman & Limbird, 2001).
• Fusion inhibitors (Fl's): Interfere with the binding, fusion and entry of an HIV into a human cell. By blocking this step in HIV's replication cycle, it slows the progression from HIV infection to AIDS (Wikipedia, 2008) • Chemokine coreceptor antagonists (CCR's): Prevent the entry of HIV
into target cells by binding to co-receptors on the surface of CD4 cells, blocking a required step in viral entry (Coffey, 2008).
• Integrase inhibitors (ll's): Bind a viral enzyme, known as integrase, and interfere with the incorporation of reverse-transcribed HIV DNA into the chromosomes of host cells (Coffey, 2008).
There are different kinds of trade name products on the market, as shown in table 1.3.
Table 1.3 Examples of ARV drugs currently on the market (HIV INSITE, 2008)
Drug Class Drug Name Examples of Trade
Names Abbreviations NRTI Abacavir Didanosine Emtricitabine Lamivudine Stavudine Tenofovir Zalcitabine Zidovudine Ziagen® Videx® Emtriva® Epivir® Zerit® Viread® Hivid® Retrovir® ABC ddl FTC 3TC d4T TDF ddC AZT/ZDV NNRTI Delavirdine Efavirenz Nevirapine Rescriptor® Stocrin® Viramune® DLV EFV NVP PI Amprenavir Indinavir Nelfinavir Ritonavir Saquinavir Agenerase® Crixivan® Viracept® Norvir® Invrase® APV IDV NFV RTV SQV
Fl Enfluvirtide Fuzeon® ENF
CCR Maraviroc Celsentri® MVC
II Raltegravir Isentress® RAL
1.9.2 Prophylaxis for HIV exposure
For prophylaxis of HIV exposure, known as the PEP (Post-Exposure Prophylaxis) packs, the following is recommended by the National Department of Health, as illustrated in table 1.4.
Table 1.4 PEP drug regimen (National Department of Health, 2004)
Drug Dose Frequency Duration
Zidovudine (AZT) 300 mg Lamivudine (3TC) 150 mg
12-hourly 28 days
Lopinavir / Ritinovir in cases 400 mg/100 mg of high exposure
1.9.3 Adult ARV treatment
According to the Department of Health of the Republic of South Africa (2003), the indicator of the need for antiretroviral treatment is based on the clinical assessment and the CD4 count. The lower the CD4 count and the higher the viral load, the higher the risk of AIDS and the more urgent the need for treatment.
Table 1.5 Criteria for initiation of antiretroviral therapy in adults and children (National Department of Health, 2004)
Non-pregnant adults and adolescents Children under 6 years
• CD4 < 200 cells/mm3 and / or symptomatic,
irrespective of stage; or
• WHO stage IV AIDS defining illness, irrespective of CD4 count; and
• Patient prepared and wilting to comply with taking antiretroviral drugs.
• CD4 < 15% and symptomatic; or • WHO Paediatric Stage III AIDS
defining illness, irrespective of CD4; and
• At least one responsible person capable of administering child's medication.
The aim of antiretroviral treatment primarily is to decrease HIV-related morbidity and mortality. With treatment the patient should experience fewer HIV-related illnesses, the patient's CD4 count should rise and remain above the baseline count, the patient's viral load should become undetectable (< 400 copies/mm2) and remain undetectable on Antiretroviral treatment (ART) (National Department of Health, 2004).
Two ART regimens are recommended for use in the South African public sector. Patients who fail both regimens will be referred for individual evaluation by ART specialists. New developments in ART will determine options for
salvage therapy (National Department of Health, 2004). Patients are first put on the first regimens. The first line regimen for adults is shown in table 1.6.
Table 1.6 First line regimens for adults: Regimens 1a and 1b (National Department of Health, 2004)
Regimen 1a Regimen 1b
All men, as well as women on injectable
contraception
Women who are unable to guarantee reliable contraception while on therapy Stavudine (d4T), 40 mg, 12-hourly or 30 mg,
12-hourly if < 60 kg PLUS
Lamivudine (3TC), 150 mg, 12-hourly PLUS Efavirenz (AFV), 600 mg at night.
