US008937074 B2

(12) United States Patent (10) Patent No.: US 8,937,074 B2 Meyer (45) Date of Patent: Jan. 20, 2015

(54) ENHANCEMENT OF THE ACTION OF 9/4866 (2013.01); A61K 31/133 (2013.01); ANT-INFECTIVE AGENTS AND OF A6 IK3I/4409 (2013.01); A61 K3I/496 CENTRAL AND PERPHERAL NERVOUS (2013.01); A61 K3I/4965 (2013.01); A61 K SYSTEMIAGENTS ANDTRANSPORTATION 38/02 (2013.01); A61 K39/39533 (2013.01); OF NUCLECACID SUBSTANCES CI2N 15/63 (2013.01) USPC ...... 514/256; 514/258.1 (71) Applicant: North West University, Potchefstroom (58) Field of Classification Search (ZA) None See application file for complete search history. (72) Inventor: Petrus Johannes Meyer, George (ZA) (56) References Cited (73) Assignee: North West University, Potchefstroom (ZA) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,633,284 A 5/1997 Meyer patent is extended or adjusted under 35 6,416.740 B1* 7/2002 Unger ...... 424,952 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 13/709,596 DE 2647 671 A1 4f1978 WO 9606152 A2 2, 1996 (22) Filed: Dec. 10, 2012 * cited by examiner (65) Prior Publication Data Primary Examiner — Alton Pryor US 2013/0336990 A1 Dec. 19, 2013 (74) Attorney, Agent, or Firm — Rothwell, Figg, Ernst & Manbeck, P.C. Related U.S. Application Data (63) Continuation of application No. 10/345.204, filed on (57) ABSTRACT Jan. 16, 2003, now Pat. No. 8,329,685, which is a The invention provides a method of enhancing the action of a continuation-in-part of application No. pharmaceutical agent selected from the group consisting of PCT/ZAO1/00098, filed on Jul. 19, 2001, and a the anti-infective agents, the group comprising of the antimi continuation-in-part of application No. crobial agents, the anthelmintic agents and the anti-ectopara PCT/ZAO1/00099, filed on Jul. 19, 2001, and a sitic agents, but excluding coaltar Solution and H1-antagonist continuation-in-part of application No. antihistamines, and from the group consisting of the CPNS PCT/ZAO1/00100, filed on Jul. 19, 2001. agents selected from the group of compounds acting on the central or peripheral nervous system, but excluding coal tar (30) Foreign Application Priority Data Solution and H1-antagonistantihistamines and also excluding anti-inflammatory, analgesic and antipyretic agents and also Jul. 19, 2000 (ZA) ...... 2OOO/3643 provides an enhanced method for the administration of a Jul. 19, 2000 (ZA) ...... 2000/3644 nucleic acid Substance to the cells of an animal, a plant or a micro-organism. The method is characterized in that the (51) Int. Cl. agent or nucleic acid Substance is formulated with an admin AOIN 43/54 (2006.01) istration medium which comprises a solution of nitrous oxide AOIN 43/90 (2006.01) gas in a pharmaceutically acceptable carrier solvent for the A6 IK3I/7088 (2006.01) gas and which administration medium includes at least one A6 IK9/00 (2006.01) fatty acid or ester or other suitable derivative thereof selected A6 IK 47/02 (2006.01) from the group consisting of oleic acid, linoleic acid, alpha A6 IK9/48 (2006.01) linolenic acid, gamma-linolenic acid, arachidonic acid, A6 IK3I/33 (2006.01) eicosapentaenoic acid C20:5c)3], decosahexaenoic acid A6 IK3I/4409 (2006.01) C22:603, ricinoleic acid and derivatives thereof selected A6 IK3I/496 (2006.01) from the group consisting of the C1 to C6 alkyl esters thereof, A6 IK3I/4965 (2006.01) the glycerol-polyethylene glycol esters thereof and the reac A6 IK38/02 (2006.01) tion product of hydrogenated natural oils composed largely of A 6LX39/395 (2006.01) ricinoleic acid based oils, such as castor oil with ethylene CI2N 15/63 (2006.01) oxide. The formulations of Such agents or Substances form (52) U.S. Cl. part of the invention. CPC ...... A61 K3I/7088 (2013.01); A61 K9/0019 (2013.01); A61 K47/02 (2013.01); A61 K 18 Claims, 8 Drawing Sheets

U.S. Patent Jan. 20, 2015 Sheet 2 of 8 US 8,937,074 B2

Zone inhibition by different therapies

Submicron lipid Free active vesicle Saline aSSOCiated active

SS Aureus OP Aeruginosa

Figure 2 U.S. Patent Jan. 20, 2015 Sheet 3 of 8 US 8,937,074 B2

AbSOrbance/Cut- CD4 T-cell and viral growth Off ratioS 14 2

10 l Lind 1:256 - O. 1 nMAZT

8 ss. s indi 1.512 + O. nMAZT DAZT (1nM) 6 OAZT (0.1nM) 4. Control

2

O 2 3 Lindi 1.256 - O.1nMAZT

FIGURE 3 U.S. Patent Jan. 20, 2015 Sheet 4 of 8 US 8,937,074 B2

Viability of Mel1 cells 90% 80% 70% 60% D E 50% sO 40% g 30% 20% 10% O% g CN as S. SQ CN CD s 9. Cells CN 5

s N

d s S Fungi Therapeutic s O) C

FIGURE 4 U.S. Patent Jan. 20, 2015 Sheet 5 of 8 US 8,937,074 B2

Zone inhibition of yeasts and molds Zone inhibition measured S -

C Albicans Control T Mententagrophytes h E FOCOSSum

Control Nano lipid vesicle with active

Figure 5 U.S. Patent Jan. 20, 2015 Sheet 6 of 8 US 8,937,074 B2

Bioavailability:Rifamzaloid

Time (minutes)

-0-MZL Rifamzaloid HRifampin

Figure 6 U.S. Patent Jan. 20, 2015 Sheet 7 of 8 US 8,937,074 B2

Bioavailability of Rifampicin in the MZL formulated Rifamzaloid vs its comparator Rifampin BIOAVAILABLITY OF IZONAZID

O 60 120 180 240 3OO

Time (minutes)

mNH in MZL formula as NH in Rifafour Figure 7 U.S. Patent Jan. 20, 2015 Sheet 8 of 8 US 8,937,074 B2

