Drugs in Clinical Pipeline: Artefenomel

Artefenomel  [4-(2-{4-[(1s,1''R,3''S,4s,5'r,5''S,7''S)-Dispiro[cyclohexane-1,3'-[1,2,4]trioxolane-5',2''-tricyclo[3.3.1.13,7]decan]-4-yl]phenoxy}ethyl)morpholine] is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. The outstanding efficacy and prolonged blood concentrations of Artefenomel are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death. In vitro, Artefenomel is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC₅₀ values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, Artefenomel completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, Mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, Artefenomel exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. These exceptional antimalarial and pharmacokinetic properties led to its selection as a clinical drug development candidate. Artefenomel has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients [1].

References:

1. Charma, S. A.; et. al. Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc Natl Acad Sci U S A 2011, 108(11), 4400-4405.

Drugs in Clinical Pipeline: Tafenoquine

Tafenoquine [N-[2,6-Dimethoxy-4-methyl-5-[3-(trifluoromethyl)phenoxy]quinolin-8-yl]pentane-1,4-diamine] is a Primaquine analogue. It is a member of the 8-aminoquinoline group of drugs that includes Primaquine and seldom used drug, Pamaquine. Tafenoquine is being manufactured by GlaxoSmithKline, where the drug is being investigated as a potential treatment for malaria, as well as for malaria prevention [1]. Tafenoquine is a long-acting analogue of primaquine. For non-immune travelers travelling to endemic areas for few weeks, Tafenoquine, has the potential to be the drug of choice. Besides, on leaving the endemic region, Tafenoquine need not be continued as it has an extended half-life and potential transmission blocking properties. Hence there are minimum chances of treatment failure due to non-compliance.

The distinct advantages of Tafenoquine are:

1. Proven activity against blood and liver stage parasites.

2. It has a long half life.

3. Better compliance.

4. Potential of being the drug of choice for travelers to endemic areas for short periods.

5. The drug can be stopped immediately upon leaving endemic area.

6. Chemoprophylactic properties against both P. falciparum and P. vivax.

7. The drug has a potential for radical cure of P. vivax.

8. Moreover, it has additional gametocidal and sporontocidal activity.

In December 2013, US Food and Drug Administration (US-FDA) granted "breakthrough therapy" designation for the investigational antimalarial drug Tafenoquine. Co-developers GlaxoSmithKline and the Medicines for Malaria Venture, have credited this to the ability of Tafenoquine to target Plasmodium vivax malaria, including a form that lies dormant in the liver and causes relapse of infection within weeks to months of the initial mosquito bite. An estimated 70 million to 390 million clinical cases of uncomplicated malaria caused by P. vivax occur annually with significant public health and economic consequences in South and East Asia, Latin America, and the horn of Africa. The drug, administered in a single dose, would be indicated for both malaria treatment and relapse prevention.

Common Name: Tafenoquine

Synonyms: SB-252263; SB252263; SB 252263; WR-238605; WR238605; WR 238605

IUPAC Name: N-[2,6-Dimethoxy-4-methyl-5-[3-(trifluoromethyl)phenoxy]quinolin-8-yl]pentane-1,4-diamine

CAS Number: 106635-80-7

Mechanism of Action: 

Indication: Anti-Malarial

Development Stage: Phase III

Company: GlaxoSmithKline/Medicines for Malaria Venture

References:
1. Prashar, L.; et. al. Tafenoquine: A New 8-Aminoquinoline. Medical Journal of Zambia 2009, 36(4), 187-190.

How Blood Group O Protects Against Malaria

It was the pioneer work by Oliver Gonzalez (1944) where it was postulated that plasmodia possess antigens equivalent to the blood-group antigens A and B of man. The work referred showed that the anti-A titre of Group O subjects, who had often had attacks of Plasmodium vivax and Plasmodium falciparum malaria, was far greater than in control Group O subjects; and also that the anti-B titre of the infected subjects was raised, though not to the same extent as for anti-A [1]. His theory was not totally accepted nor was it denied [2]. Since, that time till early 2000, the same dilemma persisted, whether to accept this theory or totally reject it.

Though it was well documented that people with blood type O are protected against severe malaria [3], while those with other types, such as A, often fall into a coma and die. Then there were reports that suggest otherwise, such as where blood group A, B and O were equally susceptible to malaria infection, AB blood group had less number of persons with malaria parasites. A significantly lower frequency of Plasmodium falciparum was observed among individuals with blood groups A and O. In other two blood groups B and AB, no difference in P. vivax and P. falciparum proportions were observed. A two-year study showed that the frequency of repeated attacks between all blood groups was similar [4].