Stavudine (d4T), 40 mg, 12-hourly or 30 mg, 12-hourly if < 60 kg PLUS
Lamivudine (3TC), 150 mg, 12-hourly PLUS Nevirapine (NVP 200 mg daily for first 2 weeks, increasing to 200 mg, 12-hourly.
Patients who continue to fail virologically, despite demonstrated adherence, may change to the second line regimens (National Department of Health, 2004). A patient's viral load is monitored and the ART is adjusted where necessary, according to the viral load. The second line regimen for adults is shown in table 1.7 and the viral load monitoring in table 1.8.
Table 1.7 Second line regimens for adults (National Department of Health, 2004)
Second Regimen
Zidovudine (AZT), 300 mg, 12-hourly PLUS
Didanosine (ddl), 400 mg daily on empty stomach PLUS Lopinavir/ Ritanovir (LPV/r), 400/100 mg, 12-hourly.
Table 1.8 Viral load monitoring (National Department of Health, 2004)
Viral load (VL) Response
< 400 copies/mm2 • 6-monthly viral load monitoring continues.
• Routine adherence support.
400-5000 copies/mm2 • Begin step-up adherence package. Review at next 6-month
viral load check.
• If < 400, return to routine 6-monthly monitoring and adherence support.
• If still between 400 and 5000, continue with step-up adherence package. Repeat viral load at 6 months.
> 5000 copies/mm2 • Begin step-up adherence package. Review at next 6-month
viral load check.
• If < 400, return to routine 6-monthly monitoring and adherence support.
• If between 400 and 5000, continue with step-up adherence package. Repeat viral load at 6 months.
• If > 5000, despite stepped up adherence support switch to second-line therapy only if adherence is > 80%.
1.9.4 Children ARV treatment
In Maseru, Lesotho, an eight year old boy suffered from AIDS, tuberculosis and pneumonia and weighed only 8 kg. With the help of the Paediatric-AIDS clinic in Maseru, Bokang received the necessary medicine for these conditions in May 2007. After only 6 months he gained 9 kg and is now living a healthy life like that of a normal child. Although still on ARV medicine, his life quality has improved (Gorman, 2007). This illustrates the very important role that ARV medicine plays in the fight against HIV and AIDS. Likewise, the mortality rate for children with AIDS at Baylor's Paediatric clinic in Botswana, fell from nearly 5% in 2003 to 0.3% in 2006 with the use of ARVs (Gorman, 2007).
Researchers showed that children respond faster and better to ARVs than adults, making it essential not to ignore poor youngsters with AIDS. In Africa only 1 in 20 children who need ARVs receive treatment. In addition, fewer than 1 in 10 HIV positive mothers receive the drugs necessary to help prevent the transmission of the virus to their newborns (Gorman, 2007).
At present, the majority of children are diagnosed on the basis of symptomatic HIV disease and the positive HIV antibody test of the mother of the child. The
child of an HlV-infected mother acquires HIV antibodies from his / her mother during pregnancy. These may persist in his / her blood until 15-18 months of age, even if the child is not infected with HIV. A child may thus test HIV positive, without actually being infected. When using the HIV antibody test, therefore, it is impossible to tell whether a newborn infant has already been infected with HIV or not.
Additional techniques thus exist for the detection of the virus in children under the age of 18 months. HIV infection can be diagnosed in most infected infants by the age of 6 weeks by using DNA PCR technique (National Department of Health, 2004). The paediatric first- and second-line therapies are summarised in tables 1.9 to 1.11.
Table 1.9 Paediatric first-line therapy (National Department of Health, 2004)
6 months - 3 years > 3 years a n d > 10 kg • Stavudine (d4T) • Lamivudine (3TC) • Lopinavir/Ritonavir • Stavudine (d4T) • Lamivudine (3TC) • Efavirenz
Table 1.10 Reasons to move to second-line ART in children (National Department of Health, 2004)
Virological Clinical Immunological
« Rebound of viral load to • Persistent oral thrush, • A persistent decline I baseline. which is refractory to in the CD4% > 2
treatment. months in the • A detectable viral load may be absence of TB.
tolerated in children, providing • New evidence of WHO absence of TB. that growth and elevated CD4 Stage 3 disease.
count are sustained.