Bioavailability of IZONIAZID BOAVAILABLITY OF PYRAZINAMIDE 5O

45

3 5

3 O

2 5

2 O

5

1 O

O - -op- r O 30 60 120 180 24O 3OO 360 Time (minutes) PZA in MZL formula Figure 8 Bioavailability of Pyrazinamide US 8,937,074 B2 1. 2 ENHANCEMENT OF THE ACTION OF of these conditions are caused by viral infections, the disclo ANT-INFECTIVE AGENTS AND OF sures in these patents do not refer to that fact. CENTRAL AND PERPHERAL NERVOUS It is also disclosed in these patents that, in addition to the SYSTEMIAGENTS ANDTRANSPORTATION coal tar Solution the composition may also contain an H1-an OF NUCLECACID SUBSTANCES tagonistantihistamine (e.g. diphenhydramine hydrochloride) and may in that form be used in the treatment of atopic and This application is a divisional of U.S. Ser. No. 10/345.204 allergic conditions manifesting in skin irritations such as filed Jan. 16, 2003 (which will issue as U.S. Pat. No. 8.329, eczema, dermatitis and ringworm. The latter of these condi 685 on Dec. 11, 2012), which is a continuation-in-part of PCT tions is caused by a fungal infection. Again the disclosures in International Applications PCT/ZAO1/00098, PCT/ZAO1/ 10 issue do not refer specifically to that fact. O0099 and PCT/ZAO1/00100, all filed on Jul.19, 2001 and all It further disclosed an alternative composition in which the designating the U.S., which claims the benefit of South Africa coal tar solution formulation is further provided with collagen Patent Application Nos. 2000/3643 and 2000/3644 filed on and lanolin and this formulation was found to be useful in the Jul.19, 2000, the disclosure of which are incorporated herein 15 treatment of persons Suffering from acne Vulgaris. Bacteria in their entirety by reference. are involved in the condition but no mention was made of such involvement in those patents. FIELD OF THE INVENTION Since coal tar solution is in itself not known to be an anti-viral, anti-fungal or antibacterial agent, and has merely This invention relates to pharmaceutical preparations been mentioned as being weakly antiseptic, the aforemen (which expression is herein intended to include veterinary tioned disclosures would not have been understood as Sug preparations) for use in combating infective organisms gesting that the nitrous oxide and fatty acid combination has afflicting the animal body (which expression is herein any beneficial effect on the anti-viral or anti-fungal or anti intended to include the human body). bacterial activity of any recognised anti-viral oranti-fungalor This invention also relates to pharmaceutical preparations 25 anti-bacterial agent or to have disclosed that such properties (which expression is also herein intended to include veteri are displayed by coal tar solution. As will appear below the nary preparations) for use in treating afflictions of the animal enhancement of the anti-bacterial, anti-fungal or anti-viral body affecting the central and/or peripheral nervous system properties of known agents lie at the very heart of this inven of an animal (which expression is again herein intended to tion. include the human body) in need of treatment. 30 Within the context of the disclosure in the abovementioned This invention further relates to preparations for use in patent family the notional addressee most likely would, as did transporting nucleic acid substances to and into cells of a the inventor, have understood the role of the coal tar solution plant or an animal body (which expression is again herein to sooth the itching and to assist in the repairing and healing intended to include the human body), either in vivo or invitro, of the skin which was damaged as a result of the infections/ or of a micro-organism. This invention is more particularly 35 conditions in issue. concerned with the administration of Substances based on No agent known for having an effect on the central or nucleic acid to patients for the purpose of the transportation peripheral nervous system, was amongst the active ingredi thereof through the body of the patient and the ultimate deliv ents specifically mentioned in these patents. Mention was erythereof into cells either generally or of a targeted cell type. made therein that coal tar Solution (also known as Liquor It therefore finds application in what is colloquially referred 40 Picis Carbonis) may be used as a Supplementary active ingre to as gene therapy as will become evident from the disclosure dient and that the resultant preparation is suitable for use in below. The invention is also concerned with the transfection the treatment, interalia, of psoriasis, eczemaceous conditions of plants and microorganisms with DNA thereby to affect the and dermatitis. It is also disclosed in these patents that, in genetic properties of Such plant or organism. addition to the coal tar Solution the composition may also 45 contain an H1-antagonist antihistamine (e.g. diphenhy BACKGROUND TO THE INVENTION dramine hydrochloride) and that such a preparation may ben eficially be used in the treatment of atopic and allergic con In EP 93912877.3 and U.S. Pat. No. 5,633,284 and their ditions manifesting in skin irritations such as eczema and equivalents the applicant disclosed that dermatological or dermatitis. The aforementioned conditions have since the topical compositions comprising the combination of nitrous 50 date of the above patents come to be regarded by some writers oxide NO and at least one fatty acid, or lower alkyl ester on the topic as manifestations in the skin of underlying dis thereof in a dermatologically acceptable carrier medium are orders which have or include a neurological, or immunologi useful in the treatment of a variety of skin, muscle and joint cal, or some even suggest a neuro-immunological cause. disorders. It also disclosed therein that such combinations These patents however do not refer to that explanation. It may beneficially also include additional active ingredients. 55 further gives no hint that the effects obtained are the result of The following active ingredients are specifically men any form of systemic operation of any of the components of tioned in this regard: coal tar Solution, collagen, nicotina the formulation. Coal tar liquid is not known to be an agent mide, nicotinic acid, lanolin, vitamin E, methyl salicylate, acting on the central nervous system. Insofar as these patents arnica and H1-antagonist antihistamines of which only may be seen to proffer any explanation at all for the results diphenhydramine chloride is specifically mentioned. 60 obtained, which is not what the applicant contends for, it is Known anti-bacterial, anti-viral or anti-fungal agents were Suggested that such explanation would, within the context of not amongst the active ingredients specifically mentioned in the disclosures therein, point to a local effect of Soothing the patents but mention was made therein that coal tar Solu irritation and of repairing of skin and associated tissue dam tion (also known as Liquor Picis Carbonis) may be used (as a age as a result of the conditions mentioned therein. These are Supplementary active ingredient and that the resultant prepa 65 psoriasis, shingles, fever blisters, chicken poX, acne, chil ration is suitable for use in the treatment of, inter alia fever blains, eczema, chloasmas, alopecia, dermatitis, ringworm blisters, herpes simplex, shingles and chickenpox. While all and burn wounds. US 8,937,074 B2 3 4 The concept of introducing heterologous genetic material While these applications do disclose a systemic effect in into the cells of an organism for the purpose of allowing Such that the formulations are, for example, topically applied or genetic material to be incorporated into the cellular DNA of orally administered to have an effect, say, in an affected joint Such organism is well known. It is now routinely exercised in or muscle, the specification again fails to disclose the mode or vitro and various methods have been developed for the intro 5 route of transport of the active ingredient from its site of duction of such heterologous genetic material into the cells of administration to its locus of effect, and speculation in this an organism. regard would probably be based on ex post facto hindsight. The most established of these methods is commonly It has now surprisingly been found that the aforesaid referred to as the gene gun technique. By this technique medium and media related thereto has the ability remarkably 10 to enhance the action of known anti-infective agents. The genetic material is quite literally shot into the cells of the expression “anti-infective agents' as used herein is intended organism. While some of the cells do not survive the blast, to have its extended meaning and to include the antimicrobial those that do survive are caused to proliferate. Some of these agents, the anthelmintic agents and the anti-ectoparasitic proliferating survivors will have incorporated into its own agents, but to exclude coal tar Solution and H1-antagonist genome the genetic material shot into it in the form of the 15 antihistamines. DNA. The gene so incorporated may eventually be expressed The exclusion of coal tar Solution and H1-antagonist anti to yield the product coded for by the DNA so introduced into histamines from the ambit of the present invention is intro the organism. It is also known to make use of other forms of duced without thereby conceding that the aforementioned vectors by which a gene which is required to be expressed is patents and applications contain any disclosure of any anti introduced into an organism. Such vectors include viruses. infective agent properties of Such excluded compounds, or While these techniques work adequately in the appropriate that such properties are obvious in the light of the disclosures in vitro environment on lower organisms such as bacteria, in Such patents or applications. Such inferences are specifi they are not regarded as being generally suitable for imple cally denied. The exclusion is introduced simply to avoid mentation in Vivo for the introduction of genetic material into what is anticipated to be a potential obstacle to the grant of a a living animal. Such as man. The harshness of the gene gun 25 patent in respect of an insignificant part of potential Subject technique by its very nature renders that technique generally matter which part in itself is not considered worth contesting unsuitable for in vivo application. It is further generally during examination as it might unduly delay the implemen regarded to be advisable not to expose animals or human tation in practice of the significant features of the present beings unnecessarily to viral infections, let alone where Such invention. It is expected that the remaining bulk of the subject infections are by transgenic viruses, the full genetic nature 30 matter of the present invention will greatly contribute to the and potential for mutation of which may be unknown. accessibility of medicines for the treatment of a large range of There has thus been a long-felt need for an appropriate infections, including secondary infections in HIV-compro process by which genetic material may be introduced into mised patients, at significantly reduced costs. selected cells there to express and yield a desired medicinally The expressions “anthelmintic agents' and “anti-ectopara active Substance. 35 sitic' agents are further intended to cover both agents which This need is sought to be addressed, inter alia by the dis serve to destroy and those which serve to inhibit the prolif closure contained in U.S. Pat. No. 6,258,789 (German, et al.) eration of helminths or ecto-parasites. Those expressions are and the patents cited during the examination thereof. German hence also intended to be understood in the wider sense of et al discloses a method of delivering a secreted protein into these terms. The expression “antimicrobial agents' is simi the bloodstream of a mammalian Subject by introducing into 40 larly intended to be understood in the wider sense of that word the gastro-intestinal tract of a mammalian Subject, by oral and hence to have the meaning ascribed thereto in The administration, a construct comprising a nucleic acid mol McGraw-Hill Dictionary of Scientific and Technical Terms ecule encoding the desired secreted protein and a promoter 2" Ed 1978, namely all chemical compounds that either sequence operably linked to the nucleic acid molecule, destroy or inhibit the growth of microscopic and Sub micro wherein said construct is not packaged in a viral particle, and 45 scopic organisms. This term is further specifically intended to wherein the method involves introducing the nucleic acid include all the compounds falling within the Pharmacological construct into the intestinal epithelial cells of the animal in Classification 20 set out as part of Regulation 5(1) of the question so that secretion of the protein takes place at that General Regulations made in terms of the South African locus to be available for absorption into the body from the Medicines and Related Substances Control Act, Act 101 of digestive tract. 50 1965, as well as the active ingredients of all products falling No agent based on nucleic acid was amongst the active within class 18 of the pharmacological classification ingredients specifically mentioned in the patents mentioned employed in the Monthly Index of Medical Specialities above. (“MIMS) published by Times Media in South Africa. It is In WO97/17978 and U.S. Pat. No. 6,221,377 and in corre thus intended to include: sponding patents and pending patent applications in other 55 the anti-bacterial agents (including both antibiotics and jurisdictions the present applicant disclosed that the action of Substances other than antibiotics Such as the Sulfonamides, analgesic, anti-inflammatory and anti-pyretic drugs may be the erythromycins and other macrollides, the aminoglyco enhanced by administering Such drugs in conjunction with a cides, the tetracyclines, the chloramphenicols and the quino medium which comprises nitrous oxide and at least one long lones); chain fatty acid selected from the group consisting of oleic 60 the anti-fungal agents; acid, linoleic acid, alpha-linolenic acid, gamma linolenic the anti-viral agents (including anti-retroviral agents); acid, arachidonic acid, and any of the C to C alkyl esters of the anti-protozoal agents; Such long chain fatty acids, mixtures of such acids and mix the tuberculostatics; tures of Such esters. The medium may comprise the mixture the anti-leprotics; known as Vitamin F Ethyl Ester and may optionally further 65 the germicides; comprise eicosapentaenoic acid C20: 5c)3 and decosa and hexaenoic acid C22: 6c)3. the spirochaeticides. US 8,937,074 B2 5 6 The Surprising finding of enhancement of action of the R200 for a new patient with ordinary TB. Such patients gen anti-infective agents referred to above is made against the erally also require to be hospitalised for long periods of time background of the fact that there appears to be no earlier (usually between six and eighteen months), adding signifi suggestion in the literature to the effect that either nitrous cantly to the cost of their treatment oxide or the long chain fatty acids used in the formulation 5 In an attempt to reduce discontinuance of TB-treatments referred to above, and hence also not the combination of which has been implicated in reinfections and the develop these, has any effect whatsoever on the sensitivity of any ment of resistant strains, the practice of directly observed micro-organism to any anti-infective agent. treatment or DOTS has been resorted to, with some, but based The present invention is specifically, though not exclu on the foregoing quotes, not complete success. sively aimed at the enhancement of the action of anti-myco 10 bacterial agents, and particularly those used in the treatment Iron, heavy metals, and excessive alcohol consumption (an of patients infected with Mycobacterium tuberculosis inherent feature of some identified high incidence TB com (M.Tb.). This organism is one of the most significant human munities) generate harmful reactive oxygen species which pathogens. It is responsible for an estimated seven million have been shown to be involved in the auto-oxidation of new cases of tuberculosis annually, and an estimated