FIELD OF THE INVENTION ;
The present invention relates to novel drug delivery systems based on
nanosomal systems.
In this study samples of cholesterol containing nanosomal Amphotericin B in
saline have been studied with cryo-electron microscopy (cryoEM). A fresh and a
two year old sample have been analyzed unsonicated as well as after
sonication. Analysis of the cryoEM images included size distribution and
overall morphology, such as lamellarity (unilamellar versus multilemellar) and
aggregation.
BACKGROUND OF THE INVENTION :
Rising incidences of Systemic and Topical Mycosis caused by different genus of
yeast fungi and dermatophytes in both immuno-deficient and immuno
competent patients remain an important and inadequately addressed medical
problem. Resulting mortality and alarming prolonged morbidity is of great
concern. With all the drugs discovered thus far, there have been problems of
limited spectrum, poor potency, limitations of suitable formulations, adverse
drug reactions and life-threatening toxicities and quite often a combination of
some or all of the above problems. Polyene aminoglycoside group of antibiotics
appeared most promising broad-spectrum and potent. However, toxicities of
most of these compounds prevented there inclusion as therapeutics.
Amphotericin B has been the only polyene Macrolide that stayed in wide
clinical use despite the fact that various formulations such as sodium
deoxycholate miceller suspension, liposomal, lipid complex and lipid colloidal
dispersion, all contain life-threatening nephrotoxicity to varying extant.
The objectives in formulating Amphotericin B, are to remove nephrotoxicity,
make stable preparation, ensure effectiveness at low doses and free of toxicity
and adverse drug reactions even at high doses.
Large number of patents granted/filed and publications describing
Amphotericin B formulations exist. Except for the below described four
formulations no other formulations are operational as they have not been able
to make such other formulations adequately nephro/safe.
In all the known Amphotericin B formulations, the constituents are selected
from large list of options of phospholipids/lipids and stabilizers. No
preparation is infused in saline as Amphotericin B is known to precipitate in
saline.
1. Conventional Amphotericin B : Amphotericin B - Deoxycholate
colloidal suspension in 5% Dextrose,
Efficacy 33% Nephrotoxicity 67%
Amphotericin B is insoluble in aqueous medium. The problem was
marginally overcome in late 1950s by dissolving Amphotericin B in
deoxycholate and formulating as micellar suspension in 5% dextrose in
water. Amphotericin B precipitates in NaCl and thus neither Amphotericin B
in deoxycholate was prepared in NaCl nor diluted in saline. Furthermore, this
suspension was lyophilized to render stability to the preparation.
2. Liposomal Amphotericin B diluted in 5% Dextrose
Efficacy 77% Nephrotoxicity 20%
A Liposomal Amphotericin B made up of combination of soya
phosphatidylcholine hydrogenated, distearoylphosphatidylglycerol,
cholesterol and alpha tocopherol in 4.5% sucrose and disodium
succinate hexahydrate as buffer was selected from amongst number of
combinations of phospholipids, sterols, membrane stabilizing sugars and
their varying ratios. Despite use of membrane stabilizing sucrose, this
preparation was lyophilized to overcome instability. This preparation of
Liposomal Amphotericin B is reported to precipitate in saline and thus
dilution in /contact with saline is strongly forbidden.
United States Patent 4766046 describes that:
Due to the size and, instability of Amphotericin B liposomes, it has not been
possible to prepare and store small-diamater Amphotericin B liposomes
without a significant (several fold) size increase over a several week storage
period. As a result, due to different in vivo liposome uptake and drug release
and toxicity properties which are related to liposome size, it has been difficult
to control and evaluate the therapeutic index of stored Amphotericin B
liposomes. The size instability problem is particularly serious where liposome
sizes greater than l-2microns are attained, since cholesterol-containing
Amphotericin B liposomes are substantially more toxic than smaller, original
size liposomes. The size stability problem has been solved heretofore only by
administering the sized liposomes shortly after preparation. This, of course, is
an impractical approach to drug delivery in the usual clinical setting.
3. Amphotericin B Lipid Complex diluted in 5% Dextrose
Efficacy 34% Nephrotoxicity 63%
This preparation is composed of Amphotericin B, synthetic
phospholipids viz. Dimyristoylphosphatidylcholine and Dimyristoyl-
phosphatidylgycerol. The aqueous suspension is diluted in 5% dextrose
before administration. Neither the efficacy nor toxicity profile is improved
over conventional Amphotericin B.
4. Amphotericin B Colloidal Dispersion diluted in 5% Dextrose
Efficacy 46% Nephrotoxicity 40%
Amhotericin B and Sodium Cholesteryl Sulphate lyophilized with
Tromethamine, Disodium edentate dehydrate and Lactose
monohydrate HC1.
Glucose present in Amphotericin B Lipid formulations markedly reduces the
beneficial effect of the topically applied formulation. The inhibitory mechanism
of glucose is implied to be related to high viscosity introduced by glucose or to
changes introduced by glucose on the lipid/water interface of colloidal particles
(Crowe JH et. al. 1988 Biochim. Biophys Acta.947:367-384). Even therapeutic
success of Liposomal Amphotericin B at best is 77% which is sub par to
present invention.
It was therefore, envisaged to replace dextrose with saline in this invention
which has additional advantage of reducing Amphotericin B nephrotoxicity. In
view of the well known and documented fact that Saline causes precipitation of
Amphotericin B1-4, lipid composition and lipid to drug ratio were uniquely
designed to ascertain that Amphotericin B in the nanosomes is immobilized to
prevent precipitation of the nanosomal drug.
OBJECTS OF THE INVENTION :
An object of this invention is to provide a novel formulation of cholesterol
containing nanosomes stabilized in saline suspension with reduced
nephrotoxicity.
Another object of this invention is to propose a novel formulation of cholesterol
containing nanosomes stabilized in saline suspension having enhanced
antifungal activity.
Still another object of this invention is to propose a formulation of cholesterol
containing nanosomes stabilized in saline suspension which is very stable.