Table 1.11 Paediatric second-line therapy (National Department of Health, 2004)
6 months - 3 years > 3 years a n d > 10 kg • Zidovudine (AZT) • Didanosine • Nevirapine • Zidovudine (AZT) • Didanosine • Lopinavir / Ritonavir
Didanosine must be taken alone, on an empty stomach, at least an hour before a meal. Tablets should be dissolved in at least 30 ml of water. For Regimen 2 where there has been prior exposure to nevirapine or efavirenz, lopinavir / ritonavir can be substituted as in Regimen 1 (Department of Health, 2004). The dosage and frequency of ART in children, according to the National Department of Health (2004), is shown in table 1.12.
Table 1.12 Dosage and frequency of ART in children (National Department of Health, 2004)
Drugs Formulation Dosage Comments
Nucleoside reverse transcriptase inhibitors (NRTIs)
Zidovudine Suspension: 10 mg/m€ 90-180 mg/m2 ids
Didanosine
(ddl) Suspension: 10mg/mt 90-120 mg/m2 bd
Half hour before meals.
Stavudine Suspension: 1 mg/m£ 1 mg/kg bd Abacavir i Suspension: 20 mg/mE 8 mg/kg bd Lamivudine Suspension: 10mg/m£ 4 mg/kg bd
Non- nucleoside reverse transcriptase inhibitors (NNRTIs)
Nevirapine Suspension: 10mg/m£ 120-200 mg/m2 bd Skin rash.
Efavirenz Capsules: 50, 100 and 200 mg 13-14.99 kg 15-19.99 kg 20-24.99 kg 25-32.49 kg 32.5-39.99 !■ 39.99 kg: 6 200 mg d, 250 mg d, 300 mg d, 350 mgd, ^g: 400 mg d, DOkgd Give at night.
Protease inhibitors (Pis)
Ritonavir Suspension: 80 mg/mt Start 250 mg/m2/dose bd,
increase by 50 mg/m2 every 2-3
days up to 400 mg/m2 bd. If < 2
years of age 450 mg/m2 bd.
Take with food, bitter.
Neifinavir Suspension: 50 mg/1 g 55 mg/kg bd. ritonavir
Give 2 hours before ddl.
1.9.5 Nutritional support for children
Specific nutritional conditions in children should be treated appropriately (National Department of Health, 2004):
• Multivitamin syrup (containing vitamins A, B, C and D), oral, 5 m£/dose once daily for 5 days per week.
• Ferrous gluconate, oral, 0.5 mt/kg/dose once daily for 5 days per week, for iron deficiency.
• Folic acid, 2.5 mg/dose once daily for 5 days.
1.10 CONCLUSION
HIV infection poses an enormous challenge worldwide. HIV and AIDS have become of the leading causes of death in children, especially in the developing world. Infected children in developing countries live in areas where treatment is not always feasible. Currently regulatory bodies concentrate on suitable treatment regimes for children and in the process the proper and children compliant dosage form is often neglected. Very few fixed dose combinations exist in the form of oral solution or suspension. Only single component suspensions are available for paediatric use. The development of a safe, effective, inexpensive and paediatrically friendly formulation is therefore the ultimate goal for those concerned with antiretroviral treatment.
PHYSICO-CHEMICAL AND PHARMACOLOGICAL
PROPERTIES OF ANTIRETROVIRAL DRUGS
"Live as if you were to die tomorrow. Learn as if you were to live forever."
Mahatma Gandhi
-2.1 INTRODUCTION
The chemical and physical properties of various antiretroviral drugs (ARVs) are discussed in this chapter in order to present the different classifications, physical - and solubility data that are important to consider when formulating Pheroids™ and pro-Pheroids.