three 15 Rifampicin, an antibiotic anti-mycobacterial agent used in million deaths worldwide. Of particular concern is the emer the treatment of tuberculosis, thereby generating more radical gence of tuberculosis (TB) as an increasing cause of morbid species. These free radicals have been implicated in the liver ity and mortality among persons compromised by human toxicity experienced with use of Rifampicin. immune-deficiency virus (HIV) infection. These problems associated with TB have led to the inves Although the prevalence of tuberculosis in developed tigations associated with the present invention. countries declined in the first few decades of the 1900s, this It has also surprisingly been found that the aforesaid trend has reversed and an increased incidence of tuberculosis medium has the unexpected property that it displays a has been reported in many countries. Africa alone is estimated remarkable ability to enhance the action of known agents to have approximately 170 million TB patients. In South affecting the central and/or peripheral nervous system, Such Africa the incidence of tuberculosis is also rising and is at 25 known agents being other than the group which consists of different levels in different population groups. The chapter on coal tar Solution, H1 antagonist antihistamines, the analge Tuberculosis in the 1999 edition of South African Health sics, the anti-inflammatories and the antipyretics. These Review (available at http://www.hst.org.za/sahr?) opens with agents will hereinafter collectively be referred to as “CPNS the shocking statement that: “Despite the availability of effec agents' which term is for purposes of this specification tive and affordable treatment, the number of South Africans 30 intended to embrace those biologically active compounds dying from tuberculosis continues to increase'. It is echoed which perform their action on the central or peripheral ner by the summary of the startling overview in which it is vous system of the human or animal body, but to exclude recorded that “A reported incidence of 254 cases per 100 000 recognised anti-inflammatory, analgesic and antipyretic Sub for the period 1996-1998 combined with low cure rates, indi stances and also excluding coal tar Solution and H1 agonist cate that the epidemic is still out of control. Rising levels of 35 antihistamines. Subject to these exclusions it therefore HIV infection and multi-drug resistant TB (MDRTB) repre includes the compounds which fall within Class 1 of the sent additional threats to TB control efforts.” The number of Pharmacological Classification set out as part of Regulation reported cases of pulmonary TB (PTB) is reported in the 5(1) of the General Regulations made under The Medicines Review to have risen from 90628 to 110016 new reported and Related Substances Control Act, Act 101 of 1965, and the cases per year over the period 1996 to 1998, the estimated 40 active ingredients of all products falling within Class 1 of the report rate having increased over the same period from 64% to pharmacological classification currently employed in the 71%. Some of the provinces in South Africa contributed Monthly Index of Medical Specialities (“MIMS) published significantly to the national average TB incidence figure of by Times Media in South Africa, but excluding the analgesic, 254 referred to above. The quoted Review reflects the figure anti-inflammatory and anti-pyretic compounds which, falls for Eastern Cape as 388, for the Northern Cape as 360, the 45 within the above two partially overlapping classes. Examples Free State as 338 and the Western Cape as about 500 per of Some of the compounds falling within the group is 100000, almost double the national average. It has been described in greater detail below. reported elsewhere that the highest incidence of TB in the The exclusion of coal tar Solution and H1-antagonist anti country is found in certain communities in the Western Cape histamines, and of anti-inflammatories, analgesics and anti where the estimated incidence is as high as 1400 per 100000. 50 pyretics from the ambit of the present invention is introduced Re-infection of patients is an ever-increasing problem and without thereby conceding that the aforementioned patents has been shown to be a function of reactivation of TB in and applications contain any disclosure of any CPNS agent patients not completing their therapy. It is also often associ properties of Such excluded compounds, or that Such proper ated with the appearance of drug resistant M.Tb. in the ties are obvious in the light of the disclosures in Such patents patient. The exact mechanism whereby drug resistance devel 55 or applications. Such inferences are specifically denied. The ops in mycobacteria is not yet fully understood, but the eco exclusion is introduced simply to avoid what is anticipated to nomic consequences thereof are a reality. The occurrence of be a potential obstacle to the grant of a patent in respect of a drug resistant strains of M.Tb., generally known as multi part of the subject matter which part in itself is not considered drug resistant Tuberculosis (“MDRTB) is also referred to in worth contesting during examination as it might unduly delay the aforementioned South African Health Review 1999. It 60 the implementation in practice of the significant features of states: “Accurate figures for MDRTB are currently not avail the present invention. It is expected that the remaining bulk of able but surveys in three provinces (Western Cape, Mpuma the Subject matter of the present invention will greatly con langa and Gauteng) indicate a rate of 1% in new MDRTB tribute to the accessibility of medicines for the treatment of a cases, 4% in retreatment cases. This translates to at least 2000 large range of ailments. newly active cases of MDRTB in South Africa each year. 65 It has now further surprisingly been found that the afore MDRTB is extremely expensive to treat R25 000 to R30 said medium and other similar media have the unexpected 000 per patient for the drugs alone as opposed to less than property that it may also be used as a delivery vehicle adapted US 8,937,074 B2 7 8 to be used for the delivery, into the nucleus of, or generally to, vide pharmaceutical preparations of Such agents which an animal cell, of nucleic acid compounds. preparations have enhanced action compared to the action of It was pointed out in WO97/17978 referred to above that known formulations containing the same agents. nitrous oxide is a natural gas which is also produced syntheti It is a further object of the present invention to provide a cally, and also known by the trivial name "laughing gas” method and formulation for the transportation and delivery of which has been in use for many years as an inhalation anaes nucleic acid Substances into cells of an animal, a plant or a thetic and analgesic, particularly in dentistry. micro-organism. The term “animal' is herein intended to be Its was further stated that nitrous oxide has been reported to interpreted in its wide meaning to include manand insect. The have a synergistic or potentiating effect on halothane and term “micro-organism' is herein intended to include single other gaseous anaesthetics See Goodman & Gilman's The 10 and multi-cellular organisms such as parasites. The invention Pharmacological Basis of Therapeutics 8th Ed. 1990 pp. 298 is thus concerned with all forms of life in which nucleic acid 300. Since Such known synergism or potentiation is based on the substances in the form of DNA or RNA determine the genetic use of nitrous oxide administered by inhalation, and since the properties of that form of life. use of nitrous oxide on its own as an anaesthetic and analgesic 15 These objects stem from the observations made by present has likewise been in the form of an inhalation agent, the use of applicant in respect of a selection of agents falling within the nitrous oxide for all these purposes have been confined to group of active agents as herein defined, which can advanta hospitalised patients or, at best, to treatments carried out by geously be formulated with nitrous oxide and an oil based on medical practitioners in their consulting rooms, or treatments long chain fatty acids disclosed herein, to elicit a more potent carried out by or under Supervision of a nurse in charge of a response, for each agent according to its own inherent prop home-care patient. erties, or to evoke Such response more rapidly than it does Nitrous oxide is known to be soluble in water and it has when used by conventional administration of the agent in been reported that at 20°C. and 2 atm pressure one liter of the 1SSC. gas dissolves in 1.5 liters of water, see The Merck Index 10th Ed. p. 6499. 25 BRIEF DESCRIPTION OF DRAWINGS Nitrous oxide is also known for its use as a propellant gas, mainly as a Substitute for propellant gases such as chlorof FIGS. 1-8 describes the aspects of the invention. luorocarbons, and more particularly to produce a food prod uct mousse Such as whipped cream or chocolate mousse or STATEMENTS OF THE INVENTION quick-breaking foams for hair treatment preparations. See in 30 this regard U.K. Patent 1033299, U.K. Patent 1105919 and (i) Anti-Infectives European Patent Application EPA-0123827. None of these According to the present invention there is provided a prior publications suggests that the nitrous oxide gas plays method of enhancing the action of an anti-infective agent any other role than a physical one, i.e. to expand on being characterised in that the agent is selected from the group depressurised and thereby to create a mousse or foam. In fact 35 comprising antimicrobial agents, the anthelmintic agents and it is typically regarded as an inert in these applications and the anti-ectoparasitic agents, but excluding coal tar Solution useful due to the fact that it is colourless, odourless and and H1-antagonistantihistamines, comprising the step of for tasteless but soluble in water and oils. mulating the agent with an administration medium which There appears to be no suggestion in the literature, other comprises a solution of nitrous oxide gas in a pharmaceuti than the applicants own prior patents and patent applications 40 referred to above, that aqueous Solutions of nitrous oxide cally acceptable carrier solvent for the gas and which admin might have any effect on man or animals. As far as the present istration medium includes at least one fatty acid or ester or applicant knows, it has also never been suggested that nitrous other suitable derivative thereof selected from the group con oxide may be used in conjunction with any anti-infective sisting of oleic acid, linoleic acid, alpha-linolenic acid, agent to enhance the known action of Such agent. 45 gamma-linolenic acid, arachidonic acid, eicosapentaenoic It is known in the pharmaceutical field to formulate active acid C20: 5c)3], decosahexaenoic acid C22: 6 (03, ricino ingredients in so-called liposomal formulations. Unlike the leic acid and derivatives thereof selected from the group con present invention which is based on formulations containing sisting of the C1 to C6 alkyl esters thereof, the glycerol long chain fatty acids and esters thereof the liposomes are polyethylene glycol esters thereof and the reaction product of based on a clearly distinguishable group of compounds 50 hydrogenated natural oils composed largely of ricinoleic acid namely the phospholipids, and generally also contain choles based oils, such as castor oil with ethylene oxide. terol as a stabilising agent and may further contain liso According to a further aspect of the present invention there lecitein. These compounds or classes form no part of the is provided a pharmaceutical preparation comprising an anti present invention and, in case it is necessary to do so, are infective agent characterised in that it is selected from the specifically excluded from the group of long chain fatty acids 55 group comprising antimicrobial agents, the anthelmintic and derivatives thereof incorporated in the method or formu agents and the anti-ectoparasitic agents, but excluding coal lation of the invention. tar Solution and H1-antagonistantihistamines, which agent is formulated with an administration medium which comprises OBJECTS OF THE INVENTION a solution of nitrous oxide in a pharmaceutically acceptable 60 carrier Solvent for the gas and which includes at least one fatty It is an object of the present invention to provide a method acidorester or other suitable derivative thereof selected from of enhancing the known action of anti-infective agents and to the group consisting of oleic acid, linoleic acid, alpha-lino provide pharmaceutical preparations of Such anti-infective lenic acid, gamma-linolenic acid, arachidonic acid, eicosap agents which preparations have enhanced action compared to entaenoic acid C20: 5c)3], decosahexaenoic acid C22: the action of known formulations containing the same agents. 65 6c)3, ricinoleic acid and the derivatives thereof selected from It is another object of the present invention to provide a the group consisting of the C1 to C6 alkyl esters thereof, the method of enhancing the action of CPNS agents and to pro glycerol-polyethylene glycol esters thereof and the reaction US 8,937,074 B2 10 product of hydrogenated natural oils composed largely of The essential fatty acid, or ester thereof, component of the ricinoleic acid based oils, such as castor oil, with ethylene composition preferably comprises a mixture of esters of the oxide. fatty acids listed above. Thus, in the most preferred form of The administration medium preferably includes the eicosa the invention the fatty acid component of the composition is pentaenoic acid C20: 5c)3 and/or decosahexaenoic acid constituted by the complex known as Vitamin F and in this C22: 6003 as additional long chain fatty acids to at least one regard it is preferred to make use of the ester form of Vitamin of the other components of the carrier medium defined above. F known as Vitamin F Ethyl Ester. This product is commer The reaction product of hydrogenated natural oils com cially available under the trade description of Vitamin FEthyl posed largely of ricinoleic acid based oils with ethylene oxide Ester CLR 110 000 Sh.L. U./g from CLR Chemicals Labo is preferably produced from castor oil of which the fatty acid 10 content is known to be predominantly composed of ricinoleic ratorium Dr. Kurt Richter GmbH of Berlin, Germany. The acid. This product is known as PEG-n-Hydrogenated Castor typical fatty acid distribution of this product is as follows: Oil. A range of such products is marketed by BASF under the trade description of Cremophor RH grades. Glycerol-poly ethylene glycol ester of ricinoleic acid is also marketed by the 15 same company but under the trade description of Cremophor EL. The carrier solvent for the nitrous oxide gas may be water or any of the pharmaceutically acceptable alcohols, ethers, oils or polymers such as a polyethylene glycolor the like. The oil may be organic or mineral oil. The organic oil may be an essential oil based on long chain fatty acids having between chain fatty acids known as eicosapentaenoic acid C20:5c)3 14 and 22 carbon atoms in the fatty acid. The oil may also be and decosahexaenoic acid C22:6c)3. Such a product com of either natural or synthetic origin and, if of natural origin, it bination is available from Roche Lipid Technology under the may be either plant oil or animal oil. As plant oils those rich 25 trade name “Ropufa 30 n-3 oil”. in gamma linolenic acid GLA are preferred and as animal It has been found by microscopic studies that the formula oil dairy cream may be used. tion of the anti-infective agents with a medium as herein In the preferred form of the invention the solution is an described gives rise to the formation of minute, generally aqueous solution saturated with nitrous oxide. Preferably the spherical bodies, within which, or attached to which the water is deionised and purified to be free of microbes. 