Understanding the mechanisms behind this has been one of the main goals of malaria research. Although it has been known for two decades that human red cell ABO blood group affects the ability of malaria parasites to form rosettes [5], the precise details of the interaction remained obscure. Rosettes are clusters of infected red blood cells binding to uninfected red blood cells. 

Rosetting is characterized by the binding of P. falciparum-infected red blood cells (RBCs) to uninfected RBCs to form clusters of cells that are thought to contribute to the pathology of falciparum malaria by obstructing blood flow in small blood vessels.

Many previous work had shown that rosetting parasites form larger, stronger rosettes in non-O blood groups (A, B or AB) than in group O RBCs. Furthermore, the percentage of infected RBCs forming rosettes is significantly lower in fresh clinical isolates derived from group O than in non-O patients. It appears that this is because the A and B antigens are receptors for rosetting on uninfected RBCs, being bound by a parasite protein called PfEMP1 which is expressed on the surface of infected RBCs. Rosettes still form in group O RBCs (albeit smaller and weaker than in non-O RBC) through the involvement of other RBC molecules which act as alternative receptors for rosetting. Thus, it was reasoned that if rosetting contributes directly to the pathogenesis of severe malaria and is reduced in blood group O RBCs, then group O individuals should be protected against life-threatening malaria [6].

The life-saving effect of group O is thought to occur due to the impaired ability of Plasmodium falciparum parasites to form rosettes in group O blood. Rosetting occurs due to specific members of the P. falciparum Erythrocyte Membrane Protein one (PfEMP1) family of variant antigens, on the surface of infected red cells, binding to receptors, including the A and B blood group trisaccharides, on uninfected red cells. Rosetting contributes to microvascular obstruction in severe malaria, leading to hypoxia, acidosis, organ dysfunction and death [7].

In 2012, Vigan-Womas et. al. provided an insight to the mechanism explaining how rosetting malaria parasites bind to the blood group A sugars. The team used a comination of in vitro functional studies, insights from crystallography and computational docking studies to examine the binding site for interaction between the malaria parasite rosetting ligand, PfEMP1 and the group A-trisaccharide (GalNAc-α1,3(Fuc-α1,2)Gal). They expressed the extracellular domains from a rosette-mediating PfEMP1 variant (Palo Alto VarO) as recombinant proteins, and found that the N-terminal head-structure region (known as NTS-DBLα-CIDR), bound the group A-trisaccharide. Furthermore, they crystallized the PfEMP1 N-terminal region to analyze its structure, and used computer docking to identify potential binding sites for the A-trisaccharide [8].

The salient findings were:

1. VarO rosetting shares with other rosetting lines three generic characteristics, namely an extreme sensitivity to sulphated glycosaminoglycans, the need for human serum and a marked ABO blood group preference characterised by reduced binding to group O RBCs. 

2. The experimental data indicate that the major determinant affecting VarO rosetting efficiency is indeed the ABO blood group. 

3. On exploring VarO-infected RBCs (iRBCs) binding characteristics using a monovariant culture of the Palo Alto 89F5 clone, in which greater than 90% of the iRBCs were positively selected to express PfEMP1-VarO, it was observed that VarO-iRBCs preferentially bind to blood group A compared to blood group B, which itself is preferred to blood group O.

4. More detailed RBC subgroup analysis showed preferred binding to group A1, weaker binding to groups A2 and B, and least binding to groups Ax and O.

Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα1. The authors hypothesized that RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins. This opens novel opportunities for intervention against severe malaria.

In 2015, another aspect was explored to this ever expanding mechanism, the RIFINs [9]. Their presence has been reported as members of rif (repetitive interspersed family), clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum. Their high copy number, sequence variability, and red cell surface location indicated an important role for RIFINs in malaria host–parasite interaction but the mechanism was still missing [10]. The researchers now have attempted the same.

Though sequestration and rosetting in individuals with severe Plasmodium falciparum malaria has been attributed to P. falciparum erythrocyte membrane protein 1 (PfEMP1) still few questions remained:

a: Antibodies to PfEMP1 disrupt rosettes of parasites grown only in blood group O RBCs, not group A RBCs. 

b: The majority of P. falciparum strains and fresh clinical isolates prefer group A RBCs for rosetting. 

Researchers found that enzymatic removal of PfEMP1 from the iRBC surface reduced rosetting in blood group O but not blood group A, indicating that PfEMP1 may not be the only molecule responsible for RBC binding and rosette formation. A second family of antigens found at the iRBC surface is RIFINs. These polypeptides are encoded by 150 rif genes and comprise the largest family of antigenically variable molecules in P. falciparum. Given that the function of RIFINs is unknown and that they are resistant to enzyme degradation and upregulated in rosetting parasites, researchers speculated that RIFINs contribute to the rosetting and sequestration of P. falciparum mediated by blood group A antigen.