2.2 ABACAVIR SULPHATE
Figure 2.1 Abacavir sulphate (C14H18N60)2.H2S04 (USP, 2007). 2.2.1 CHEMICAL PROPERTIES
Abacavir sulphate is a white to off-white solid (Moffat et a/., 2004). • Molecular formula: (Ci4Hi8N60)2.H2S04.
• CAS registry number: [188062-50-2]. • Additional name(s): Ziagen.
• Molecular weight: 670.74.
N
A
NJ-V
• Melting point: 165°C.
• Solubility: Soluble in water at 25°C, c = 0.15 in methanol. • pKa: 5.01 (Merck & Co., Inc., 2006; Moffat et a/., 2004).
2.2.2 PHARMACOLOGY
Abacavir is a nucleoside reverse transcriptase inhibitor (NRTI) and is converted intraceilularly in stages to carbovir triphosphate. This tnphosphate halts the DNA synthesis of retroviruses, including HIV, through competitive inhibition of re-including HIV, through competitive inhibition of reverse transcriptase and incorporation into viral DNA (Hardman & Limbird, 2001;
Pharmaceutical Press, 2002).
2.2.3 PHARMACOKINETICS
• Bioavailability: Abacavir is rapidly absorbed following oral
administration, with bioavailability between 83 and 96%, depending on the formulation.
• Half-life: Elimination half-life is 0.9-1.7 hours.
• Volume of distribution: Apparent volume of distribution is 0.65 - 0.95 l/kg.
• Clearance: The mean apparent clearance in adults is 0.56 (single 1200 mg dose) to 1.94 (100 mg dose) «/h/kg. In children (2 to 5 years) 0.08 (8 mg/kg body weight dose) to 0.12 (4 mg/kg dose) «/h/kg and (6 to 13 years) 0.03 - 0.05 «/h/kg.
• Protein binding: Approximately 50%. It also crosses the blood-brain barrier.
• Abacavir undergoes intracellular metabolism into the active antiviral metabolite carbovir triphosphate (Hardman & Limbird, 2001).
• There is no significant metabolism of abacavir by hepatic cytochrome P450 isoenzymes (Moffat et a/., 2004; Pharmaceutical Press, 2002).
2.2.4 ADVERSE EFFECTS
• Life-threatening hypersensitivity reactions have been reported during the first 6 weeks of treatment. Hypersensitivity commonly include fever, rash, cough, dyspnoea, malaise, headache and gastrointestinal disturbances, particularly nausea and vomiting, diarrhoea and abdominal pain. Caution is needed as hypersensitivity may be misdiagnosed as influenza, respiratory disease, or gastroenteritis.
• Other adverse effects associated with abacavir include pancreatitis and elevated blood glucose and triglyceride concentrations. There may be raised liver enzyme values.
• Lactic acidosis, sometimes fatal and usually associated with severe hepatomegaly and steatosis, has been reported in patients receiving nucleoside reverse transcriptase inhibitors (Moffat et al., 2004; Pharmaceutical Press, 2002).
2.2.5 PAEDIATRIC AND ADULT DOSE
Children: Between 3 months and 16 years: 8 mg per kg body-weight twice daily up to a maximum of 600 mg daily.
2.3 NEVIRAPINE
o
Figure 2.2 Nevirapine C15H14N4O (Pharmaceutical Press, 2002). 2.3.1 CHEMICAL PROPERTIES
Nevirapine has two polymorphic forms, i.e. anhydrous and a hemihydrate. Both forms are white to almost white powders (Pharmaceutical Press, 2002). Recently five solvate forms of nevirapine were also reported (Caira et a/., 2008).
Molecular formula: C15H14N4O.
CAS registry number: [129618-40-2].
CAS name(s):
11-Cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2',3'-e][1,4]diazepin-6-one.
Additional name(s): BI-RG-587. Molecular weight: 266.30 (anhydrous).
Percent composition: C 67.65%; H 5.30%; N 21.04%; 0 6.01%. Melting range: 247 - 249°C.