30 active ingredient is contained in a stable form and from which When the formulation containing the anti-infective agent it is delivered at the site of action namely on or inside the to be enhanced by means of the nitrous oxide is to be in a infective agent. liquid (including an encapsulated liquid) presentation for oral The anti-infective agent utilised in the method or formula administration or in a nasal or bronchial or pulmonary spray tion according to the present invention may comprise any one or in the form of an injectable formulation, such formulation 35 or more of the vast spectrum of anti-infective agents as herein may incorporate, as part of the administration medium, water defined. or acceptable other liquid into which the nitrous oxide is In a preferred form of the invention the anti-infective agent dissolved and in which the fatty acid or ester thereof is either is selected from the group comprising: dissolved or Suspended or emulsified along with the anti the anthelmintics infective agent to be enhanced by being formulated therewith. 40 the anti-ectoparasiticides Likewise, where the anti-infective agent is to be adminis the anti-bacterial agents (including both antibiotics and tered to the patient as a topical, buccal or vaginal cream or Substances other than antibiotics); ointment, or as a Suppository, the formulation used in making the antifungal agents; up Such cream, ointment, or Suppository may incorporate, the anti-viral agents; along with the anti-infective agent to be enhanced, a quantity 45 the anti-protozoal agents; of water or other liquid containing, and preferably Saturated the tuberculostatics; with, nitrous oxide, the long chain fatty acid or ester thereof the anti-leprotics; and the anti-infective agent formulated therewith, and, fur the germicides; ther, Such additional excipients and carriers as are conven and tionally used in the pharmaceutical trade in making up Such 50 the spirochaeticides. dosage forms. From amongst these anti-infective agents this invention is The carrier Solvent for the nitrous oxide gas may thus in an particularly concerned with the anti-bacterials and the tuber alternative formulation according to the invention be essen culostatics. These classes of agents overlap to some extent. tially non-aqueous and composed of least one fatty acid or The anti-infective agent may in a specific application of the ester thereof selected from the group consisting of oleic acid, 55 invention comprise an antimicrobial of the class of com linoleic acid, alpha-linolenic acid, gamma-linolenic acid, pounds known as the tuberculostatics or anti-mycobacterial arachidonic acid, eicosapentaenoic acid C20: 5c)3, decosa compounds and may specifically be selected from the group hexaenoic acid C22: 603, ricinoleic acid and derivatives consisting of Rifampicin, Isoniazid, Pyrazinamide, Ethambu thereof selected from the group consisting of the C1 to C6 tol and combinations of any two or more of these. alkyl esters thereof, the glycerol-polyethylene glycol esters 60 It is a further aspect of the invention that the formulation, thereof and the reaction product of hydrogenated natural oils and specifically, though not exclusively, the anti-TB formu composed largely of ricinoleic acid based oils with ethylene lation of the invention, may be prepared to be adapted for oxide, required to be part of the formulation. pulmonary administration. In the case of the anti-TB formu A formulation Suited to transdermal application whether as lation it will thereby bring the formulation into contact with an ointment, cream or lotion or in the form of a skin patch 65 the pathogen at a primary locus thereof and without passage providing a reservoir for the formulation is also a preferred through, or absorption from the digestive tract and possible form of the formulation according to the invention. Subsequent passage through the liver. US 8,937,074 B2 11 12 The invention has not yet been demonstrated by empirical SULPHASALAZINE work to be applicable to all the agents listed below. However 1) Inflammatory bowel diseases and in rheumatoid in respect of Such anti-infective agents which have already arthritis been formulated with the aforementioned administration SUCCNYLSULPHATHIAZOLE medium of the invention, and evaluated by different methods 1) Gastrointestinal infections for the anticipated enhancement of anti-infective action, no PHTHALYLSULPHATHIAZOLE negative result has as yet been seen despite the chemical 1) Gastrointestinal infections diversity of the anti-infective agents which has been investi 7) SULPHONAMIDE COMBINATIONS: gated. The applicant thus confidently expects, on the basis of CO-TRIMOXAZOLE (Trimethoprim+Sulphamethox the observations in respect of products representing a range of 10 azole)— 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, classes of Such agents, that the invention will find general Osteomyelitis, Cellulitis and other diseases that are application across the entire spectrum of anti-infective agents Methicillin-sensitive (Staphylococcus aureus) embraced by the term as herein defined and of which some 2) Pneumonia, Arthritis, Sinusitis, Otitis that are Peni examples are set out below. It is part of the applicant's present 15 cillin-sensitive (Streptococcus Pneumoniae) postulations by which it seeks to find an understanding of the 3) Meningitis and Bacteremia (Listeria monocytoge invention and to which it does not wish to be bound at this nes) stage, that while the administration medium of the present 4) Urinary tract infections, Bacteremia, Other infec invention serves to transport the anti-infective agent formu tions (Escherichia coli) lated therewith most efficiently through the human or animal 5) Urinary tract and other infections (Enterobacter body, that medium also plays an important role in transfer species) ring, by an as yet unexplained mechanism, the anti-infective 6) Typhoid fever, Paratyphoid fever, Bacteremia, agent through the outer membranes of and into the pathogenic Acute gastroenteritis (Salmonella) organism thereby to cause an effective anti-infective dose of 7) Acute gastro-enteritis (Shigella) the agent rapidly to be achieved and to be maintained in the 25 8) Otitis media, Sinusitis, Pneumonia, Epiglottitis, organism until it finally succumbs to the effect of the anti Meningitis (Haemophilus influenzae) infective agent. 9) Chancroid (Haemophilus ducreyi) It is in this respect that the applicant believes that the 10) Brucellosis (Brucella)+Gentamicin present invention will find general application despite the vast 11)Yersiniosis (Yersinia enterocolitica) 30 12) Cholera (Vibrio cholerae) list of agents mentioned below. The following table sets out 13) Meningitis (Flavobacterium meningosepticum) examples of the specific anti-infective agents with which this 14) Melioidosis (Pseudomonas pseudomalilei) invention is concerned will now be identified with reference 15) Granuloma inguinale (Calymmatobacterium to the broad classes in which they fall and, in some cases, also granulomatis) with reference the respective indications for which such 35 16) Legionnaires disease (Legionella Pneumophila) agents are indicated and, in some cases, further also with 17) Pulmonary lesions, Brain abscess, Lesions of reference to the infective agent giving rise to the indication to other organs (Nocardia asteroides) be addressed which products comprise the following: 18) Lymphogranuloma Venereum, Trachoma, Inclu A SULPHONAMIDES sion conjunctivitis blennotthea. Non-specific ure 1) Short-acting 40 thritis, Cervicitis (Chlamydia trachomatis) SULPHAPYRIDINE 19). Pneumonia in impaired host Mild or moderate 1) Inflammatory bowel diseases and in rheumatoid disease, Moderately severe or severe disease arthritis (Pneumocystis carinii) SULPHADIAZINE B. QUINOLONES 1) Nocardiosis (Nocardia species) 45 B1 FIRST GENERATION 2) Toxoplasmosis--Pyrimethamine NALIDIXIC ACID (oxolinic acid) 3) Long term prophylaxis of rheumatic fever 1) Urinary tract infections SULPHADIMIDINE CINOXACIN SULPHAFURAZOLE 1) Urinary tract infections 2) Medium-acting: 50 PIPEMIDIC ACID SULPHAMETHOXAZOLE PIROMIDIC ACID 3) Long-acting: ACROSOXACIN SULPHADIMETHOXINE QUINOLONES SULPHAMETHOXYDIAZINE B2 SECOND GENERATION SULPHAMETHOXYPYRIDAZINE 55 CIPROFLOXACIN 4) Ultra-Long-acting: 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, SULFADOXINE Osteomyelitis, Cellulitis and Other infections Me SULFAMETOPYRAZINE thicillin-sensitive and Methicillin-resistant (Sta 5) Topical sulphonamide: phylococcus aureus)+Rifampin SILVER SULPHADIAZINE 60 2) Urinary tract infection (Enterococcus) 1) Antibacterial in patients with burns 3) Otitis, Sinusitis, Pneumonia (Moraxella catarrha MAFENIDEACETATE lis) 1) Antibacterial in patients with burns 4) Penicillin-sensitive and Penicillinase-producing SULPHACETAMIDE gonococcuS (Neisseria gonorrhoeae (gonococ Other sulphonamides: 65 cus)) SULPHAGUANIDINE 5) Carrier state (Post-treatment) (Neisseria meningiti 1) Gastrointestinal infections dis (meningococcus)) US 8,937,074 B2 13 14 6) Urinary tract infection and other infections, Bacte NITROFURANTOIN remia (Escherichia coli) 1) Urinary tract infection (Escherichia coli) 7) Urinary tract and other infections (Proteus mirabi MENAZOPYRIDINE lis) 1) Is notaurinary antiseptic but it does have an analgesic 8) Urinary tract and other infections (Enterobacter action on the urinary tract and alleviates symptoms of species) dysuria, frequency, burning and urgency. 9) Urinary tract infection, Pneumonia, Bacteremia D. PENCILLIN (Pseudomonas aeruginosa) NARROW SPECTRUM 10) Urinary tract infection (Klebsiella pneumoniae) BENZYLPENICILLIN (Penicillin G) acid-labile 11) Typhoid fever, Paratyphoid fever, Bacteremia, 10 1) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, Acute gastroenteritis (Salmonella) Cellulitis, Erysipelas, Pneumonia, Bacteremia, 9. Toxic shock-like syndrome and Other systemic 12) Acute gastroenteritisY (Shigella) infections (Streptococcus pyogenes Group A) 13) Otitis media, Sinusitis, Pneumonia (Haemophilus 2) Endocarditis, Bacteremia (Streptococcus (viridans influenze) 15 group)+Gentamicin. 14) Chancrois (Haemophilus ducreyi) Bacteremia, Endocarditis, Meningitis. (Streptococ 15) Plague (Yersinia pestis) cus agalactiae Group B)+tAminoglycoside. 16)Yersomopsis Sepsis (Yersinia enterocolitica) 4) Bacteremia, Endocarditis, Brain and other 17) Tualermia (Francisella tularensis) abscesses, Sinusitis (Streptococcus (anaerobic 18) Cholera (Vibrio tularensis) 2O species) 19) Enteritis (Campulobacter jejuni) 5) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin 20) Bacteremia, Endocarditis (Campylbacter fetus) Sensitive and Penicillin-Resistant Endocarditis, 21) Legionnaires disease (Legionella pneumophila) Meningitis, other serious infections Penicillin 22) Disseminated disease in AIDS (Mycobacterium Sensitive (Streptococcus pneumoniae pneumo avium-intracellulare)+Clarithromycin; ththambu- 25 coccus) tol; +Clofamizine 6) Endocarditis or other serious infections (bacter OFLOXACIN emia), Urinary tract infections (Enterococcus)+ 1) Penicillin-Sensitive and Penicillinase-Producing Gentamicin gonococcuS (Neisseria gonorrhoeae (gonococ- 7) Penicillin-sensitive gonococcuS (Neisseria gonor cus)) 30 rhoeae)+Probenecid. 2) Urinary tract infection and other infections, Bacte- 8) Meningitis (Neisseria meningitidis meningococ remia (Escherichia coli) cus) 3) Urinary tract and Other infections (Proteus mira- 9) “Malignant pustule'. Pneumonia (Bacillus anthra bilis) cis) 4) Urinary tract and Other infections (Enterobacter 35 10) Endocarditis, Infected foreign bodies, Bacteremia species) (Corynebacterium species, aerobic and anaerobic 5) Urinary tract infection (Klebsiella pneumoniae) diphtheroids)-tan Aminoglycoside or +Rifampin 6) Typhoid fever, Paratyphoid fever, Bacteremia, 11) Meningitis, Bacteremia (Listeria monocytogenes) Acute gastroenteritis (Salmonella) tGentamicin 7) Urinary tract infection (Pseudomonas aeruginosa) 40 12) Erysipeloid (Erysipelothrix rhusiopathiae) 8)Yersiniosis, Sepsis (Yersinia enterocolitica) 13) Gas gangrene (Clostridium perfiringens and other 9) Cholera (Vibrio cholerae) species) 10) Enteritis (Campylobacter jejuni) 14) Tetanus (Clostridium tetani) 11) Bacteremia, Endocarditis (Campylobacter fetus) 15) Urinary tract infection, Bacteremia, Other infec 12) Leprosy (Mycobacterium leprae) 45 tions (Escherichia coli)+a Penicillinase inhibitor. NORFLOXACIN 16) Urinary tract and other infections (Proteus, other 1) Acute gastroenteritis (Salmonella) species)+a B-Lactamase inhibitor 2) Acute gastroenteritis (Shigella) 17) Wound infection (animal bites), Abscesses, Bac ENOXACIN teremia, Meningitis (Pasteurella multocida) LOMEFLOXACIN 50 18) Ulcerative pharyngitis, Lung abscess, Empyema, PEFLOXACIN Genital infections, Gingivitis (Fusobacterium AMIFLOXACIN nucleatum) FLEROXACIN 19) Bacteremia, Arthritis, Endocarditis, Abscesses LEVOFLOXACIN (Streptobacillus moniliformis) NADIFLOXACIN 55 20) Syphilis (Treponema pallidum) RUFLOXACIN 21)Yaws (Treponema pertenue) SPARFLOXACIN 22) Stage 2-neurological, Cardiac, Arthritis (Borrelia 1) Active against Streptococcus pneumoniae and burgdorferi Lyme disease) anaerobic bacteria. 23) Relapsing fever (Borrelia recurrentis) TOSUFLOXACIN 60 24) Weil's disease, Meningitis (Leptospira) ENROFLOXACIN 25) Cervicofacial, Abdominal, Thoracic, and other C. Anti-Septic and Analgesic Agents for Urinary Tract lesions (Actinomyces israelii) Infections PHENOXYMETHYL-PENICILLIN METHENAMIN (Penicillin V) 1)Not a primary drug for the treatment of acute urinary 65 acid-stable tract infections, but it is of value for chronic Suppres 1) Pharyngitis, Scarlet Fever, Otitis Media, Sinusitis, sive treatment. Cellulitis, Erysipelas, Pneumonia, Bacteremia, US 8,937,074 B2 15 16 Toxic shock-like syndrome, and other systemic 2) Pneumonia, Bacteremia (Pseudomonas aerugi infections (Streptococcus pyogenes Group A) nosa)-tan Aminoglycoside 2) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin 3) Urinary tract infection (Pseudomonas aeruginosa) Sensitive and Penicillin-Resistant Endocarditis, 4) Variety of nosocomial and opportunistic infections Meningitis, other serious infections Penicillin (Serratia)+an Aminoglycoside sensitive (Streptococcus pneumoniae pneumo PIPERACILLIN coccus) 1) Urinary tract and other infections (Enterobacter 3) Urinary tract infections (Enterococcus) species) 4) Urinary tract infection, Bacteremia, Other infec 2) Pneumonia, Bacteremia (Pseudomonas aerugi tions (Escherichia coli)+a Penicillinase-inhibitor. 