Further object of this invention is to propose a novel formulation of cholesterol
containing nanosomes stabilized in saline suspension which is less toxic.
Still further object of this invention is to propose a novel formulation of
cholesterol containing nanosomes stabilized in saline suspension in which the
dose of Amphotericin B used is very little for its high anti-infection activity .Yet
another object of this invention is to propose a novel formulation for diverse
applications aimed at optimizing lower Amphotericin B doses for higher anti-
infective activity, further minimizing nephro / toxicity and stable preparation
for various applications such as intra-venous, ophthalmic and topical etc.
BRIEF DESCRIPTION OF THE INVENTION :
According to this invention there is provided a novel formulation for treating
fungal infection comprising: Cholesterol containing nanosomal Amphotericin B
nanosomes in a suspension.
In accordance with this invention, there is also provided a process for
preparing the novel formulation of cholesterol containing nanosomes stabilized
in Saline suspension.
DETAILED DESCRIPTION OF THE INVENTION ;
In a solution, the concentration is the number of molecules of the solute in per
unit volume of the solvent which in case of injectables and infusions or any
other liquid dose form is generally aqueous, whereas in particulate
preparations including suspensions such as colloidal suspensions, the effective
concentration of the drug is determined by the number of drug carrying
particles. Such carriers are diverse nano or micro size assemblies of lipids,
proteins or other biodegradable carriers for injectable, oral or inhalable, trans-
dermal etc. and non-biodegradable for topical preparations predominantly. In
this invention, nanosomes have an optimally high lipid to drug ratio to increase
the number of drug containing nanosomes for each unit quantity of drug and
thereby achieving an efficacy optimization by complementing with nanofication
before administration.
Optimization of lipid to drug ratio with particulate size is based on novel
concept of the present invention in increasing effective concentration of active
pharmaceuticals by increasing the number of carrier particles of the API in the
formulations post production and prior to administration. This is achieved by
reducing the size of the particles of more lipid containing formulation. In the
formulation, because of optimally higher lipid to drug ratio there are more
number of particles/mg of drug. More particles result in better/higher
distribution of drug in the body. As there is more effective concentration of
drug in suspension there is more number of molecules distributed evenly
throughout the body. This will decrease the requirement of therapeutic dose.
As the API for example Amphotericin B is toxic in higher doses, by reducing the
requirement of API, will automatically reduce the toxicity to the
animals/patients and thus leads to safer drug formulation.
The APIs in the particulate preparation are encapsulated within the multiple
layers of the carrier particles. Hydrophobic / lipophilic drugs remain
intercalated in the lipid bilayer of lipid nanosomes and therefore not released
in aqueous in-vivo environment even upon fragmentation of or breakdown of
such nanosomes. Such drugs encapsulated in lipid nanosomes are transferred
from surface of nanosomes to the target cell surface. In such an instance, the
drugs encapsulated in the inner lamellae of nanosomes are not utilized
therapeutically and end up getting phagocitosd by the cells of reticulo-
endothelial system and exited from the in-vivo therapeutic chain. The
cumulative encapsulation of the drug in the inner lamellae is several times
higher than on the nanosomes surface lamella. If all the lamellae of a multi-
lamellar nanosomes are separated to form new small multiple nanosomes the
inner lamella encapsulating the drug will thus be converted into outer drug
exposing lamella of the nanosomes, it can be logically expected that effective
concentration of the drug would increase without any addition of the API to
such mix lamellar formulation. This optimization to give higher active
concentration for achieving higher therapeutic efficacy would make it possible
by subjecting mix lamellar micro and/or nanosomes to ultrasonic disruption
on the patients' bedside. From the mix lamellar population each particle
makes available several lamella each of which is converted into several
nanosomes. By increasing the number of nanosomes, majority of the API in the
carrier particles is brought to the surface thus multiplying the therapeutic
efficacy in proportion to the increase in the number of drug carrying
nanosomes. In the present instance the lipid to drug ratio varies form 45:1 to
45:15. as shown in the table below.
Table: 1
Shape and size of different lipid preparations

More the lipid matrix : drug means-
More are the numbers of liposomes / mg of drug and thus better reach
Nanofication Process ;
A novel approach has been innovated to carry out bedside nanofication of the
drug in accordance with this invention by using an Ultrasonic processor.
This machine allows hand free operation and converts particles into smaller
and less lamellar nanosomes resulting in optimally increased therapeutic
efficacy through the mechanisms enumerated below.
At the inflammation site the capillaries develop fenestrations and become more
permeable which allow movement of cells and particulate substances to
migrate/crossover from circulating blood to the surrounding inflamed site. The
enhancement of delivery of the drugs in particulate substances to the inflamed
areas by fenestration can be facilitated by converting larger carrier particles to
nanosomes. Nanofication results in increasing the therapeutic efficacy due to
resultant increase in number of drug containing nanosomes concentration at
the target site.
Yet another important application of nanofication is to reduce the loss of the
larger particles to the phagocytic cells of the reticulo-endothelial system.
Larger particles are rapidly identified as foreign particles by the scavenger cells
and resultantly are phagocytosed and cleared from the circulation rapidly. The
rate of phagocytosis is inversely proportional to the size of drug carrying
particles. Therefore, decreasing the size by converting the large and
multilameller particles to nanosomes would increase the plasma half-life and
delay phagocytic loss thereby further adding to increase in therapeutic efficacy.
The present invention relates to a controlled release pharmaceuticals
employing an array of technologies of novel drug delivery systems based on
nanosomal systems. In this study samples of cholesterol containing nanosomal
Amphoterich B in saline have been studied with cryo-electron microscopy
(cryoEM). Fresh and two year old sample have been analyzed unsonicated as
well as after sonication. Analysis of the cryo-EM images included size
distribution and overall morphology, such as lamellarity (unilamellar versus
multilamellar) and aggregation.