Solubility: Almost insoluble in water (0.1 mg/ml at neutral pH), highly
soluble at pH < 3, lipophilic.
pKa: 2.8 (Merck & Co., Inc., 2006). 2.3.2 PHARMACOLOGY
Nevirapine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) of the dipyridodiazepinone class. Nevirapine diffuses into the cell and binds to
conformational changes that inactivate the enzyme, and potently suppress HIV replication (Hardman & Limbard, 2001; Merck & Co., Inc., 2006).
2.3.3 PHARMACOKINETICS
• Bioavailability: Oral about 93%. Absorption is not affected by food.
• Half-life: 45 hours, which decreases on multiple dosing to 20 - 30 hours over a 2 - 4 week period. Half-life is 21 hours in children aged 4 - 8 years and 28 hours in children over 8 years.
• Volume of distribution: Steady state 1.2 -1.5 i/kg. Reported as 1.54
i/kg in female and 1.38 i/kg in males.
• Clearance: Oral 0.27 - 0.52 m«/kg/min. Plasma 3.96 i/h (after a 200 mg dose).
• Protein binding: Approximately 60%.
• Nevirapine crosses the placenta and is distributed into breast milk. It is extensively metabolised by hepatic microsomal enzymes, principally by cytochrome P450 isoenzymes of the CYP3A family (Moffat et a/., 2004; Pharmaceutical Press, 2002).
2.3.4 ADVERSE EFFECTS
• The most common adverse effect is skin rash, usually occurring within the first 6 weeks of starting therapy. Severe and life-threatening skin reactions have occurred, including Stevens-Johnson syndrome, and more rarely, toxic epidermal necrolysis.
• Hypersensitivity reactions, including angioedema, urticaria and anaphylaxis have been reported.
• Abnormalities in liver function tests are also common, whereas severe hepatitis and hepatic necrosis, occasionally fatal, have occurred.
• Other common adverse effects include nausea, vomiting, diarrhoea, abdominal pain, fatigue, fever, drowsiness, myalgia and headache. • Granulocytopenia has occurred and has been observed more
2.3.5 PAEDIATRIC AND ADULT DOSE
Children aged 2 months - 8 years: 4 mg/kg body-weight once daily for 14 days, then if there is no rash, 7 mg/kg twice daily; those aged 8 - 1 6 years may be given 4 mg/kg once daily for 14 days, then 4 mg/kg twice daily, not exceeding a total dose of 400 mg daily.
Adults: 200 mg once daily for the first 14 days, then increased to 200 mg twice daily, provided that there is no rash.
If treatment is interrupted for more than 7 days, it should be reintroduced, using the lower dose for the first 14 days, as for new treatment (Pharmaceutical Press, 2002).
2.4 EFAVIRENZ
Figure 2.3 Efavirenz CUH9CIF3NO2 (Pharmaceutical Press, 2007). 2.4.1 CHEMICAL PROPERTIES
Efavirenz is a white to slightly pink, crystalline powder (Moffat et a/., 2004).
Molecular formula: C14H9CIF3NO2. CAS register number: [154598-52-4].
CAS name(s):
(4S)-6-Chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1 -benzoxazin-2-one.
Additional name(s): DMP-266, Stocrin and Sustiva. Molecular weight: 229.26.
Percent composition: C 53.27%; H 2.87%; Cl 11.23%; F 18.06%;
N4.44%; 0 10.14%.
Melting range: 139 -141 °C.
Solubility: Practically insoluble to water (< 10 g/£), c = 0.3 in
methanol.
pKa: 10.2 (Merck & Co., Inc., 2006; Moffat et a/., 2004).
2.4.2 P H A R M A C O L O G Y
Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI), which diffuses into the cell where it binds, adjacent to the active site of reverse transcriptase. This produces a conformational change in the enzyme that inhibits function (Hardman et a/., 2001).
2.4.3 PHARMACOKINETICS
• Bioavailability: Well absorbed orally, bioavailability is increased by a
high-fat meal with 50%.