10 nosa)-tan Aminoglycoside 5) Urinary tract and other infections (Proteus, other 3) Urinary tract infection (Pseudomonas aeruginosa) species)+a B-Lactamase inhibitor 4) Variety of nosocomial and opportunistic infections E. PENCILLIN (Serratia)+an Aminoglycoside BROAD SPECTRUM 15 MEZLOCILLIN AMOXICILLIN 1) Urinary tract and other infections (Enterobacter 1) Pharyngitis, Scarlet Fever, Otitis Media, Sinusitis, species) Cellulitis, Erysipelas, Pneumonia, Bacteremia, 2) Pneumonia, Bacteremia (Pseudomonas aerugi Toxic shock-like syndrome and other systemic nosa)-tan Aminoglycoside infections (Streptococcus pyogenes group A) 3) Urinary tract infection (Pseudomonas aeruginosa) 2) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin 4) Variety of nosocomial and opportunistic infections Sensitive and Penicillin-Resistant (Streptococcus (Serratia)+an Aminoglycoside pneumoniae Pneumococcus) AZLOCILLIN 3) Urinary tract and other infections (Proteus mirabi 1) Urinary tract and Other infections (Enterobacter lis 25 species) 4) Gis Media, Sinusitis, Pneumonia (Haemophilus 2) Pneumonia, Bacteremia (Pseudomonas aerugi influenzae)+Clavulanic acid. nosa)-tan Aminoglycoside 5) Wound-infection (animal bites), Abscesses, Bacte 3) Urinary tract infection (Pseudomonas aeruginosa) remia, Meningitis (Pasteurella multocida)+Clavu 4) Variety of nosocomial and opportunistic infections lanic acid 30 (Serratia)+an Amihoglycoside 6) Erythema chronica migrans-skin (Borrelia burg BACAMPICILLIN dorferi Lyme disease) TALAMPICILLIN 7) Pulmonary lesions, Brain abscess, Lesions of other PIVAMPICILLIN organs (Nocardia asteroides)+Clavulanic acid CARBENICILLIN 8) Otitis, Sinusitis, Pneumonia (Moraxella catarrha 35 lis)+Clavulanic acid APALCILLIN 9) Penicillin-Sensitive gonococcuS (Neisseria gonor CARINDACILLIN rhoeae)+Probenecid PIVMECILLINAM AMPICILLIN CARFECILLIN 1) Bacteremia, Endocarditis, Meningitis (Streptococ 40 METAAMPICILLIN cus agalactiae Group B) HETACILLIN 2) Urinary tract infection, Endocarditis, or Other seri TEMOCILLIN ous infections Bacteremia (Enterococcus) F. PENCILLIN 3) Otitis, Sinusitis, Pneumonia (Moraxella catarrha PENICILLINASE-RESISTANT PENICILLINS (isox lis)+Clavulanic acid 45 aZoly penicillins) 4) Penicillin-Sensitive gonococcuS (Neisseria gonor OXACILLIN rhoeae)+Probenecid 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 5) Meningitis, Bacteremia (Listeria monocytogenes) Osteomyelitis, Cellulitis, and Other Staphylococ 6) Urinary tract infection, Other infections, Bacter cus aureus infections methicillin-sensitive (Sta emia (Escherichia coli)+an Amihoglycoside 50 phylococcus aureus) 7) Endocarditis, Infected foreign bodies, Bacteremia CLOXACILLIN (Corynebacterium species, aerobic and anaerobic 1) Effective against Penicillinase-Producing Staphy diphtheroids)+Sulbactam lococcus aureus 8) Urinary tract and other infections (Proteus mirabi DICLOXACILLIN lis) 55 1) Effective against Penicillinase-Producing Staphy 9) Typhoid Fever, Paratyphoid Fever, Bacteremia, lococcus aureus Acute Gastroenteritis (Salmonella) FLUCLOXACILLIN 10) Acute Gastroenteritis (Shigella) 1) Effective against Penicillinase-Producing Staphy 11) Epiglottitis, Meningitis (Haemophilus influ lococcus aureus enza)+Sulbactam 60 METHICILLIN 12) Bacteremia, Endocarditis, Meningitis (Campylo 1) Effective against Penicillinase-Producing Staphy bacter fetus) lococcus aureus 13) Cervicofacial, Abdominal, Thoracic, an Other NAFCILLIN lesions (Actinomyces israelii) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, TICARCILLIN 65 Osteomyelitis, Cellulitis, and Other Staphylococ 1) Urinary tract and other infections (Enterobacter cus aureus infections methicillin-sensitive (Sta species) phylococcus aureus) US 8,937,074 B2 17 18 G. CEPHALOSPORINS 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, G1. FIRST GENERATION Cellulitis, Erysipelas, Pneumonia, Bacteremia, CEPHAZOLINACEPHRADINE Toxic shock-like syndrome and other systemic 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, infections (Streptococcus pyogenes Group A) Osteomyelitis, Cellulitis and other Staphylococcus aureus infections Methicillin-Sensitive (Staphy 3) Bacteremia, Endocarditis (Streptococcus agalac lococcus aureus) tiae Group B) 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, 4) Bacteremia, Endocarditis, Brain and other CellulitiS, Erysipelas, Pneumonia, Bacteremia, abscesses, Sinusitis (Streptococcus anaerobic spe Toxic shock-like syndrome and other systemic cies) infections (Streptococcus pyogenes Group A) 10 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni 3) Bacteremia, Endocarditis (Streptococcus agalactiae cillin-Sensitive (Streptococcus Pneumoniae Group B) Pneumococcus) 4) Bacteremia, Endocarditis, Brain and other 6) Urinary tract infection, other infections, Bacter abscesses, Sinusitis (Streptococcus anaerobic spe cies) 15 emia (Escherichia coli) 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni 7) Urinary tract and other infections (Proteus mirabi cillin-Sensitive (Streptococcus Pneumoniae lis) Pneumococcus) 8) Urinary tract infection (Klebsiella pneumoniae) 6) Urinary tract infection, other infections, Bacter 9) Pneumonia (Klebsiella pneumoniae)-tan Ami emia (Escherichia coli) noglycoside 7) Urinary tract and other infections (Proteus mirabi 10) Wound infection (animal bite), Abscesses, Bacte lis) remia, Meningitis (Pasteurella Multiocida) 8) Urinary tract infection (Klebsiella pneumoniae) 11) Ulcerative pharyngitis, Lung abscess, Empyema, 9) Pneumonia (Klebsiella pneumoniae):tan Ami Genital infections, Gingivitis (Fusobacterium noglycoside 25 nucleatum) 10) Wound infection (animal bite), Abscesses, Bacte 12) “Malignant Pustule' remia, Meningitis (Pasteurella Multiocida) Pneumonia (Bacillus 11) Ulcerative pharyngitis, Lung abscess, Empyema, anthracis) Genital infections, Gingivitis (Fusobacterium CEFROXADINE nucleatum) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 12) “Malignant Pustule' Pneumonia (Bacillus 30 Osteomyelitis, Cellulitis and other Staphylococcus anthracis) aureus infections Methicillin-Sensitive (Staphy CEPHALORIDINE lococcus aureus) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, Osteomyelitis, Cellulitis and other Staphylococcus Cellulitis, Erysipelas, Pneumonia, Bacteremia, aureus infections Methicillin-Sensitive (Staphy 35 Toxic shock-like syndrome and other systemic lococcus aureus) infections (Streptococcus pyogenes Group A) 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, 3) Bacteremia, Endocarditis (Streptococcus agalac Cellulitis, Erysipelas, Pneumonia, Bacteremia, tiae Group B) Toxic shock-like syndrome and other systemic 4) Bacteremia, Endocarditis, Brain and other infections (Streptococcus pyogenes Group A) 40 abscesses, Sinusitis (Streptococcus anaerobic spe 3) Bacteremia, Endocarditis (Streptococcus agalac cies) tiae Group B) 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni 4) Bacteremia, Endocarditis, Brain and other cillin-Sensitive (Streptococcus Pneumoniae abscesses, Sinusitis (Streptococcus anaerobic spe Pneumococcus) cies) 45 6) Urinary tract infection, other infections, Bacter 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni emia (Escherichia coli) cillin-Sensitive (Streptococcus Pneumoniae 7) Urinary tract and other infections (Proteus mirabi Pneumococcus) lis) 6) Urinary tract infection, other infections, Bacter 8) Urinary tract infection (Klebsiella pneumoniae) emia (Escherichia coli) 50 9) Pneumonia (Klebsiella pneumoniae)-tan Ami 7) Urinary tract and other infections (Proteus mirabi noglycoside lis) 10) Wound infection (animal bite), Abscesses, Bacte 8) Urinary tract infection (Klebsiella pneumoniae) remia, Meningitis (Pasteurella Multiocida) 9) Pneumonia (Klebsiella pneumoniae):tan Ami 11) Ulcerative pharyngitis, Lung abscess, Empyema, noglycoside 55 Genital infections, Gingivitis (Fusobacterium 10) Wound infection (animal bite), Abscesses, Bacte nucleatum) remia, Meningitis (Pasteurella Multiocida) 12) “Malignant Pustule' Pneumonia (Bacillus 11) Ulcerative pharyngitis, Lung abscess, Empyema, anthracis) Genital infections, Gingivitis (Fusobacterium CEFADROXIL nucleatum) 60 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 12) “Malignant Pustule' Pneumonia (Bacillus Osteomyelitis, Cellulitis and other Staphylococcus anthracis) aureus infections Methicillin-Sensitive (Staphy CEPHRADINE lococcus aureus) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, Osteomyelitis, Cellulitis and other Staphylococcus 65 Cellulitis, Erysipelas, Pneumonia, Bacteremia, aureus infections Methicillin-Sensitive (Staphy Toxic shock-like syndrome and other systemic lococcus aureus) infections (Streptococcus pyogenes Group A) US 8,937,074 B2 19 20 3) Bacteremia, Endocarditis (Streptococcus agalac 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni tiae Group B) cillin-Sensitive (Streptococcus Pneumoniae 4) Bacteremia, Endocarditis, Brain and other Pneumococcus) abscesses, Sinusitis (Streptococcus anaerobic spe 6) Urinary tract infection, other infections, Bacter cies) emia (Escherichia coli) 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni 7) Urinary tract and other infections (Proteus mirabi cillin-Sensitive (Streptococcus Pneumoniae lis) Pneumococcus) 8) Urinary tract infection (Klebsiella pneumoniae) 6) Urinary tract infection, other infections, Bacter 9) Pneumonia (Klebsiella pneumoniae)-tan Ami emia (Escherichia coli) 10 noglycoside 7) Urinary tract and other infections (Proteus mirabi lis) 10) Wound infection (animal bite), Abscesses, Bacte 8) Urinary tract infection (Klebsiella pneumoniae) remia, Meningitis (Pasteurella Multiocida) 9) Pneumonia (Klebsiella pneumoniae):tan Ami 11) Ulcerative pharyngitis, Lung abscess, Empyema, noglycoside 15 Genital infections, Gingivitis (Fusobacterium 10) Wound infection (animal bite), Abscesses, Bacte nucleatum) remia, Meningitis (Pasteurella Multiocida) 12) “Malignant Pustule' Pneumonia (Bacillus 11) Ulcerative pharyngitis, Lung abscess, Empyema, anthracis) Genital infections, Gingivitis (Fusobacterium PIVCEPHALEXIN nucleatum) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 12) “Malignant Pustule' Pneumonia (Bacillus Osteomyelitis, Cellulitis and other Staphylococcus anthracis) aureus infections Methicillin-Sensitive (Staphy CETATRIAZINE lococcus aureus) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, Osteomyelitis, Cellulitis and other Staphylococcus 25 Cellulitis, Erysipelas, Pneumonia, Bacteremia, aureus infections Methicillin-Sensitive (Staphy Toxic shock-like syndrome and other systemic lococcus aureus) infections 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, (Streptococcus pyogenes Group A) Cellulitis, Erysipelas, Pneumonia, Bacteremia, 3) Bacteremia, Endocarditis (Streptococcus agalac Toxic shock-like syndrome and other systemic 30 tiae Group B) infections (Streptococcus pyogenes Group A) 4) Bacteremia, Endocarditis, Brain and other 3) Bacteremia, Endocarditis (Streptococcus agalac abscesses, Sinusitis (Streptococcusanaerobic spe tiae Group B) cies) 4) Bacteremia, Endocarditis, Brain and other 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni abscesses, Sinusitis (Streptococcus anaerobic spe 35 cillin-Sensitive (Streptococcus Pneumoniae cies) Pneumococcus) 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni 6) Urinary tract infection, other infections, Bacter cillin-Sensitive (Streptococcus Pneumoniae emia (Escherichia coli) Pneumococcus) 7) Urinary tract and other infections (Proteus mirabi 6) Urinary tract infection, other infections, Bacter 40 lis) emia (Escherichia coli) 8) Urinary tract infection (Klebsiella pneumoniae) 7) Urinary tract and other infections (Proteus mirabi 9) Pneumonia (Klebsiella pneumoniae)-tan Ami lis) noglycoside 8) Urinary tract infection (Klebsiella pneumoniae) 10) Wound infection (animal bite), Abscesses, Bacte 9) Pneumonia (Klebsiella pneumoniae):tan Ami 45 remia, Meningitis (Pasteurella Multiocida) noglycoside 11) Ulcerative pharyngitis, Lung abscess, Empyema, 10) Wound infection (animal bite), Abscesses, Bacte Genital infections, Gingivitis (Fusobacterium remia, Meningitis (Pasteurella Multiocida) nucleatum) 11) Ulcerative pharyngitis, Lung abscess, Empyema, 12) “Malignant Pustule' Pneumonia (Bacillus Genital infections, Gingivitis (Fusobacterium 50 anthracis) nucleatum) CEFPROZIL 12) “Malignant Pustule' Pneumonia (Bacillus 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, anthracis) Osteomyelitis, Cellulitis and other Staphylococcus CEFALEXIN aureus infections Methicillin-Sensitive (Staphy 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 55 lococcus aureus) Osteomyelitis, Cellulitis and other Staphylococcus 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, aureus infections Methicillin-Sensitive (Staphy Cellulitis, Erysipelas, Pneumonia, Bacteremia, lococcus aureus) Toxic shock-like syndrome and other systemic 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, infections (Streptococcus pyogenes Group A) Cellulitis, Erysipelas, Pneumonia, Bacteremia, 60 3) Bacteremia, Endocarditis (Streptococcus agalac Toxic shock-like syndrome and other systemic tiae Group B) infections (Streptococcus pyogenes Group A) 4) Bacteremia, Endocarditis, Brain and other 3) Bacteremia, Endocarditis (Streptococcus agalac abscesses, Sinusitis (Streptococcus anaerobic spe tiae Group B) cies) 4) Bacteremia, Endocarditis, Brain and other 65 5) Pneumonia, Arthritis, Sinusitis, Otitis media Peni abscesses, Sinusitis (Streptococcus anaerobic spe cillin-Sensitive (Streptococcus Pneumoniae cies) Pneumococcus) US 8,937,074 B2 21 22 6) Urinary tract infection, other infections, Bacter 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly emia (Escherichia coli) coside 7) Urinary tract and other infections (Proteus mirabi CEFACLOR lis) 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) 8) Urinary tract infection (Klebsiella pneumoniae) 2) Urinary tract and other infections (Proteus mirabilis) 9) Pneumonia (Klebsiella pneumoniae):tan Ami 3) Urinary tract infection (Klebsiella Pneumoniae) noglycoside 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly 10) Wound infection (animal bite), Abscesses, Bacte coside remia, Meningitis (Pasteurella Multiocida) LORACARBEF 11) Ulcerative pharyngitis, Lung abscess, Empyema, 10 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) Genital infections, Gingivitis (Fusobacterium 2) Urinary tract and other infections (Proteus mirabilis) nucleatum) 3) Urinary tract infection (Klebsiella Pneumoniae) 12) “Malignant Pustule' Pneumonia (Bacillus 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly anthracis) 15 coside CEPHALOSPORINS CEPHALOSPORINS G2 SECOND GENERATION G3 THIRD GENERATION CEFOXITIN CEFTRIAXONE 1) Penicillin-Sensitive and Penicillinase-Producing 1) Endocarditis, Bacteremia (Streptococcus Viridans gonococcuS (Neisseria gonorrhoeae) Group) 2) Gasgangrene (Clostridium perfringens and other spe 2) Meningitis (Streptococcus agalactiae Group B) C1S 3) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin-Re 3) very of nosocomial and opportunistic infections sistant (Streptococcus pneumoniae Pneumonococ 4) Ulcerative pharyngitis, Lung abscess, Empyema, cus) Genital infections, Gingivitis (Fusobacterium 25 4) Endocarditis, Meningitis. Other serious infections nucleatum) Penicillin intermediate-resistant and Penicillin-Sen CEFOTETAN sitive (Streptococcus Pneumoniae) 1) Gasgangrene (Clostridium perfiringens and other spe 5) Penicillin-Sensitive and Penicillin Producing gono cies) coccuS (Neisseria gonorrhoeae gonococcus) 2) Variety of nosocomial and opportunistic infections 30 6) Meningitis (Neisseria meningitidis meningococ CEFURCXIME AXETIL cus) 1) Otitis media, Sinusitis, Pneumonia (Haemophilus 7) Typhoid fever, Paratyphoid fever, Bacteremia (Sal influenza) monella) CEPHAMANDOLE 8) Epiglottis, Meningitis (Haemophilus influenzae) 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) 35 2) Urinary tract and other infections (Proteus mirabilis) 9) Chancroid (Haemophilus ducreyi) 3) Urinary tract infection (Klebsiella Pneumoniae) 10) Wound infection (animal bite), Abscesses, Bacter 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly emia, Meningitis (Pasteurella multocida) coside 11) Melioidosis (Pseudomonas pseudomalilei) CEFURCXIME 40 12) Bacteremia, Endocarditis, Meningitis (Campylo 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) bacter fetus) 2) Urinary tract and other infections (Proteus mirabilis) 13) Syphilis (Treponema pallidum) 3) Urinary tract infection (Klebsiella Pneumoniae) 14) Erythema chronica migrans-skin, Stage 2-neuro 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly logical, Cardiac, Arthritis (Borrelia burgdorferi coside 45 Lyme disease) CEFONICID 15) Pulmonary lesions, Brain abscess, Lesions of other 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) organs (Nocardia asteroides) 2) Urinary tract and other infections (Proteus mirabilis) CEFOTAXIME 3) Urinary tract infection (Klebsiella Pneumoniae) 1) Meningitis (Streptococcus agalactiae Group B) 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly 50 2) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin-Re coside sistant (Streptococcus pneumoniae Pneumonococ CEFORANIDE cus) 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) 3) Endocarditis, Meningitis. Other serious infections 2) Urinary tract and other infections (Proteus mirabilis) Penicillin intermediately. Resistant and Penicillin 3) Urinary tract infection (Klebsiella Pneumoniae) 55 Sensitive (Streptococcus Pneumoniae) 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly 4) Endocarditis, Meningitis. Other serious infection coside Penicillin G-Resistant (Streptococcus pneumo CEFOTIAM niae)+Rifampin or +Vancomycin 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) 5) Meningitis (Neisseria meningitidis meningococ 2) Urinary tract and other infections (Proteus mirabilis) 60 cus) 3) Urinary tract infection (Klebsiella Pneumoniae) 6) Epiglottits, Meningitis (Haemophilus influenzae) 4) Pneumonia (Klebsiella Pneumoniae)-tan Aminogly CEFTIZOXIME coside 1) Gasgangrene (Clostridium perfiringens and other spe CEFAMYCINS cies) 1) Otitis, Sinusitis, Pneumonia (Moraxella catarrhalis) 65 CEFIXIME 2) Urinary tract and other infections (Proteus mirabilis) 1) Penicillin-Sensitive and Penicillinase-Producing 3) Urinary tract infection (Klebsiella Pneumoniae) gonococcuS (Neisseria gonorrhoeae)