The following specific questions were addressed:
size distribution
overall morphology characterization
Cholesterol containing nanosomal Amphotericin B in saline samples, fresh and
two years old, were imaged using cryoEM. The samples were diluted 10 times
and imaged unsonicated and after 45 min of sonication. Sonication was done
in an ice-water-bath which kept the temperature of the sample below 8°C.
Size distribution
cryoEM of the diluted nanosome samples showed a very heterogeneous
specimen with particles of various morphologies and sizes. The size of the
particles varied from 20 nm to micrometer scale in diameter. As might be
expected, larger amounts of nanosomes with smaller diameters, 20-200 nm,
were seen in the sonicated samples compared to the unsonicated samples. No
clear differences could be observed in the cryoEM images between the old and
fresh nanosome samples, neither in the unsonicated nor in the sonicated case.
Since the samples contained particles with such a wide distribution of sizes
and shapes calculating and presenting the means diameters would not be
relevant and would only give misleading information.
Overall morphology
The nanosomes imaged by cryoEm showed well defined membranes with a
thickness of approximately 7 nm. The sonicated samples showed a higher
degree of small nanosomes which were often seen as separate particles
compared to the unsonicated samples where the nanosomes were almost
always in contact with each other. The outermost membrane(s) of the
unsonicated particles, in both the old and fresh samples, often surrounded
more than one nanosome making it hard to differentiate between neighboring
nanosomes.
Lamellarity
cryoEM showed that the nanosomes appear as both unilamellar and
multilamellar. More unilamellar nansomes were found in the sonicated
samples compared to the unsonicated samples. The number of membrane
layers surrounding the multitlamellar particles varied from 2 lamella to onion-
like structures with over 10 membrane layers. It also seems as if large particles
may entrap smaller ones where the membranes are not in close proximity.
However, it is possible that the ice thickness allow these entrapped nanosomes
to be spatially located along the z-axis.
Aggregation
cryoEM did not show many three dimensional nanosome aggregates in any of
the samples. The particles in the unsonicated samples were mainly in close
contact and it was often hard to differentiate between where one nanosome
ends and another begins since they often shared the outermost membrane
layer/layers.
Conclusion
The nanosome samples showed a very heterogeneous specimen with particles
of various morphologies and diameters ranging from 20nm to micrometer scale.
Unilamellar nanosomes with smaller diameters, 20-200 nm, were much more
frequently seen in the sonicated samples compared to the unsonicated
samples. It appears as sonication effectively ruptures the large multilamellar
particles seen in the unsonicated samples into small unilamellar nanosomes.
Nevertheless, some large multitlamellar liposomes were still observed in the
sonicated samples and they were often better separated compared to the large
multilamellar particles in the unsonicated samples. Prolonged sonication may
result in a more complete rupture of large particles.
There were no clear visible differences between the old versus fresh nanosome
samples either comparing the unsonicated or sonicated samples.
Importance of Saline in reducing AmB nephrotoxicity
Dose related toxicities particularly nephro-toxicity has been the major
impediment in parenteral administration of Amphotericin B. Amphotericin B is
known to bind to sterol rich membranes and undergo self-assembled pores
formation leading to lysis of these cells. Kidneys being rich in Cholesterol
content and Polyene Macrolides such as Amphotericin B having high affinity to
cholesterol present a complex scenario limiting use of such compounds as
therapeutic agents. Encapsulating Amphotericin B in Cholesterol containing
lipid formulation has been of inadequate advantage (see Table 2 below)

Furthermore, the apparent remedy to overcome Amphotericin B toxicity by
formulating as Saline suspension is being achieved where as earlier
Amphotericin has been known to precipitate in Saline. In this invention
Amphotericin B has been immobilized in the cholesterol containing matrix
preventing mobilization and consequent precipitation. The product of this
inventin - Cholesterol containing nanosomal Amphotericin B in Saline
suspension drastically lowers nephro-toxicity.
While Ergosterol containing nanosomes would allow higher concentrations of
Amphotericin B and would be of value for treatment of Visceral Leishmaniasis
only, as provided by this invention replacement of
Ergosterol with cholesterol in nanosomal Amphotericin B in Saline suspension
is required for potent activity against yeast, moulds and dermatophytes as well
as leishmania.
The invention to include saline as a suspension medium is based on the
hypothesis that renal function impairment caused by Amphotericin B viz
azotemia is associated with a decrease in glomerular filtration rate (GFR) and
renal blood flow, reduction in concentration ability, altered urinary
acidification, and potassium wasting. Nephrotoxicity of Amphotericin B is
related to its ionophore properties on biological membranes. (Schell R E:
Amphotericin B induced nephrotoxicity : Influence of Sodium Status (Letter).
Nephron 1992; 60:52.)
Glomerular toxicity can develop quickly after a single dose of Amphotericin B or
evolve slowly after days to weeks of Amphotericin B therapy depending on the
hydration status and underlying renal function of the patient. It is reported
that administering intravenous saline before and after Amphotericin B
infusions, a practice known as sodium loading blunts the decreases in the
Glomerular Filteration Rate caused by Amphotericin B (R Sabra 85 R A Branch
(1991) Mechanism of Amphotericin B- Induced Decrease in Glumerular
Filteration Rate in Rats. Antimicrobial Agents and Chemotherapy : 35; 2509-
2514).
LD50
By increasing the lipid to drug ratio and also the use of saline for suspending
the nanosomes has made these nanosomes very safe. During animal
preclinical toxicity studies the LD50 could not be determined as no animal
died up to 60mg/kg and above 60mg/kg could not be administered. These
nanosomes are ready to use liquid suspenson so cannot be administered as
concentrated dose and animals could not tolerate higher volume than for a
60mg dose. It is observed that neither Cholesterol without saline as in
Lipsomal Amphotericin B (refer to AmBisome Toxicity/Composition Reference
given above) nor Saline without Cholesterol as in Amphotericin B Lipid
Complex (refer to Abelcet Toxicity/Composition Reference given above) reduce
nephrotoxicity to the insignificant levels as our unique composition of
Cholesterol containing high lipid to drug ratio nanosomes in Saline
suspension. The in vitro efficacy of Cholesterol containing nanosomal
Amphotericin B in saline is many folds higher (at places 10 times) (MIC many
fold lower) than the conventional Amphotericin B against the large number of
clinical isolates of yeasts and moulds. The reason of higher activity is because
these nanosomes as well as the fungal membrane, both have similar and
favorable hydro-lipophilic environment so that the transfer of molecule from
the nanosome to the fungus is easy.