To study the function of the RIFINs, researchers investigated primary structures of RIFINs and found that the majority (~70%) belong to subgroup A (A-RIFIN) and possess an insertion of 25 amino acids at the N terminus (indel) that the B-RIFINs lack. Researchers analyzed the ability of rif gene–transfected CHO cells to bind RBCs. A-RIFIN CHO cells bound large numbers of group A RBCs (up to ~25 RBCs per CHO cell), whereas the binding of group O RBCs was less pronounced and similar to that of CHO cells expressing the N-terminal domain of PfEMP1 (DBL1α). This suggests that the group A antigen is a major receptor for A-RIFINs. RBC binding was negligible with B-RIFIN CHO cells or CHO cells expressing PfEMP1 (DBL1α of PfEMP1-FCR3S1.2var1) (control) [10]. Moreover, group A1 RBCs bound significantly better than group A2 RBCs. The researchers through various models examplified the role of RIFINs in microvascular binding of P. falciparum iRBCs. Their results hint that RIFINs may contribute to the varying global distribution of ABO blood groups in favor of blood group O.

References:

1. Oliver-Gonzalez, J.; et. al. A substance in animal parasites related to the human isoagglutinogens. J Infect Dis 1944, 74, 173-177.

2. Raper, A. B. ABO blood groups and malaria. 1967.

3. Gupta, M.; et. al. Relationship between ABO blood groups and malaria. Bull World Health Organ 1980, 58(6), 913-915.

4. Singh, N.; et. al. ABO blood groups among malaria cases from district Mandla, Madhya Pradesh. Indian J Malariol 1995, 32(2), 59-63.
5. Carlson, J.; et. al. Plasmodium falciparum erythrocyte rosetting is mediated by promiscuous lectin-like interactions. J Exp Med 1992, 176(5), 1311-1317.
6. Rowe, J. A.; et. al. Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention. Curr Opin Hematol 2009, 16(6), 480-487.
7. Alexandra, R. J. Revealing the secrets of malaria parasite interaction with blood group A sugars. Pathog Glob Health 2013, 107(2), 45.
8. Vigan-Womas, I.; et. al. Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting. PLoS Pathog 2012, 8(7), e1002781.
9. Wahlgren, M.; et. al. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria. Nat Med 2015, 21(4), 314-317.
10. Kyes, S. A.; et. al. Rifins: A second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum. Proc Natl Acad Sci U S A 1999, 96(16), 9333-9338.

"Oliver like Einstein knew something, that we still dont"

Gra17 And Gra23 Proteins As New Target For Anti-Malaria Drugs

Gra17 And Gra23 Proteins As New Target For Anti-Malaria Drugs

In a paper published in Cell Host and Microbe, the researchers describe how they identified novel drug targets while studying the way in which the parasites Toxoplasma gondii (which causes toxoplasmosis), and Plasmodium, (which causes malaria), access vital nutrients from their host cells.

Pathogens that cause malaria and tuberculosis spend a large portion of their life inside specially built compartments within their host cells. These compartments, termed as “parasitophorous vacuoles,” separate the host cytoplasm and the parasite by a membrane, and thereby protect the parasites from the host cell’s defenses. These "vacuoles" appeared to be designed in such a manner that its membrane acts as a barrier between the parasite and the host cell. This makes it more difficult for the parasite to release proteins involved in the transformation of the host cell beyond the membrane in order to spread the disease, and for the pathogen to gain access to vital nutrients.

Previous research has shown that the vacuoles are selectively permeable to small molecules, allowing certain nutrients to pass through pores in the membrane. But until now, no one has been able to determine the molecular makeup of these pores, and how they are formed.

Researchers used the most basic knowledge of survival to solve this riddle. A parasite by definition will hunt for certain key nutrients from their host. So it will evolve some mechanism to overcome these “vacuoles” membrane issues to gain access to these nutrients. The researchers discovered two proteins namely GRA17 and GRA23, secreted by the parasite Toxoplasma, which are responsible for forming these pores in the vacuole.  Though it was found by an accident (as all good researches have been ), while investigating how the parasites are able to release their own proteins out into the host cell beyond the vacuole membrane after invasion. Similar research into how the related Plasmodium pathogen performs this trick had identified a so-called “protein export complex” that transports encoded proteins from the parasite into its host red blood cell, which transforms these red blood cells in a way that is vital to the spread of malaria.

Moreover, researchers identified proteins secreted by Toxoplasma that appeared to be homologues, or of shared ancestry to, this protein export complex in Plasmodium.