US 8,937,074 B2 25 26 3) Urinary tract and other infections (Proteus, other GENTAMICIN species) 1) Urinary tract infection, other infections, Bacteremia. 4) Urinary tract infection (Pseudomonas aeruginosa) (Escherichia coli)+Ampicillin 5) Pneumonia, Bacteremia (Pseudomonas aerugi 2) Urinary tract infection and other infections (Entero nosa)+an Aminoglycoside bacter species) 6) Pneumonia (Klebsiella pneumoniae) 3) Urinary tract infection and other infections (Proteus 7) Variety of nosocomial and opportunistic infections mirabilis) (Serratia) 4) Urinary tract infection and other infections (Proteus, 8) Various nosocomial infections (Acinetobacter) other species) 10 5) Urinary tract infection (Pseudomonas aeruginosa) 9) Bacteremia, Endocarditis (Campylobacter fetus) 6) Pneumonia, Bacteremia (Pseudomonas aerugi 10) Pulmonary lesions, Brain abscess, Lesions of other nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; organs (Nocardia asteroides) +Ceftazidime; +Aztreonam, +Imipenem MEROPENEM 7) Urinary tract infection (Klebsiella pneumoniae) 1) Active against Some Imipenem-Resistant Pseudomo 15 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or nas aeruginosa Piperacillin 2) Urinary tract infection (Pseudomonas aeruginosa) 9) Variety of nosocomial and opportunistic infections I. OTHER B-LACTAMANTIBIOTICS (Serratia)+A broad-spectrum Penicillin MONOBAKTAMS 10) Various nosocomial infections (Acinetobacter) AZTREONAM 11) Sepsis (Yersinia enterocolitica) 1) Urinary tract infection, Other infections, Bacter 12), Endocarditis, Infected foreign bodies, Bacteremia emia (Escherichia coli) (Corynebacterium species; aerobic and anaerobic 2) Urinary tract and Other infections (Proteus, other diptheroids)+Penicillin G species) 13) Endocarditis or other serious infection bacteremia 3) Urinary tract infection (Pseudomonas aeruginosa) 25 (Enterococcus)+Penicillin G or Ampicillin; +Vanco 4) Pneumonia, Bacteremia (Pseudomonas aerugi mycin nosa)+an Aminoglycoside 14) Meningitis, Bacteremia (Listeria monocytogenes)+ 5) Pneumonia (Klebsiella pneumoniae) Ampicillin or Penicillin G 6) Variety of nosocomial and opportunistic infections 15) Brucellosis (Brucella)+Doxycycline (Serratia) 30 16) Tularemia (Francisella tularensis) J. AMINOGLYCOSIDE 17) Bacteremia, Endocarditis (Campylobacter fetus) TOBRAMYCIN STREPTOMYCIN 1) Urinary tract infection, other infections, Bacteremia. 1) Urinary tract infection, other infections, Bacteremia. (Escherichia coli)+Ampicillin (Escherichia coli)+Ampicillin 35 2) Urinary tract infection and other infections (Entero 2) Urinary tract infection and other infections (Entero bacter species) bacter species) 3) Urinary tract infection and other infections (Proteus 3) Urinary tract infection and other infections (Proteus mirabilis) mirabilis) 4) Urinary tract infection and other infections (Proteus, 4) Urinary tract infection and other infections (Proteus, 40 other species) other species) 5) Urinary tract infection (Pseudomonas aeruginosa) 5) Urinary tract infection (Pseudomonas aeruginosa) 6) Pneumonia, Bacteremia (Pseudomonas aerugi 6) Pneumonia, Bacteremia (Pseudomonas aeruginosa) nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; A broad-spectrum Penicillin; +Ciprofloxacin: +Ceftazidime; +Aztreonam, +Imipenem +Ceftazidime; +Aztreonam, +Imipenem 45 7) Urinary tract infection (Klebsiella pneumoniae) 7) Urinary tract infection (Klebsiella pneumoniae) 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or Piperacillin Piperacillin 9) Variety of nosocomial and opportunistic infections 9) Variety of nosocomial and opportunistic infections (Serratia)+A broad-spectrum Penicillin (Serratia)+A broad-spectrum Penicillin 50 10) Various nosocomial infections (Acinetobacter) 10) Various nosocomial infections (Acinetobacter) 11) Sepsis (Yersinia enterocolitica) 11) Sepsis (Yersinia enterocolitica) 12) Endocarditis, Infected foreign bodies, Bacteremia 12) Endocarditis, Infected foreign bodies, Bacteremia (Corynebacterium species; aerobic and anaerobic (Corynebacterium species; aerobic and anaerobic diptheroids)+Penicillin G diptheroids)+Penicillin G 55 AMICACIN 13) Bacteremia, Arthritis, Endocarditis, Abscesses 1) Urinary tract infection, other infections, Bacteremia. (Streptobacillus moniliformis) (Escherichia coli)+Ampicillin 14) Pulmonary, Milary, Renal, Meningeal, and other 2) Urinary tract infection and other infections (Entero tuberculous infections (Mycobacterium tuberculo bacter species) sis)+Rifampin or Ethambutol 60 3) Urinary tract infection and other infections (Proteus 15)Yaws (Treponema pertenue) mirabilis) 16) Plague (Yersinia pestis)+Tetracycline 4) Urinary tract infection and other infections (Proteus, 17) Tularemia (Francisella tularensis) other species) 18) Glanders (Pseudomonas mallei)+a Tetracycline 5) Urinary tract infection (Pseudomonas aeruginosa) or +Chloramphenicol 65 6) Pneumonia, Bacteremia (Pseudomonas aerugi 19). Occasionally administrate for tuberculosis (Myco nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; bacterium tuberculosis) +Ceftazidime; +Aztreonam, +Imipenem US 8,937,074 B2 27 28 7) Urinary tract infection (Klebsiella pneumoniae) 2) Urinary tract infection and other infections (Entero 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or bacter species) Piperacillin 3) Urinary tract infection and other infections (Proteus 9) Variety of nosocomial and opportunistic infections mirabilis) (Serratia)+A broad-spectrum Penicillin 4) Urinary tract infection and other infections (Proteus, 10) Various nosocomial infections (Acinetobacter) other species) 11) Sepsis (Yersinia enterocolitica) 5) Urinary tract infection (Pseudomonas aeruginosa) 12) Endocarditis, Infected foreign bodies, Bacteremia 6) Pneumonia, Bacteremia (Pseudomonas aerugi (Corynebacterium species; aerobic and anaerobic nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; diptheroids)+Penicillin G 10 13) Disseminated disease in AIDS (Mycobacterium +Ceftazidime; +Aztreonam, +Imipenem avium-intracellulare) 7) Urinary tract infection (Klebsiella pneumoniae) 14) Pulmonary lesions, Brain abscess, Lesions of the 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or other organs Piperacillin NETILMICIN 15 9) Variety of nosocomial and opportunistic infections 1) Urinary tract infection, other infections, Bacteremia. (Serratia)+A broad-spectrum Penicillin (Escherichia coli)+Ampicillin 10) Various nosocomial infections (Acinetobacter) 2) Urinary tract infection and other infections (Entero 11) Sepsis (Yersinia enterocolitica) bacter species) 12) Endocarditis, Infected foreign bodies, Bacteremia 3) Urinary tract infection and other infections (Proteus (Corynebacterium species; aerobic and anaerobic mirabilis) diptheroids)+Penicillin G 4) Urinary tract infection and other infections (Proteus, 13) For bladder irrigation+Polymyxin B other species) 14) Variety of infections of the skin and mucous mem 5) Urinary tract infection (Pseudomonas aeruginosa) branes caused by microorganisms susceptible to the 6) Pneumonia, Bacteremia (Pseudomonas aerugi 25 drug. These include infections associated with burns, nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; wounds, ulcers, and infection dermatoses. +Ceftazidime; +Aztreonam, +Imipenem K. TETRACYCLINE 7) Urinary tract infection (Klebsiella pneumoniae) TETRACYCLINE 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or 1) Sinusitis (Moraxella catarrhalis) Piperacillin 30 2) Plague (Yersinia pestis)+Streptomycin 9) Variety of nosocomial and opportunistic infections 3) Glanders (Pseudomonas mallei)+Streptomycin (Serratia)+A broad-spectrum Penicillin 4) Stage 2-Neurological, cardiac, arthritis (Borrelia 10) Various nosocomial infections (Acinetobacter) burgdorferi Lyme disease) 11) Sepsis (Yersinia enterocolitica) CHLORTETRACYCLINE 12) Endocarditis, Infected foreign bodies, Bacteremia 35 OXYTETRACYCLINE (Corynebacterium species; aerobic and anaerobic DOXYCYCLINE diptheroids)+Penicillin G 1) Erysipeloid (Erysipelothrix rhusiopathiae) 13) Effective against certain gentamicin-resistant patho 2) Gas gangrene (Clostridium perfingens & other spe gens, except enterococci cies) KANAMYCIN 40 3) Tetanus (Clostridium tetani) 1) Urinary tract infection, other infections, Bacteremia. 4) Urinary tract infection (Escherichia coli) (Escherichia coli)+Ampicillin 5) Brucellosis (Brucella)+Gentamicin or Rifampin 2) Urinary tract infection and other infections (Entero 6) Chancriod (Haemophilus ducreyi) bacter species) 7) Plague (Yersinia pestis) 3) Urinary tract infection and other infections (Proteus 45 8) Wound infection-animal bite (Pasteurella multocida) mirabilis) 9) Cholera (Vibrio cholerae) 4) Urinary tract infection and other infections (Proteus, 10) Lung abscess, empyema (Fusobacterium nuclea other species) tum) 5) Urinary tract infection (Pseudomonas aeruginosa) 11) Arthritis (Streptobacillus moniliformis) 6) Pneumonia, Bacteremia (Pseudomonas aerugi 50 12) Syphilis (Treponema pallidum) nosa)+A broad-spectrum Penicillin; +Ciprofloxacin; 13) Yaws (Treponema pertenue) +Ceftazidime; +Aztreonam, +Imipenem 14) Erythema chronica migrans-skin (Borrelia burgdor 7) Urinary tract infection (Klebsiella pneumoniae) feri Lyme disease) 8) Pneumonia (Klebsiella pneumoniae)+Mezlocillin or 15) Relapsing fever (Borrelia recurrentis) Piperacillin 55 16) Weil's disease and meningitis (Leptospira) 9) Variety of nosocomial and opportunistic infections 17) Cervicofacial, abdominal, thoracic, and other (Serratia)+A broad-spectrum Penicillin lesions (Actinomyces israelii) 10) Various nosocomial infections (Acinetobacter) 18) Non-specific urethritis (Ureaplasma urealyticum) 11) Sepsis (Yersinia enterocolitica) 19) Atypical pneumonia' (Mycoplasma pneumoniae) 12) Endocarditis, Infected foreign bodies, Bacteremia 60 20) Typhus fever, Murine typhus, Brill's disease, Rocky (Corynebacterium species; aerobic and anaerobic Mountain spotted fever, Q fever, and Rickettsialpox diptheroids)+Penicillin G (Rickettsia) 13) Orally for the prophylactic use as adjunctive therapy 21) Psittacosis (Chlamydia psittaci) in cases of hepatic coma 22) Lymphogranuloma Venereum, Trachoma, Inclusion NEOMYCIN 65 conjunctivitis (blennorrhea), Non-specific urethritis, 1) Urinary tract infection, other infections, Bacteremia. Cervicitis (Chlamydia trachomatis) (Escherichia coli)+Ampicillin 23) Pneumonia (Chlamydia pneumoniae) US 8,937,074 B2 29 30 MINOCYCLINE 11) Bacteremia, Arthritis, Endocarditis, Abscesses DEMECLOCYCLINE (Streptobacillus moniliformis) METHACYCLINE 12) Legionnaires disease (Legionella pneumophila) L. CHLORAMPHENICOL trifampin CHLORAMPHENICOL 1) Meningitis (Streptococcus agalactiae Group B) 13) Relapsing fever (Borrelia recurrentis) 2) Bacteremia, Endocarditis, Brain and other abscesses, 14) Cervicofacial, Abdominal, Thoracic and other and Sinusitis (Streptococcus anaerobic species) lesions (Actinomyces israelii). 3) Pneumonia, Arthritis, Sinusitis, Otitis, Endocarditis, 15) Non-specific Urethritis (Ureaplasma Urealyti Meningitis. Other serious infections (Streptococcus 10 cum) pneumoniae pneumonococcus). 16) 'Atypical pneumonia' (Mycoplasma pneumo 4) Meningitis (Neisseria meningitidis meningococ niae) cus) 17) Lymphogranuloma Venereum, Trachoma, Inclu 5) "Malignant pustule'. Pneumonia (Bacillus anthracis) sion conjunctivitis blennorrhea. Non-specific 6) Bacteremia (Listeria monocytogenes) 15 urethritis, Cerviciitis (Chlamydia trachomatis) 7) Erysipeloid (Erysipelothrix rhusiophathiae) 18) Pneumonia (Chlamydia pneumoniae) 8) Gasgangrene (Clostridium perfiringens and other spe 19) Erysipeloid (Erysipelothrix rhusiopathiae) cies) 20) Bordetella pertussis disease and for post-expo 9) Typhoid fever, Paratyphoid fever, Bacteremia (Sal sure prophylaxis of all household member and monella) 10) Epiglottitis, Meningitis (Haemophilus influenza) other close contacts. 