Stabilizing Nanosomes in Cholesterol & Saline
Stability of Cholesterol containing nanosomal Amphotericin B nanosomes in
Saline without any other membrane stabilizing agents, has been made
possible by the novel composition of the present invention Nansomal
Amphotericin B of the present invention by itself is stable for at least 24
months from the date of manufacture. Thus, it is definite that Cholesterol and
Saline plays a major role in increasing the stability of the composition.
EXAMPLES:
EXAMPLE 1: l-15mg drug encapsulation
In the cholesterol containing Nanosomal Amphotericin B in saline, different
amount i.e. lmg to 15mg of Amphotericin B per ml could be intercalated
successfully without increasing the amount of lipids meaning that for each of 1
to 15 mg/ml of Amphotericin B formulations, quantity of lipids is fixed 45 mg
i.e. lipid to drug ratio varies from 45:1 to 45:15.
Determination of "Drug: Lipid ratio in the Nanosomes" and "Amphotericin B in
the supernatant" of the innovated formulations:
Sample Details : Product Code
lmg/ml- NAmB-C/1
3mg/ml- NAmB-C/3
5mg/ml- NAmB-C/5
10mg/ml- NAmB-C/10
15mg/ml- NAmB-C/15
Amphotericin B assay and residual Methanol contents were determined and
results are presented below.
Observations:
(A)Amphotericin B determination in Supernatant liquid -
Centrifuged all samples and taken supernatant liquid to perform assay
for Amphotericin B. Reading at 405 nm in UV-visible spectrophotometer was
found negligible which establishes absence of Amphotericin B in the
supernatant liquid meaning that there is no unencapsulated Amphotericin B in
the innovated formulation.
(B)Drug : Lipid Ratio in Nanosomes
Determined Drug : Lipid Ratio in the Nanosomes of above formulations.
Observations are given below-
Product code Concentration Drug/Lipid Ratio
NAmB-C/1 lmg/ml 1:44.6
NAmB-C/3 3mg/ml 3:44.4
NAmB-C/5 5mg/ml 5:45.3
NAmB-C/10 10mg/ml 10:45.01
NAmB-C/15 15mg/ml 15:45.8
Example-2 : Relative nephro-toxicity of nanosomal Amphotericin B in Saline
v/s Dextrose suspension-
Inherent nephrotoxicity of Amphotericin B has been the major obstacle in
releasing full potential of this broad-spectrum and potent antifungal drug.
Despite known demerits of dextrose on the drug action as elaborated earlier
and the possibility of the advantages of saline, dextrose could not be replaced
with saline as ironically Amphotericin B is well known to precipitate in saline.
The unique design of this nanosomal Amphotericin B has allowed the stable
formulation in saline suspension by immobilizing Amphotericin B in
nanosomal matrix. To evaluate the nephrotoxicity of dextrose v/s saline the
following experiments were carried out-
Experiments were conducted on two groups of swiss albino mice, each guoup
comprised of 6 male and 6 female. One group was administered with saline
formulation and another with dextrose at a daily dose of 3mg/kg for the first 8
days which was further escalated to 5mg/kg/day for 9th and 10th. Blood was
collected on alternate days from half the number of each group of animals and
serum creatinine determined from day 2 to day 11 daily.
INDIVIDUAL ANIMAL CLINICAL CHEMISTRY DATA
Nanosomal Amphotericin B in 5% dextrose Dose : 3mg / kg/day
5 mg / kg/day since day 8
Table-3

Animal ID - Mb4949 - Found dead on day 2 after blood sampling
Animal ID - Mb4948 - Found dead on day 6 after blood sampling
Table-4
Nanosomal Amphotericin B in Normal Saline Dose : 3mg / kg/day
5 mg / kg/day since day 8

No significant differences were observed in serum creatinine levels in either
group which necessitated experiments with higher doses and longer duration.
In this experiment two groups of mice each comprised of 50 mice of equal
number of males and females. The daily dose was replaced by alternate day
administration of a dose of 10mg/kg, one group was given nanosomal
Amphotericin B in 5% dextrose and another in normal saline. For the
determination of serum creatinine levels, blood was collected on weekly basis.
In the dextrose group no significant differences were seen up to 2nd week and
even subsequently up to six weeks the rise in serum creatinine levels was not
significant. Increase in serum creatinine beyond doubling of baseline is seen in
2 animals in 4th week, 3 animals in 5th week and one animal in 6th week. In one
animal in the 3rd week and in another in5th week death appears to be related
to renal toxicity of the drug. Over all the nephrotoxicity was seen in 14% of the
animals.
In the saline group the serum creatinine levels remain consistant throughout
the experiment duration of 6 weeks. Only in one animal the rise in serum
creatinine level exceeded the doubling of the baseline after 6 weeks of
administration. The overall nephro-toxicity is seen in only 2% of cases. 5 of the
mice died during the experiment but the death does not seem to be related to
the drug.
Example-3 :Stabilizing Nanosomes in cholesterol 85 saline
Stability of cholesterol containing Nanosomal Amphotericin
B in saline without any other membrane stabilizing agents, for two years has
been made possible by unique composition reported in this invention.
Real time stability test on finished product
Product : Cholesterol containing Nanosomal Amphotericin B in
Saline.