• Halflife: Elimination halflife for single dose is 52 76 hours, and 40 -55 hours in multiple doses.
• Volume of distribution: About 16 - 6 1 % is excreted in faeces and 14 - 34% in urine.
• Clearance: Mean plasma clearance 0.18 L/h/kg.
• Protein binding: > 90% bound to plasma proteins, mainly albumin (Moffat etal., 2004; Gibbon, 2003).
2.4.4 ADVERSE EFFECTS
• The most common adverse effects are skin rashes and Central nervous system (CNS) disturbances. Mild rashes may resolve on continued treatment, but severe forms may occur, whilst erythema multiform and Stevens-Johnson syndrome have been reported occasionally.
• CNS symptoms include dizziness, headache, insomnia or somnolence, impaired concentration and abnormal dreaming. Psychotic-like symptoms and severe acute depression have also been reported.
• Other adverse effects include nausea and diarrhoea, fatigue and pancreatitis.
• Raised liver enzyme values have occurred, particularly in patients with viral hepatitis.
• Raised serum-cholesterol and -triglyceride concentrations have been reported (Pharmaceutical Press, 2002).
2.4.5 PAEDIATRIC AND ADULT DOSE
Children: Over 3 years of age (10-14 kg), 200 mg once daily; (15 -19 kg), 250 mg once daily; (32.5 - 39 kg), 400 mg once daily; (> 40 kg), 600 mg once daily.
Adults: 600 mg once daily. Dosing at bedtime is recommended during the first 2 - 4 weeks of therapy to improve tolerability (Pharmaceutical Press, 2002).
2.5 LAMIVUDINE
NHj>
N I)
HO.
Figure 2.4 Lamivudine C8HnN303S (Pharmaceutical Press, 2007). 2.5.1 CHEMICAL PROPERTIES
Lamivudine forms white crystals a white solid when recrystallised from methanol or ethyl acetate (Moffat et a/., 2004).
• Molecular formula: C8H-HN3O3S. • CAS registry number: [134678-17-4].
• CAS name(s): (2R-cis)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1 H)-pyrimidinone.
• Additional name(s): (-)-2'-deoxy-3'-thiacytidine; 3TC; and 3'thia-2', 3'-dideoxycytidine.
• Molecular weight: 229.26.
• Percent composition: C 41.91%; H 4.84%; N 18.33%; 0 20.94%; S 13.99%.
• Melting range: 160-162°C.
• Solubility: Approximately 70 mg/m« in water at 20°C, c = 0.38 in methanol (Merck & Co., Inc., 2006; Gibbon, 2003).
2.5.2 PHARMACOLOGY
Lamivudine is a nucleoside reverse transcriptase inhibitor (NRTI) and is converted intracellularly in stages to the triphosphate. This triphosphate halts the DNA synthesis of retroviruses, including HIV, through competitive
inhibition of reverse transcriptase and incorporation into viral DNA. Lamivudine is also active against the hepatitis B virus. Resistance to lamivudine has been reported in isolates of HIV and hepatitis B virus. The manufacturers recommend that lamivudine should not be used during the first three months of pregnancy and it is recommended not to be used with zidovudine (Hardman et al., 2001; Pharmaceutical Press, 2002).
2.5.3 PHARMACOKINETICS
• Bioavailability: 80 - 87%. Absorption is delayed, but not reduced by ingestion with food.
• Half-life: 5 - 7 hours (after single dose), which may increase in the presence of renal impairment.
• Volume of distribution: Approximately 100 £.
• Clearance: About 70% is excreted unchanged in the urine.
• Protein binding: < 36% (Moffat et al., 2004; Pharmaceutical Press, 2002).
• Lamivudine crosses the blood-brain barrier with a ratio of Cerebrospinal fluid (CSF) to serum concentrations of about 0.12, whilst it crosses the placenta and is distributed into breast milk.
• Lamivudine is metabolised intracellularly to the active antiviral triphosphate. Hepatic metabolism is low and it is cleared mainly unchanged by renal excretion (Pharmaceutical Press, 2002).