11) Brucellosis (Brucella) 21) Tetanus in patients who are allergic to penicillin 12) Plague (Yersinia pestis) (Clostridium tetani) 13) Sepsis (Yersinia enterocolitica) CLARITHROMYCIN 14) Tularemia (Francisella tularensis) 25 1) Legionnaires disease (Legionella pneumophila) 15) Cholera (Vibrio cholerae) 2) Atypical pneumonia' (Mycoplasma pneumoniae) 16) Glanders (Pseudomonas mallei)+Streptomycin 3) Pneumonia (Chlamydia pneumoniae) 17) Melioidosis (Pseudomonas pseudomallei) 18) Meningitis (Campylobacter fetus) 4) Enteritis (Campylobacter jejuni) 19) Ulcerative pharyngitis, Lung abscess and Empyema, 30 5) Disseminated disease in AIDS (Mycobacterium Genital infections, Gingivitis (Fusobacterium avium-intracellulare)+Ethambutol; Clofazimine; nucleatum) +Ciprofloxacin 20) Bacteremia, Arthritis, Endocarditis, Abscesses 6) Erythema chronica migrans-skin (Borrelia burg (Streptobacillus moniliformis) dorferi Lyme disease) 21) Typhus fever, Murine typhus, Brill's disease, Rocky 35 7) Modest activity against H. influenzae and N. gon mountain spotted fever, Q fever, Rickettsialpox (Rick orrhoeae ettsia) 8) Good activity against M. catarrhalis 22) Psittacosis ornithosis (Chlamydia psittaci) 9) Enhanced activity against Some protozoa (e.g., THIAMPHENICOL Toxoplasma gondii, Cryptoporidium and Plasmo AZIDAMPHENICOL 40 M. ERYTHROMYCIN AND OTHERS dium spp. MACROLIDES: 10) Regimens for the treatment of peptic ulcers ERYTHROMYCIN related to H. pylori infection 1) Abscesses, Bacteremia, Endocarditis, Pneumonia 11) Lepromatous leprosy (Mycobacterium leprae)+ Osteomyelitis, Cellulitis and other Staph. aureus 45 minocycline infections Methicillin-Sensitive (Staphylococcus AZITHROMYCIN aureus) 1) Otitis media, Sinusitis, Pneumonia (Haemophilus 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, influenzae) Cellulitis, Erysipelas, Pneumonia, Bacteremia, 2) Enteritis (Campylobacter jejuni) toxic shock-like syndrome, and other systemic 50 infections (Streptococcus pyogenes Group A) 3) Legionnaires disease (Legionella pneumophila) 3) Bacteremia, Endocarditis, Brain and other 4) Erythema chronica migrans-skin (Borrelia burg abscesses, Sinusitis (Streptococcus anaerobic spe dorferi Lyme disease) cies) 5) "Atypical pneumonia' (Mycoplasma pneumoniae) 4) Penicillin-Sensitive gonococcuS (Neisseria gonor 55 6) Lymphogranuloma Venereum, Trachoma, Inclu rhoeae) sion conjunctivitis blennorrhea. Non-specific 5) "Malignant pustule'. Pneumonia (Bacillus anthra urethritis, Cerviciitis (Chlamydia trachomatis) cis) 6) Pharyngitis, Laryngotracheitis, Pneumonia, and 7) Pneumonia (Chlamydia pneumoniae) other local lesions, Carrier state (Corynebacterium 60 8) Less active against Streptococcus spp. And Entero diptheriae) cocci 7) Bacteremia (Listeria monocytogenes) 9) Active against M. catarrhals, Pasteurella multo 8) Chancroid (Haemophilus ducreyi) cida, Fusobacterium spp., N. gonorrhoeae 9) Enteritis (Campylobacter jejuni) 10) Enhanced activity against Mycobacterium avium 10) Ulcerative pharyngitis, Lung abscess, Empyema, 65 intracellulare, as well as against protozoa (e.g. Genital infections, Gingivitis (Fusobacterium Toxoplasma gondii, Crytosporidium and Plasmo nucleatum) dium spp. US 8,937,074 B2 31 32 11) Toxoplasmosis encephalitis and diarrhoea due to BACITRACIN Cryptosporidium 1) Infected eczema, Infected dermal ulcers ROXITHROMYCIN 2) Suppurative conjunctivitis and infected corneal ulcer N. LINCOMYCIN when they are cause by susceptible bacteria CLINDAMYCIN 5 3) Eradication of nasal carriage of Staphylococci 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 4) Antibiotic-associated diarrhoea (Clostridium difficile) Osteomyelitis, Cellulitis and other Staphylococcus RP 5.9500 aureus infections Methicillin-Sensitive (Staphylo 1) Is a good inducer of the methylase enzyme that medi COccus aureus). 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, ates MLS resistance Cellulitis, Erysipelas, Pneumonia, Bacteremia, Toxic 10 2) Are Synergistic and therefore, erythromycin-resistant shock-like syndrome, and other systemic infections organisms frequently are susceptible to RP 59500 in (Streptococcus pyogenes Group A) vitro 3) Bacteremia, Endocarditis, Brain and other abscesses, GLYCYLCYCLINES (Tetracycline Antibiotic deriva Sinusitis (Streptococcus anaerobic species) tives) 4) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin 15 1) They inhibit some tetracycline-resistant organisms Sensitive and Penicillin-Resistant(Streptococcus 2) Also appear to be active against multiply drug-resis pneumoniae) tant strains of Staphylococci, pneumacocci and Van 5) Pharyngitis, Laryngotracheitis, Pneumonia, Other comycin-resistant enterococci local lesions (Corynebacterium diphteriae) GLYCOPEPTIDE AND OTHER: 6) Gasgangrene (Clostridium perfiringens and other spe VANCOMYCIN cies) 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, 7) Tetanus (Clostridium tetani) Osteomyelitis, Cellulitis and other Staphylococcus 8) Enteritis (Campylobacter jejuni) aureus infections Methicillin-Sensitive and 9) Ulcerative pharyngitis, Lung abscess, Empyema, Methicillin Resistant (Staphylococcus aureus) Genital infections, Gingivitis 25 2) Pharyngitis, Scarlet fever, Otitis media, Sinusitis, 10) Pneumonia in impaired host Mild or moderate dis Cellulitis, Erysipelas, Pneumonia, Bacteremia, ease and moderately severe or severe disease (Pneu Toxic shock-like syndrome, and other systemic mocystis carinii)+Primaquin infections (Streptococcus pyogenes Group A) 11) Treatment of infections with anaerobes, especially 3) Endocarditis, Bacteremia (Streptococcus Viridans those due to B. fragilis 30 Group) 12) Intra-abdominal or pelvic abscesses and peritonitis+ 4) Bacteremia, Endocarditis (Streptococcus agalac an Aminoglycoside or +Penicillin or +Cephalothin tiae Group B) 13) Topically or orally for acne Vulgaris and forbacterial 5) Pneumonia, Arthritis, Sinusitis, Otitis Penicillin Vagionosis Resistant (Streptococcus pneumoniae). SPECTINOMYCIN 35 6) Endocarditis, Meningitis. Other serious infections 1) Penicillin-Sensitive and Penicillinase-Producing Penicillin intermediately Resistant (Streptococ gonococcuS (Neisseria gonorrhoeae) cus pneumoniae)+Rifampin 2) In pregnancy when patients are intolerant to B-Lac 7) Endocarditis, Meningitis, Other serious infections tams and when quinolones are contraindicated Penicillin G-Resistant (Streptococcus pneumo 3) Recommended as an alternative regimen in patients 40 niae)+Rifampin or +Cefotaxime who are intolerant or allergic to B-Lactam antibiotics 8) Endocarditis or other serious infection bacter and quinolones emia (Enterococcus)+Gentamincin POLYMYXIN B (Polymyxin B Sulfate) 9) Urinary tract infection (Enterococcus) 1) Available for ophthalmic, otic and topical use in com 10) Endocarditis, Infected foreign bodies, Bacteremia bination with a variety of other compounds. 45 (Corynebacterium species, aerobic and anaerobic 2) Infections of the skin, mucous membranes, eye, and diptheroids) ear due to polymyxin B-sensitive microorganisms 11) Tetanus (Clostridium tetani) 3) External otitis, frequently due to pseudomonas 12) Antibiotic-associated colitis (Clostridium diffi 4) Infection of corneal ulcers (Pseudomonas aerugi cile) nosa) 50 13) Meningitis (Flavobacterium meningosepticum) 5) Pneumonia (Pseudomonas) 14) Pseudomembranous colitis COLISTIN (Colisten Sulfate) 15) Staphylococcal infections in patients who are 1) Diarrhoea caused by bacteria susceptible to the drug allergic to penicillins and cephalosporins in infants and children O. Drugs Used in the Treatment of Tuberculosis, Mycobac RAMOPLANIN (glycopeptide) 55 terium avium Complex, and Leprosy 1) Treatment of acne and skin infections, and to reduce AMIKACIN, nasal carriage of Staphylococci AMINOSALICYCLIC ACID Active against Bacteroides spp. AZITHROMYCIN TEICOPLANIN (glycopeptide) CAPREOMYCIN 1) Osteomyelitis, Endocarditis caused by Methicillin 60 CEFOXITIN Resistant and Methicillin-Susceptible Staphylococci, CIPOFLOXACIN Streptococci and Enterococci CLARITHROMYCIN 2) Bacteremia, Endocarditis methicillin susceptible CLOFAZIMINE (Staphylococcus aureus)+an Aminoglycoside genta DAPSONE mycin 65 DOXYCYCLINE 3) Enterococcal endocarditis+Gentamicin ETHAMBUTOL 4) Endocarditis+Vancomycin ETHIONAMIDE US 8,937,074 B2 33 34 IMIPENEM ITRACONAZOLE ISONIAZID ITRACONAZOLE, KANAMYCIN ITRACONAZOLE, MINOCYCLINE ITRACONAZOLE, OFLOXACIN IV MICONAZOLE OFLOXACIN KETOCONAZOLE PYRAZINAMIDE KETO-CONAZOLE RIAMPIN KETO-CONAZOLE RIFABUTIN KETO-CONAZOLE RIFAMPIN 10 STREPTOMYCIN KETOCONAZOLE, MICONAZOLE SULFONAMIDE TRIMETHOPRIM-SULFAMETHOXAZOLE, MICONAZOLE P. OTHER TREATMENTS MICONAZOLE NYSTATIN RIFAMPIN 15 NAFTIFINE 1) Abscesses, Bacteremia, Endocarditis, Pneumonia, NYSTATIN Osteomyelitis, Cellulitis. Other Methicillin-Sensi NYSTATIN tive Methicillin-Resistant (Staphylococcus aureus)+ SPOROTRICHOSIS Ciprofloxacin or +Trimethoprim-sulfumethoxazole SYSTEMIC when methicillin-resistant TERBINAFINE 2) Endocarditis, Meningitis. Other serious infection TERBINAFINE Penicillin-Intermediately resistant (Streptococcus TERCONAZOLE pneumonia (pneumonococcus))+Vancomycin TIOCONAZOLE 3) Endocarditis, Meningitis. Other serious infection TOPICAL CLOTRIMAZOLE Penicillin G-resistant (Streptococcus pneumoniae 25 UNDECYLENATE (pneumonococcus))+Cefotaxime or +Vancomycin R OTHER TREATMENTS 4) Carrier state (post-treatment) (Neisseria meningitidis POLIEEN ANTIEBIOTICS (meningoccoccus)) AMPHOTERICIN B 5) Pharyngitis, Laryngotracheitis, Pneumonia, Other 1) Deep infection (Candida species)+Flucytosine local lesions (Corynebacterium diphtheriae) 30 2) Disseminated (non-meningeal), Meningitis (Coc 6) Endocarditis, Infected foreign bodies, Bacteremia cidioides immitis) (Corynebacterium species, aerobic and anaerobic 3) Chronic Pulmonary disease, Disseminated (Histo (diphtheroids))+Penicillin G plasma capsulatum) 7) Brucellosis (Brucella)+Doxycycline or +Trimethop 4) All the Blastomyces brasiliensis infections 1. 35 5) All the paracocidioides brasiliensis infections fol 8) Meningitis (Flavobacterium meningosepticum) lowed up by a sulfonamide 9) Legionnaires disease (Legionella pneumophila)+ 6) Extracutaneous (Sporothrix schenckii) Erythromycin 7) Invasive (Aspergillus species) Q. ANTI-FUNGALAGENTS 8) All the infections of the mucormycosis Agents AMPHOTERICIN B 40 9) Pulmonary (Cryptococcus neoformans) AMPHOTERICIN B 10) Meningitis (Cryptococcus neoformans)-tElucy AMPHOTERICIN B tosine AMPHOTERICIN B NYSTATIN AMPHOTERICIN B 1) Cutaneous or vaginal thrush, oral thrush (Candida AMPHOTERICIN B 45 species) AMPHOTERICIN B FLUCYTOSINE AMPHOTERICIN B, 1) Deep infections (Candida species)+Amphotericin BUTOCONAZOLE B CICLOPIROX Meningitis (Cryptococcus neoformans)+Amphoteri CICLOPIROX 50 cin B CLOTRIMAZOLE POVIDONE ODINE CLOTRIMAZOLE Povidone-iodine is an iodophore which is used as a CUTANEOUS disinfectant and antiseptic mainly for the treatment ECONAZOLE of contaminated wounds and pre-operative prepa ECONAZOLE 55 ration of the skin and mucous membranes as well as EXTRACUTANEOUS for the disinfection of equipment. FLUCONAZOLE S. IMIDAZOLES AND TRIAZOLES FLUCONAZOLE KETOCONAZOLE FLUCONAZOLE 1) Cutaneous or vaginal thrush, Oral thrush, (Candida FLUCONAZOLE 60 species) FLUCONAZOLE, 2) Chronic pulmonary disease (Histoplasma capsula FLUCYTOSINE tum) GRISEOFULVIN 3) All Blastomyces dermatitidis infections. HALOPROGIN 4) All Paracoccidioides brasiliensis infections INTRATHECAL 65 ITRACONAZOLE IODIDE, ITRACONAZOLE 1) Cutaneous or vaginal thrush, Oral thrush (Candida ITRACONAZOLE species) US 8,937,074 B2 35 36 2) Disseminated (non-meningeal), Meningitis (Coccid NAFTIFINE ioides immitis) 1) Treatment of Tinea cruris and Tinea corporis 3) Disseminated (Histoplasma capsulatum) 2) Cutaneous candidiasis and Tinea versicolor 4) Cutaneous, Extracutaneous (Sporothrix schenckii) TERBINAFINE 5) Invasive (Aspergillus species) 1) Tinea corporis, Tinea cruris, Tinea pedis 6) Chronic pulmonary disease (Histoplasma capsula 2) Cutaneous candidiasis and Tinea versicolor tum) 3) Treatment of ringworm and in some cases of onycho 7) All paracoccidioides brasiliensis infections mycosis 8) All Blastomyces dermatitidis infections V. MISCELLANEOUSANTI-FUNGALAGENTS 10 UNDECYLENIC ACID FLUXONAZOLE 1) Treatment of various dermatomycoses, especially 1) Cutaneous or vaginal thrush, Oral thrush, Deep infec Tinea pedis tion (Candida Species) 2) Treatment of diaper rash, Tinea cruris and other minor 2) Disseminated (non-meningeal), Meningitis (Coccid dermatologic condition’s ioides immitis) 15 BENZOIC ACID AND SALICYLIC ACID 3) Chronic pulmonary disease (Histoplasma capsula 1) Treatment of Tinea pedis and sometimes used to treat tum) Tinea capitis 4) Meningitis (Cryptococcus neoformans) PROPIONIC ACID AND CAPRYLIC ACID CLOTRIMAZOLE 1) Treatment of the dermatomycoses 1) Oral thrush (Candida Species) POTASSIUM IODIDE GRISEOFULVIN 1) Cutaneous (Sporothrix schenckii) 1) Mycotic disease of the skin, hair and nails due to W. ANTI-FUNGALAGENTS FOR OPHTHALMICUSE Microsporum, Trichophyton or Epidermophyton NATAMYCIN 2) Tinea capitis (M. canis, M. audouini, T. Schoenleinii Fungal blepharitis, conjunctivitis, keratitis and T. verrucosum) 25 IMIDAZOLES CLOTRIMAZOLE 3) "Ringworm” of the glabrous skin, Tinea cruris and Fungal keratitis tinea corporis (M. canis, T. rubrum, T verrucosum ECONAZOLE and E. floccosum) Fungal keratitis 4) Tinea of the hands (T. rubrum, T. mentagraphytes) FLUCONAZOLE 5) Tinea of the beard (Trichopyton species) 30 Fungal keratitis 6) Athlete's foot’ or epidermophytosis involving the KETOCONAZOLE skin and nails (T. mentagraphytes and the hyperkera Fungal keratitis totic type to T. rubrum). MICONAZOLE T. TOPICAL ANTI-FUNGALAGENTS Fungal keratitis, endophthalmitis Imidazoles and Triazoles for Topical Use 35 PYRIMIDINES CLOTRIMAZOLE FLUCYTOSINE 1) Dermatophyte infections, Cutaneous candidiasis, Fungal keratitis Vulvovaginal candidiasis POLYENES ECONAZOLE AMPHOTERICIN B MICONAZOLE 40 Fungal keratitis, endophthalmitis 1) Tinea pedis, Tinea cruris, Tinea versicolor X. ANTI-VIRAL AGENTS 2) Vulvovaginal candidiasis ANTI-HERPESVIRUS AGENTS: 3) Some vaginal infections caused by Candida gla ACYCLOVIR brata 1) Genital disease, Keratoconjunctivitis, Encephali TERCONAZOLE 45 tis, Neonatal HSV. Mucocutaneous HSV in 1) Vaginal Candidiasis immuno-compromised host (Herpes simplex BUTOCONAZOLE virus) 1) Vaginal Candidiasis 2) Herpes Zoster or varicella in immuno-compro TIOCONAZOLE mised host, pregnancy, Varicella or herpes Zoster in 1) Candida Vulvovaginitis 50 normal host (Varicella Zoster virus) OXICONAZOLE VALACYCLOVIR 1) Infections caused by the common pathogenic der 1) Genital herpes or localised herpes