Shelf Life : This Nanosomal Amphotericin B is stable at least 24
months from the date of manufacturing
Proposed Expiry : 24 months
Storage : Store at 2-8 °C
Control Batches :Stability study testing of Nanosomal Amphotericin B was
carried out on 3 batches
Batch No Manufacturing Date Expiry Date
50F07-147 08/2007 07/2009
50F07-148 08/2007 07/2009
50F07-149 08/2007 07/2009
Storage Condition : For long term stability study the products are kept at
2-8 °C
Testing Intervals : The stored samples are withdrawn at predetermined
Intervals, the intervals are as follows-
0 month
3 month
6 month
12 month
18 month
24 month
Real time stability study data of Cholesterol containing Nanosomal
Amphotericin B in Saline
Batch No : 50F07-147
Manufacturing Date : 08/2007
Expiry Date : 07/2009
Table-6

Real time stability study data of Cholesterol containing Liposomal
Amphotericin B in Saline
Batch No : 50F07-148
Manufacturing Date : 08/2007
Expiry Date : 07/2009
Table-7

Real time stability study data of Cholesterol containing Nanosomal
Amphotericin B in Saline
Batch No : 50F07-149
Manufacturing Date : 08/2007
Expiry Date : 07/2009
Table-8

Example-4: Comparison of in-vitro activity of Conventional Amphotericin B and
Cholesterol containing Nanosomal Amphotericin B in Saline (N Amphotericin B)
The experiments were carried out to determine antifungal spectrum and MIC of
Nanosomal Amphotericin B vis-a-vis commonly used antifungals viz.
Amphotericin B, Voriconazole, Itraconazole and Fluconazole to ascertain
efficacy against pathogenic yeasts and moulds including dermatophytes.
Following clinical isolates were tested:

The in-vitro activity of cholesterol containing Nanosomal Amphotericin B in
saline is many folds higher (in some cases 10 times & MIC much lower) than
the conventional Amphotericin B against the large number of clinical isolates of
yeasts and moulds. Furthermore, Amphotericin B was hitherto known for not
being effective against dermatophtes while Nanosomal Amphotericin B is
effective against dermatophtes viz. Trichophyton rubrum, T. tonsurans, T.
mentagrophytes, Microsporum gypseum, and Epidermophyton floccosum. The
reason of higher activity may be because these nanosomes as well as the fungal
membrane, both have similar and favorable hydro-lipophilic environment so
that the transfer of Amphotericin B molecule from the nanosome to the fungus
is easy.
The observations also support the possibility of lowering the dose when
Amphotericin B is administered as cholesterol containing nanosomes.
Conventional Amphotericin B which is colloidal suspension of the drug in
Sodium Deoxycholate is administered as a daily dose of lmg/kg body wt./day
while its commercially available lipid formulations developed for overcoming
dose limiting toxicity have doses ranging from 3-6mg/kg body wt./day. High
doses effect treatment economics and make the drug unaffordable while the
Cholesterol containing Nanosomal Amphotericin B would help lower
therapeutic dose and in turn make treatment affordable.
Dermatophytes;
1. Trichophyton rubrum, T. tonsurans, T. mentagrophytes.
2. Microsporum gypseum
3. Epidermophyton floccosum
Fungi causing Skin Infection:
1. Candida, Aspergillus, Mucor
Species resistant to Azoles:
1. Candida albicans N Amphotericin B : Active (0.03-0.25)
Amphotericin B : Active (0.125-1)
Fluconazole : Variable (0.125-64)
Voriconazole : Variable (0.03-8)
Itraconazole : Variable (0.03-8)
2. Cryptococcus neoformans N Amphotericin B : Active (0.03-0.25)
Amphotericin B : Active (0.25-1)
Fluconazole : Variable (0.125-16)
Voriconazole : Variable (0.03-16)
Itraconazole : Intermediate (0.03-4)
3. Aspergillus flavus, N Amphotericin B : Active (0.06-0.5)
A.fumigatus Amphotericin B : Active (0.5-2)
Fluconazole : Resistant (32-64)
Voriconazole : Active (0.125-1)
Itraconazole Variable (0.03-16)
4. Aspergillus oryzae N Amphotericin B : Active (1-2)
Amphotericin B : Intermediate (2-4)
Fluconazole : Resistant (64)
Voriconazole : Intermediate (0.5-4)
Itraconazole : Active (0.125-0.5)
5. Fusarium spp. N Amphotericin B : Active (0.06-0.5)
Amphotericin B : Active (0.5-1)
Fluconazole Resistant (16-64)
Voriconazole Intermediate (0.5-4)
Itraconazole Resistant (8-32)
6. Pseudallescheria boydii N Amphotericin B : Active (0.125-1)
Amphotericin B Active (0.5-1)
Fluconazole Resistant (8-32)
Voriconazole : Active
Itraconazole : Active
7. Rhizopus arrhizus, R. pusilus N Amphotericin B : Active (0.125-0.5)
Amphotericin B : Active (0.5-2)
Fluconazole : Resistant (8-64)
Voriconazole Variable (1-8)
Itraconazole : Resistant (8-16)
8. Absidia corymbifera N Amphotericin B : Active (0.06)
Amphotericin B : Active (0.5)
Fluconazole : Resistant (64)
Voriconazole : Variable (2-16)
Itraconazole : Resistant (16)
9. Mucor spp. N Amphotericin B Active (0.5-1)
Amphotericin B Active (1-2)
Fluconazole : Resistant (64)
Voriconazole Resistant (8-16)
Itraconazole : Resistant (0.03-8)
10. Apophysomyces elegans N Amphotericin B : Active (1)
Amphotericin B : Active (2)
Fluconazole : Resistant (64)
Voriconazole Intermediate (8)
Itraconazole Resistant (16)
11. Curxmlaria spp. N Amphotericin B : Active (0.03-0.125)
Amphotericin B : Active (0.125-1)
Fluconazole : Resistant (8-64)
Voriconazole : Active (1-2)
Itraconazole : Resistant (8-16)
12. Alternaria spp. N Amphotericin B Active (0.25)