2.5.4 ADVERSE EFFECTS
• Gastrointestinal track (GIT) adverse effects: abdominal pain, nausea, vomiting, diarrhoea, etc;
• Headache, fever, rash, alopecia, malaise, insomnia, cough, and nasal symptoms;
• Neutropenia and anaemia (usually when given in combination with
zidovudine), thrombocytopenia and increases in liver enzymes have occurred and
• Lactic acidosis and hypersensitivity (Pharmaceutical Press, 2002).
2.5.5 PAEDIATRIC AND ADULT DOSE
Lamivudine is used in the treatment of both HIV and hepatitis B.
Children with HIV infection between 3 months to 12 years: 4 mg/kg twice daily to a maximum of 150 mg twice daily.
Adults with HIV infection: 150 mg twice daily.
Adults with chronic hepatitis B: 100 mg once daily (Pharmaceutical Press, 2002).
2.6 STAVUDINE
p
Figure 2.5 Stavudine - CioH12N204 (Pharmaceutical Press, 2007). 2.6.1 CHEMICAL PROPERTIES
Stavudine is a white to off-white, crystalline powder (Pharmaceutical Press, 2007).
• Molecular formula: C10H12N2O4. • CAS registry number: [3056-17-5].
• CAS name(s): 1-[(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H,3H)-dione; and
1-(2,3-dideoxy-(3-D-glycero-pent-2-enofuranosyl)-5-methylpyrimidine-2,4(1 H,3H)-dione (D4T). • Additional name(s): d4T. • Molecular weight: 224.2. • Percent composition: C 41.91%; H 4.84%; N 18.33%; 0 20.94%; S 13.99%. • Melting range: 160 - 162°C.
• Solubility: Soluble in water, c = 0.7, and sparingly soluble in ethanol (Merck & Co., Inc., 2006).
2.6.2 PHARMACOLOGY
Stavudine is a nucleoside reverse transcriptase inhibitor (NRTI), related to thymidine. After passive diffusion into the cell, stavudine must be phosphorylated into the active form, stavudine triphosphate. Stavudine triphosphate inhibits reverse transcriptase and terminates the DNA chain
(Hardman & Limbard, 2001; Pharmaceutical Press, 2002).
2.6.3 PHARMACOKINETICS
• Bioavailability: 86% (adults); 6 1 % - 78% (children).
• Half-life: Elimination 1.0 - 1.5 hours (single dose); intracellular stavudine triphosphate 3.5 hours (measured in vitro); 0.82 -1.24 hours (children).
• Volume of distribution: 0.88 - 1.06 «/kg (adults); 16.8 - 19 Urn2
(children).
• Clearance: Oral 0.46 - 0.6 Mi/kg (adults); 212 - 256 m*/min/m2
(children).
• Protein binding: Negligible (Moffat et ai, 2004).
2.6.4 ADVERSE EFFECTS
• Dose related peripheral neuropathy;
• Raised liver enzyme concentrations may occur during treatment; • Pancreatitis has been reported and fatalities have occurred;
• Asthenia, chest pain, hypersensitivity reactions and an influenza-like syndrome, dizziness, headache and insomnia;
• Gastrointestinal track (GIT) adverse effects: abdominal pain, constipation, diarrhoea, nausea and vomiting;
• Anorexia, neutropenia and thrombocytopenia and
• Lactic acidosis, lymphadenopathy and neoplasm (Pharmaceutical Press, 2002).
2.6.5 PAEDIATRIC AND ADULT DOSE
Children: Over 3 months of age and weighing < 30 kg: 1 mg/kg every 12 hours; > 30 kg: adult dose.
Adult dose: < 60 kg: 30 mg every 12 hours; > 60 kg: 40 mg every 12 hours. The use of stavudine in combination with zidovudine is not recommended.
Dosage reduction is recommended for patients with renal impairment (Pharmaceutical Press, 2002).
2.7 CONCLUSION
The chemical and physical properties of various ARVs were discussed in this chapter and important data to consider in the formulation of Pheroids™ and pro-Pheroids were presented.