In the next step, researchers planned experiments to find out what these proteins do to help the parasite survive. When researchers stopped these proteins from functioning, they found it made no difference to the export of proteins from the parasite beyond the vacuole. This meant that these proteins are involved in something more important that protein export, which might be small molecule transfer (as in survival important). So, in step two researchers added dyes to the host cell, and again knocked out the two proteins, the researchers found that it prevented the dyes flowing into the vacuole. More significantly though, when the researchers expressed a Plasmodium export complex gene in the modified Toxoplasma, they found that the dyes were able to flow into the vacuole once again, suggesting that this small-molecule transport function had been restored.

The authors summed up their works in these important statements:

1. Crucially, since these proteins are only found in the parasite phylum Apicomplexa, to which both Toxoplasma and Plasmodium belong, they could be used as a drug target against the diseases.

2. This is a really strong potential drug target for restricting the access of these parasites to a set of nutrients.

3. In addition to malaria, the technique could also be used to target the parasite Eimeria, which affects cattle and poultry, among other animals, and therefore has a huge economic cost.

Article Citation: Gold, D. A.; et. al. The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Cell Host Microbe 2015, 17(5), 642-652.

SAYE: An Antimalarial Phytomedicine from Burkina Faso

SAYE: An Antimalarial Phytomedicine from Burkina Faso

Burkina Faso (formerly called the Republic of Upper Volta) is a landlocked country in West Africa around 274,200 square kilometres (105,900 sq mi) in size. It is surrounded by six countries: Mali to the north; Niger to the east; Benin to the southeast; Togo and Ghana to the south; and Ivory Coast to the southwest. Its capital is Ouagadougou.

Like many other African countries, Burkina Faso faces malaria as one of the biggest human killer disease. The humane laws in Burkina Faso officially recognizes traditional medicine as part of the health system (Loi No 23/94/ADP). The national policy on traditional medicine aims to integrate traditional medical practices and medicinal products derived from the traditional pharmacopoeia into the national health care system in order to improve access to medicines for the whole population.

SAYE sounds like CHAI, thats North-Indian word for TEA

Using this life-promoting law, Dr. Zéphirin Dakuyo promotes/sells mixture named SAYE, which literally means “jaundice” in the local Dioula language. SAYE is manufactured by mixing the three dried and coarsely chopped ingredients in the proportions given in Table. It is sold in a box of 175 g of the chopped, dried plant parts. Patients are instructed to mix 3 tablespoons of the dried plant material in two glasses of water, boil the mixture for 5 minutes, filter it, and drink it. Adults should drink one large glass three times a day, and children age 7 years and older are advised to drink half a glass three times a day for 5 days.

Component		Amount per box (g)		Amount per batch (kg)
C. planchonii Hook. f. ex Planch. (Bixaceae) rhizome		115		230
Cassia alata L. (Caesalpiniaceae) leaves		25		50
Phyllanthus amarus Schumach. (Euphorbiaceae) whole plant		35		70

Dr. Zéphirin Dakuyo was the first pharmacist to be posted in Banfora Hospital in Southwest Burkina Faso, in 1983. At the time, chloroquine was the first-line treatment for malaria, but it has since been abandoned because of high levels of drug resistance. He soon received feedback from patients that they did not like chloroquine but preferred to treat themselves with herbal medicines, in particular the roots of N'Dribala (Cochlospermum planchonii). However, they did not have time to collect this medicinal plant themselves, so Dr. Dakuyo, with support from the hospital staff, started to harvest and package it for the patients. The medicine was sold at the hospital to patients with malaria and was also provided to community health workers to supply to patients.

Dr. Dakuyo received feedback from patients that SAYE was even more effective than N'Dribala for treating malaria, and patients started buying it for this condition. In 1986, Dr. Dakuyo also developed capsules of powdered SAYE because he found that many patients did not have time to boil the herbs every day.

In 1993, Dr. Dakuyo left the hospital to start his own pharmacy; he also set up a small factory for producing the herbal medicine. As demand increased and he began producing other herbal products, the size of the factory gradually increased. In 2001 it was registered as a company, Phytofla. In 2005, SAYE and N'Dribala both received an official license from the Ministry of Health for the treatment of uncomplicated malaria. Annual sales now stand at 60,000 boxes of SAYE and 25,000 boxes of N'Dribala. Although Artemisinin-based Combination Therapies (ACTs) are widely available in Burkina Faso, they are expensive and are believed to have some adverse effects. Therefore, many adult patients still prefer to use SAYE, sometimes in combination with a modern medicine and sometimes alone.

Article citation: Dakuyo, Z.; et. al. SAYE: The Story of an Antimalarial Phytomedicine from Burkina Faso. The Journal of Alternative and Complementary Medicine 2015, 21(4), 187-195.

Herbal medicine is Ayurveda