AmphotericinB : Active (1-2)
Fluconazole Resistant (32-64)
Voriconazole : Active (0.5-1)
Itraconazole Active (1-2)
13. Cladophialophora bantiana N Amphotericin B Active (0.25-0.5)
AmphotericinB : Active (0.5-1)
Fluconazole : Resistant (32-64)
Voriconazole Active (0.25-2)
Itraconazole : Active (0.5-2)
14. Phialophora verrucosa N Amphotericin B : Active (2)
Amphotericin B Intermediate (4)
Fluconazole : Resistant (16)
Voriconazole Active (1)
Itraconazole : Resistant (16)
15. Scytalidum dimidatum N Amphotericin B : Active (0.03)
Amphotericin B : Active (0.5)
Fluconazole : Resistant (32)
Voriconazole : Active (0.5)
Itraconazole Resistant (16)
16. Sporothrix schenckii N Amphotericin B : Active (0.125-0.25)
Amphotericin B : Active (0.5-1)
Fluconazole : Resistant (8-64)
Voriconazole : Variable (0.03-16)
Itraconazole : Variable (0.03-16)
17'. Penicillium marneffei N Amphotericin B Active (0.125-0.5)
Amphotericin B : Active (0.25-1)
Fluconazole : Resistant (32-64)
Voriconazole : Active (0.25-1)
Itraconazole Active (1-2)
18. Trichophyton rubrum N Amphotericin B : Active 0.06-0.125
Amphotericin B Active (0.25-1)
Fluconazole : Variable/Resistant (4-32)
Voriconazole : Active (0.25-0.5)
Itraconazole : Resistant (8-16)
19. Trichophyton mentagrophytes N Amphotericin B Active (0.125-0.5)
Amphotericin B Active (0.5-2)
Fluconazole Resistant (32-64)
Voriconazole : Active (0.5-1)
Itraconazole Active (1-2)
20. Microsporum gypseum N Amphotericin B : Active (0.125)
Amphotericin B : Active (0.5)
Fluconazole : Resistant (64)
Voriconazole : Active (0.125-0.5)
Itraconazole : Active (0.25-0.5)
21. Paeciliomyces spp. N Amphotericin B : Variable (0.25-16)
Amphotericin B : Intermediate (1-4)
Fluconazole : Resistant (64)
Voriconazole : Variable (0.125-8)
Itraconazole Variable (0.06-16)
Fluconazole Resistant species: Microsporum gypseum, Trichophyton
mentagrophytes, Trichophyton rubrum, Penicillium marneffei, Sporothrix
schenckii, Scytalidum dimidatum, Phialophora verrucosa, Cladophialophora
bantiana, Alternaria spp., Curvularia spp., Apophysomyces elegans, Mucor spp.,
Absidia corymbifera, Rhizopus arrhizus, R. pusilus, Pseudallescheria boydii,
Fusarium spp., Aspergillus flavus. A. fumigatus, A. oryzae, Paeciliomyces spp.
Fluconazole Variable species: Candida albicans, Cryptococcus neoformans.
Voriconazole Resistant species: Mucor spp.
Voriconazole Variable species: Sporothrix schenckii, Absidia Corymbifera,
Rhizopus arrhizus, R. pusilus, Cryptococcus neoformans, Candida Albicans,
Paeciliomyces spp.
Itraconazole Resistant species: Trichophyton rubntm, Curvularia spp., Fusarium
spp., Absidia Corymbifera, Mucor spp., Apophysomyces elegans, Curvularia
spp., Rhizopus arrhizus, R. pusilus, Phialophora verrucosa, Scytalidum
dimidatum.
Itraconazole Variable species: Sporothrix schenckii, Aspergillus flavus, A.
fumigatus, Candida albicans, Paeciliomyces spp.
Nanosomal Amphotericin B variable species: Paeciliomyces spp.
Example-5: TEM & Freeze Fracture SEM pre and post sonication -
Effect of Lipid: Drug ratio on particle count /ml, Effect of sonication on particle
count /ml -
The Nanosome samples showed a very heterogeneous specimen with particles
of various morphologies and diameters ranging from 20 nm to micrometer
scale. Unilamellar nanosomes with smaller diameters, 20 to 200 nm, were
much more frequently seen in the sonicated samples compared to the
unsonicated samples. It appears as sonication effectively breaks the large
mulitlamellar particles seen in the unsonicated samples into small unilamellar
nanosomes. Nevertheless, some large mulitlamellar particles were still observed
in the sonicated samples and they were separated compared to the large
mulitlamellar particles in the unsonicated samples. Prolonged sonication may
result in a more complete breaking of large particles.
There were no clear visible differences between the old versus fresh samples
either comparing the unsonicated or sonicated samples. The samples, hence,
seem to be stable over the time period (2 years) investigated as shown in figures
1 86 2.
Example-6: Topical ophthalmic use of Saline Suspension of Cholesterol
containing Nanosomal Amphotericin B-
The cholesterol containing Nanosomal Amphotericin B in saline is also studied
topically in eyes and found to be safe and effective. Aspergillus fumigatus
keratitis model treated with Nanosomal and conventional Amphotericin B of
different concentration and untreated controls. The results show that half the
concentration of Cholesterol containing Nanosomal Amphotericin B in Saline is
as effective as conventional Amphotericin B full concentration.
Evaluation of efficacy and toxicity of Cholesterol containing Nanosomal
Amphotericin B in Saline formulation during treatment of experimental fungal
keratitis in rabbit.
Objectives:
a. To evaluate the efficacy of topical Nanosomal Amphotericin B in the
treatment of induced fungal keratitis in experimental rabbit model.
b. To compare the efficacy of Nanosomal Amphotericin B at 0.1% and 0.05%
concentration with conventional Amphotericin B at 0.1% concentration.
c. To assess any ocular toxicity in rabbit due to treatment with Nanosomal
Amphotericin B and compare the same with toxicity due to topical application
of Amphotericin B at 0.1% concentration.
Methods
Subjects: New Zealand White rabbits - 72
Fungal isolate: Aspergillus fumigatus (ATCC 13073), Candida albicans,
Fusarium solani
Aspergillus fumigatus and Fusarium solani were grown on a potato dextrose
agar slant at 30° C for 3-10 days. A conidial suspension was prepared by gently
swabbing the culture with a sterile swab and transferring it to 3-4 ml of sterile
saline in a 15 ml conical tube. Final concentration of conidia was adjusted to
obtain 106 conidia / ml. Candida albicans was grown on a potato dextrose agar
plate for 24 hours at 35° C. Five colonies >lmm in diameter was picked and
suspended in 5 ml of 0.85% sterile saline in a sterile 15 ml conical tube. The
suspension was vortexed and cells counted using a hemocytometer. Working
suspension of yeast cells was prepared in sterile saline to achieve a final
concentration of 106 CFU/ml.
Induction of keratitis and treatment
A total of 72 rabbits were used in the study of which 60 rabbits were infected
with Aspergillus fumigatus inoculum of which 22 rabbits were infected using
the contact lens model, while 38 were infected using the intrastromal
technique. Eight Rabbits were infected with clinical isolate of Candida albicans
( 4 rabbits with 108 yeast/ ml and 2 with 109 cells/ ml by intrastromal
inoculation and 2 rabbits were infected by using contact lens model with 109
cells/ml). Four rabbits were infected with clinical isolate of Fusariwn Solani
with inoculum dose of (106 spores/ ml) by intrastromal technique.
Induction of keratitis using contact lens: Rabbits were anesthetized with
intramuscular ketamine and xylazine. Corneal anesthesia was given using
topical proparacaine 0.5%. The nictitating membrane of the right eye was
removed by sharp dissection. A 7-mm filter paper disk moistened with 99%
isopropyl alcohol (Merck, USA) was placed on the center of the cornea for 30s
and the corneal epithelium was removed atraumatically. The eye was rinsed
with sodium lactate solution to remove any remaining traces of isopropyl
alcohol. A grid pattern of abrasions was made on the central cornea. The fungal
inoculum was transferred to the denuded cornea using a large-bore pipette tip
and the inoculum was retained in the cornea by placing the sterile contact lens
(diameter, 14.0 mm) (Pure vision, Bosch and Lomb, Ireland). To prevent contact
lens extrusion the lids were closed by performing tarsorrhaphy with 5-0 silk
sutures. The eyes were examined after 48 hours by removing contact lens and
subsequently after every 48 h. Corneal button from each of these rabbits were
obtained and subjected to microbiological and histopathological investigation.
Induction of keratitis by intrastromal injection of inoculum: Rabbits were
anesthetized with intramuscular ketamine and xylazine. Corneal anesthesia
was given with topical Proparacaine 0.5%. 20 1 of fungal inoculum. (106
spores/ lm) was injected intrastromally using a bent 30 G insulin needle
under slit lamp guidance. The rabbits were examined after every 2 days for
signs of keratitis.
Evaluation of antifungals: Since persistent infection was seen with the model
of intrastromal injection of inoculum, the treatment study was carried out with
this model. For the treatment rabbits were randomly divided into four groups of
each containing 4 rabbits.
The groups inoculated with Aspergillus fumigatus were:-
Group 1) Treated with 0.1% Nanosomal Amphotericin B,
Group 2) Treated with 0.1% conventional Amphotericin B,
Group 3) Treated with 0.05% Nanosomal amphotericin B
Group 4) Sterile normal saline instillation (untreated controls).
The infections before and after therapy were graded by giving the composite
score for different clinical signs determined using with slit lamp microscope.
Clinical scores were tabulated for each groups and their mean was taken.
Results
Contact lens model: For the initial standardization of fungal keratitis, of 22
rabbits used eight rabbits were infected with inoculum containing only spores
suspension of Aspergillus fumigatus by following contact lens model. However,
this did not give any clinical or microbiological evidence of infection (both
smear and cultures were negative, Table - 1). Subsequently fourteen rabbits
were infected with a mixture of spores and mycelium, which gave consistent
infection in the rabbits as seen in table -1. Although there was consistent
clinical infection, the severity of infection reduced after five days. Hence it was
not possible to start the treatment on 5th day. Therefore, the intrastromal
injection model was adopted for subsequent experiments and the treatment
with antifungals was started on the fifth day of infection.
Table-9
Results of induction of fungal keratitis in rabbit eyes using contact lens model

Evaluation of antifungal therapy using intrastromal model: Untreated
rabbits had a mean score of 16.1 ± 4.1 SD on day 15. However, 0.1%
Nanosomal Amphotericin-B treated rabbits had a mean score of 8.6 ± 2.37 SD,
which was statistically significant when compared to the untreated group
(p<0.001). Similarly, 0.05% Nanosomal amphotericin-B and 0.1% conventional
amphotericin-B treated group had a mean scores of 8.8 ± 2.37 SD and 8.4 ± 2.
0 SD respectively.
There was significant difference in the healing when the rabbits were treated
with all the three drugs as compared to the untreated group and was
statistically significant (p<0.001). However, the composite clinical score of .05%
Nanosomal formulation is similar to .1% of conventional drug for Aspergillus
keratitis.
Example-7: Experimental evidence of innovatively designed "Phospholipids-
Cholesterol Nanosome mediated Dermal Delivery of Amphotericin B"
Amphotericin B well known for not being absorbed through skin has limited
development of an effective topical formulation that has been overcome by this
invention by encapsulating Amphotericin B in innovated
Cholesterol/Phospholipid Nanosomes. The penetration and dermal delivery of
nanosomes encapsulated Amphotericin B may well be accounted to the
Cholesterol-Phospholipid Nanosome specific advantages, i.e., interaction of
phospholipids with intercellular lipids; presence of moisture in conjunction
with lipids; and the changes in physicochemical properties, such as solubility
and partitioning of the Amphotericin B molecules as desired. Further, as
observed, the achieved retention of Amphotericin B in skin on topical
application of nanosomal Amphotericin B in innovated formulation is one of the
most sought after behavior for improved drug-receptor interaction as well as for
protracted action of Amphotericin B.
The results, as obtained after study of the skin permeation behavior and
fluorescent marked photographic analysis, convincingly points towards the
superiority of the Nanosomal Amphotericin B formulation vis-d-vis
conventionally prepared Amphotericin B cream. This forms the basis of the
novelty in the Nanosomal Amphotericin B for topical dermal delivery of
Amphotericin B.
Objectives:
To study the influence of Cholesterol containing nanosomes in Saline
formulation on the dermal delivery of Amphotericin B in comparison to
conventional Amphotericin B cream-
Development of suitable study medium for permeation studies
Method development and validation for analysis
In-vitro permeation studies using Franz diffusion cell
Determination of drug-skin retentively
Monitoring of drug transportation (fluorescence marked studies)
Amphotericin B comes under the BCS class IV drugs, hence is difficult to
penetrate into any biological barrier including skin. The various reasons
includes are as follows:
Drug specific problems
Solubility
Partitioning
Skin specific problem (Tough horny keratin barrier)
Drug-Skin interactions
Improper interactions due to the difference in physicochemical properties of the
drug and skin
Despite earlier attempts to make useful topical application, the bulky molecule
of Amphotericin B, is difficult for absorption through the skin. As a result,
none could successfully achieve the goal. The fundamental problem of molecule
is its physico-chemical property as well as skin barrier. This provides a rational
to explore the potential of nanosomal system for topical delivery. Herein the
hypothesis is based on the principle that the drug within the aqua-lipoidal
vesicular milieu would acquire a different physicochemical set of properties to
interact favorably, in-order to migrate deeper into the skin layers. The moisture
within the vesicles, along with lipidic-molecule, is the key role player in the
improved drug transfer vis-a-vis conventional systems. Besides this, integration
of phospholipids with skin lipids helps build conducive milieu for improved
delivery.
Methodology
Diffusion and Retention studies - Skin permeation of Amphotericin B using
different carrier systems were studied using a Franz diffusion cell. The effective
permeation area of the diffusion cell and receptor cell was 3.14 cm2 and
volumes of the respective cells were 10 ml and 30 ml. The temperature of
receptor fluid was maintained at 32 ± 1°C. The receptor compartment
contained Briz-35 (5%) + Docusate Sodium (DOS) (1%) in distilled water to
facilitate sink condition.
Abdominal skin of male Laca mice (4 to 6 weeks old) was mounted, after hair
removal and de-fatting the skin, between the donor and receptor compartment.
Formulation equivalent to 358.5 ug (nanosomal/conventional cream) was
applied on donor compartment, after equilibrating the skin with sink medium
for 2 hr. Samples (1 ml) were withdrawn through the sampling port of the
receptor compartment, with replacement, at predetermined interval and
analyzed by UV spectrophotometer after suitable dilution.
Fluorescent marked migration study- Inherent fluorescent character of the
Amphotericin B has been exploited to visualize the migration and localization of
the drug with time. Laca mice were shaved using hair removing cream a day
before the study. Mice were scarified humanely at predetermined interval and,
immediately skin was washed with PBS pH 7.4 and stored in 10% formalin at -
20 °C till cryo-microsectioning. The sections were viewed under fluorescence
microscope with F2 filter.
Observations
Diffusion and Retention studies- A range of solvent systems were tried and
finally a system composed of Briz-35 (5%) + Docusate Sodium (1%) was chosen
as a sink media. The objective of this study was to asses the penetration,
retention and permeation of the drug into, at and across the skin layers
respectively. The major observation is that there is appreciable drug retention
within the layers of skin after nanosomal application (i.e. 1.291 ± 0.04) vis-?-vis
conventional cream (0.142 ± 0.05). However, with regards to permeation, the
drug Amphotericin B failed to permeate across the skin layers (in both the
cases of nanosomal and conventional systems) is insignificant as shown in
figure 3.
Fig. 3. Fluorescent transverse section of skin after the application of (a)
conventional Amphotericin B cream at 0.5 hr; (b) conventional Amphotericin B
cream at 1.0 hr; (c) conventional Amphotericin B cream at 2.0 hr; (d)
nanosomal Amphotericin B formulation at 0.5 hr (e) nanosomal Amphotericin
B formulation at 1.0 hr (f) nanosomal Amphotericin B formulation at 2.0 hr.
Table-11
Drug release and retention of Amphotericin B-

Fluorescent marked migration study- The fluorescent marked study (after
application of conventional and nanosomal formulations) to monitor the
penetration of drug in the skin layer has been shown in fig. 1. It comprises the
monitoring at different time intervals, i.e., 0.5, 1.0, 2.0 hrs. The study revealed
an appreciable difference at these intervals. The most remarkable difference
was found after 2 hrs of study.
The outcome of permeation behavior studies (Franz diffusion cell) and
penetration studies (fluorescent marked skin-histology studies) points to the
ability of the nanosomal vesicles to improve the delivery of Amphotericin B. As
shown by the drug retention data as well as 2 hr picture of skin-histology, the
Amphotericin B contained in the nanosomes is able to penetrate appreciably in
comparison to conventional drug formulation. The poor permeation of
nanosomal Amphotericin B as well as conventional drug formulation reveals
that the drug does not get across the skin layer and hence is not fit for the
transdermal drug Amphotericin B delivery. Henceforth, the Amphotericin B in
nanosome has shown a good potential for dermal delivery. The poor
penetration even serves as an advantage, as it does not allow the Amphotericin
B to be systemically absorbed.
WE CLAIM:
1. A novel formulation for treating fungal infections comprising: Cholesterol
containing Amphotericin B in a suspension.
2. The formulation as claimed in claim 1, wherein the lipid to Amphotericin B
ratio varies from 45:1 to 45:15.
3. The formulation as claimed in claim 1, wherein the suspension used is
Saline.
4. The formulation as claimed in claim 1, wherein the Amphotericin B is
sonicated to convert the particles into smaller and less lamellar nanosomes
within diameters 20-200 nm.

A novel formulation for treating fungal infections comprising Cholesterol
containing Nanosomal Amphotericin B in a Saline Suspension