Zoster matophytes FAMCICLOVIR SULCONAZOLE 1) Varicella or herpes Zoster in normal host (Varicella 1) Infections caused by the common pathogenic der 55 Zoster virus) matophytes PENCICLOVIR U. OTHER ANTI-FUNGAL AGENTS FOR TOPICAL 1) It is inhibitory for hepatitis B virus USE FOSCARNET CICLOPIROX OLAMINE 1) Retinitis in patients with AIDS (Cytomegalovirus) 1) Cutaneous candidiasis, Tinea corporis, Tinea cruris, 60 2) Mucocutaneous HSV in immuno-compromised Tinea pedis, Tinea versicolor host (Herpes simplex virus) 2) Dermatomycoses and candidal infections 3) Herpes Zoster or varicella in immuno-compro HALOPROGIN mised host, pregnancy (Varicella Zoster virus) 1) Tinea pedis, Tinea cruris, Tinea corporis, Tinea GANCICLOVIR manuum and Tinea versicolor 65 1) Retinitis in patients with AIDS (Cytomegalovirus) TOLNAFTATE IDOXURIDINE 1) Tinea pedis 1) Keratoconjunctivitis (Herpes simplex virus) US 8,937,074 B2 37 38 SORIVUDINE GENTAMICIN SULFATE (GARAMYCIN, GENOTIC, 1) Herpes Zoster in HIV-infected adults GENT-AK, GENTACIDIN) TRIFLURIDINE 1) Conjunctivitis, blepharitis, keratitis 1) Keratoconjunctivitis (Herpes simplex virus) NORFLOXACIN(CHIBROXIN) VIDARABINE 1) Conjunctivitis 1) Encephalitis, neonatal herpes (Herpes simplex SULFACETAMIDE SODIUM virus) (AK-SULF, BLEPH-10, CETAMIDE, SULF-10, 2) Zoster or varicella in immuno-compromised ISOPTO CETAMIDE, OPHTHACET, SULAMYD patients SODIUM) Y. ANTI-RETROVIRAL AGENTS 10 1) Conjunctivitis, blepharitis, keratitis SULFISOXAZOLE DIOLAMINE (GANTRISIN) ZIDOVUDINE 1) Conjunctivitis, blepharitis, keratitis 1) AIDS, HIV antibody positive and CD4 countless than POLYMYXIN B COMBINATIONS 500/mm (Human immuno-deficiency virus) 1) Conjunctivitis, blepharitis, keratitis DIDANOSINE 15 TETRACYCLINE HYDROCHLORIDE (ACHROMY 1) Advanced HIV infections in adults and children over CIN) 6 months 1) Conjunctivitis, blepharitis STAVUDINE TOBRAMYCIN SULFATE (TOBREX) 1) AIDS, HIV antibody positive and CD4 countless than 1) Conjunctivitis, blepharitis, keratitis 400/mm (Human immuno-deficiency virus) (ii) CPNS Agents ZALCITABINE According to the present invention there is further provided 1) AIDS a method of enhancing the action of a pharmaceutical agent 2) HIV infection and CD4 count less than 300/mm selected from the group consisting of the CPNS agents Z. OTHER ANTI-VIRAL AGENTS selected from the group of compounds acting on the central or AMANTADINE 25 peripheral nervous system, but excluding coaltar Solution and 1) Influenza (Influenza A) H1-antagonistantihistamines and also excluding anti-inflam RIMANTADINE matory, analgesic and antipyretic agents, comprising the step 1) Influenza (Influenza A) of formulating the agent with an administration medium INTERFERONS ALFA which is characterised in that it comprises a solution of 1) Genital papilloma (Human papilloma virus) 30 nitrous oxide gas in a pharmaceutically acceptable carrier RIBAVIRIN Solvent for the gas and which administration medium 1) Pneumonia and bronchiolitis infancy (Respiratory includes at least one fatty acid or ester or other suitable syncytial virus) derivative thereof selected from the group consisting of oleic AA.NEWERAGENTS UNDER CLINICAL DEVELOP acid, linoleic acid, alpha-linolenic acid, gamma-linolenic MENT 35 acid, arachidonic acid, eicosapentaenoic acid C20: 5c)3. LAMIVUDINE decosahexaenoic acid C22: 603, ricinoleic acid and deriva PROTEASE INHIBITORS tives thereof selected from the group consisting of the C1 to ACYCLIC NUCLEOSIDE PHOSPHONATES C6 alkyl esters thereof, the glycerol-polyethylene glycol AB. ANTI-VIRAL AGENTS FOR OPHTHALMICUSE esters thereof and the reaction product of hydrogenated natu IDOXURIDINE (HERPLEX) 40 ral oils composed largely of ricinoleic acid based oils such as 1) Herpes simplex keratitis castor oil with ethylene oxide. TRIFLURIDINE (VIROPTIC) According to a further aspect of the present invention there 1) Herpes simplex keratitis is provided a pharmaceutical preparation comprising a phar VIDARABINE (VIRA-A) maceutical agent which is a CPNS agent selected from the 1) Herpes simplex keratitis 45 group of compounds acting on the central or peripheral ner 2) Herpes simplex conjunctivitis Vous system, but excluding coal tar Solution and H1-antago ACYCLOVIR (ZOVIRAX) nist antihistamines and also excluding anti-inflammatory, 1) Herpes Zoster ophthalmicus analgesic and antipyretic agents, comprising the step of for 2) Herpes simplex keratitis mulating the agent with an administration medium which is FOSCARNET (FOSCAVIR) 50 characterised in that it comprises a solution of nitrous oxide 1) Cytomegaloviris retinitis gas in a pharmaceutically acceptable carrier solvent for the GANCICLOVIR(CYTOVENE) gas and which administration medium includes at least one 1) Cytomegaloviris retinitis fatty acid or ester or other suitable derivative thereof selected AC. Topical Antibacterial Agents Commercially Available from the group consisting of oleic acid, linoleic acid, alpha for Ophthalmic Use 55 linolenic acid, gamma-linolenic acid, arachidonic acid, BACITRACINZINC (AK-TRACIN) eicosapentaenoic acid C20: 5c)3, decosahexaenoic acid 1) Conjunctivitis, blepharitis C22: 6 (03, ricinoleic acid and derivatives thereof selected CHLORAMPHENICOL (AK-CHLOR, CHLORO from the group consisting of the C1 to C6 alkyl esters thereof, MYCETIN, CHLOROPTIC, OCU-CHLOR) the glycerol-polyethylene glycol esters thereof and the reac 1) Conjunctivitis, keratitis 60 tion product of hydrogenated natural oils composed largely of CHLORTETRACYCLINE HYDROCHLORIDE (AU ricinoleic acid based oils such as castor oil with ethylene REOMYCIN) oxide. 1) Conjunctivitis, blepharitis The administration medium may further include eicosap CIPROFLOXACIN HYDROCHLORIDE (CILOXAN) entaenoic acid C20:5c)3 and/or decosahexaenoic acid C22: 1) Conjunctivitis, keratitis 65 6c)3 as additional long chain fatty acids. ERYTHROMYCIN (AK-MYCIN, ILOTYCIN) The carrier solvent for the nitrous oxide gas may be water 1) Blepharitis, conjunctivitis or any of the pharmaceutically acceptable alcohols, ethers, US 8,937,074 B2 39 40 oils or polymers such as, for example a polyethylene glycol. Cs: 21.7% The oil may be organic or mineral oil. The organic oil may be Cs: 34.8% an essential oil based on long chain fatty acids having Cs: 28.0% between 14 and 22 carbon atoms in the fatty acid. The oil may >Cs: 1.6% also be of either natural or synthetic origin and, if of natural unknown: 2.1% origin, it may be either plant oil or animal oil. As plant oils It is further preferred to add to the formulation the long those rich in gammalinolenic acid GLA are preferred and as chain fatty acids known as eicosapentaenoic acid C20:5c)3 animal oil dairy cream may be used. and decosahexaenoic acid C22:6c)3. Such a product com In the preferred form of the invention the solution is an bination is available from Roche Lipid Technology under the aqueous solution saturated with nitrous oxide. 10 trade name “Ropufa 30 n-3 oil”. The water is preferably de-ionised water and free of It has been found by microscopic studies that the formula microbes. tion of active agents with a medium as herein described gives When the CPNS agent to be enhanced by means of the rise to the formation of minute, generally spherical bodies, in nitrous oxide is in a liquid formulation, Such formulation may which the active ingredient is contained in a stable form and incorporate as part of the administration medium water or 15 from which it is delivered at the site of action. acceptable other liquid solvent into which the nitrous oxide The CPNS agent utilised in the method or formulation and fatty acid or ester thereof had been dissolved or sus according to the present invention may comprise any one or pended or emulsified along with the CPNS agent to be more of the vast spectrum of CPNS agents as herein defined. enhanced by being formulated therewith. When the formula From amongst the CPNS agents falling within the scope of tion containing the CPNS agent to be enhanced by means of the definition set out above this invention is particularly con the nitrous oxide is to be in a liquid (including an encapsu cerned with the following Sub-classes of compounds: lated liquid) presentation for oral administration or in a nasal Central nervous system stimulants including central ana or bronchial or pulmonary spray or in the form of an inject leptics, psycho analeptics (antidepressants), respiratory able formulation, Such formulation may incorporate, as part stimulants, hallucinogenic medicines; of the administration medium, water or acceptable other liq 25 Central nervous system depressants including anaesthet uid into which the nitrous oxide is dissolved and in which the ics, sedatives, hypnotics, barbiturates, non-barbiturates, anti fatty acid or ester thereof is either dissolved or suspended or convulsants, (including anti-epileptics), tranquillisers (in emulsified along with the CPNS agent to be enhanced by cluding phenothiazines and their derivatives, rauwolfia, being formulated therewith. diphenylmethane and its derivatives, alkyl diols and their Likewise, where the CPNS agent is to be administered to 30 derivatives), centrally acting muscle relaxants; the patient by being applied as a topical, buccal or vaginal Local anaesthetics; cream or ointment, or as a cutaneous patch, or in the form of Medicines affecting autonomic functions including micro-depots, or as an intravenous, intramuscular or Subcu adrenomimetics (sympathomimetics), adrenolytics (sym taneous injection, or as a Suppository, the formulation used in patholytics), cholinomimetics (cholinergics), cholinolytics making up Such cream, ointment, patch, depots or injectable 35 (anticholinergics) (including anti-Parkinsonism prepara formulation or Suppository may incorporate, along with the tions), Ganglion blockers, anti-emetics and anti-vertigo CPNS agent to be enhanced, a quantity of water or other preparations, decongestants, -hydroxytryptamine (serotonin) liquid containing, and preferably saturated with, nitrous and serotonin antagonists, and anti-Alzheimers agents. oxide, the long chain fatty acid or ester thereof and a CPNS The invention has not yet been demonstrated by empirical agent conjugated therewith, and, further, such additional 40 work to be applicable to all the agents or classes of agents excipients and carriers as are conventionally used in the phar referred to herein. However in respect of such CPNS agents maceutical trade in making up Such dosage forms. which have already been formulated with the aforementioned The carrier solvent for the nitrous oxide gas may thus be administration medium of the invention, and evaluated by essentially non-aqueous and composed of the at least one different methods for the anticipated enhancement of action, fatty acid or ester thereof selected from the group consisting 45 no negative result has as yet been observed despite the chemi of oleic acid, linoleic acid, alpha-linolenic acid, gamma-lino cal diversity of the CPNS agents which has been investigated. lenic acid, arachidonic acid, eicosapentaenoic acid C20: The applicant thus confidently expects on the basis of these 5(03, decosahexaenoic acid C22: 603, ricinoleic acid and preliminary observations that the invention will find general derivatives thereof selected from the group consisting of the application across the entire spectrum of CPNS agents C1 to C6 alkyl esters thereof, the glycerol-polyethylene gly 50 embraced by these terms as herein defined and of which some col esters thereof and the reaction product of hydrogenated examples are set out herein. natural oils composed largely of ricinoleic acid based oils It is part of the applicant’s present postulations by which it with ethylene oxide, required to be part of the formulation. seeks to find an understanding of the invention and to which The essential fatty acid, or ester thereof, component of the it does not wish to be bound at this stage, that while the composition preferably comprises a mixture of esters of the 55 administration medium of the present invention serves to fatty acids listed above. Thus, in the most preferred form of transport the CPNS agent formulated therewith most effi the invention the fatty acid component of the composition is ciently through the human or animal body, that medium also constituted by the complex known as Vitamin F and in this plays an important role in transferring, by an as yet unex regard it is preferred to make use of the ester form of Vitamin plained mechanism, the CPNS agent through the membranes F known as Vitamin F Ethyl Ester. This product is commer 60 of and into the cells thereby to cause an effective CPNS cially available under the trade description of Vitamin FEthyl intercellular and/or intracellular concentration of the agent Ester CLR 110 000 Sh.L. U./g from CLR Chemicals Labo rapidly to be achieved, and to be maintained. ratorium Dr. Kurt Richter GmbH of Berlin, Germany. The It is in this respect that the applicant believes that the typical fatty acid distribution of this product is as follows: present invention will find general application despite the vast Acetophenazine, Chlorpromazine, Chlorprothixene, 10 Flupenthixol, Fluphenazine, Mesoridazine, Meth rated, Caffeine and Sodium Benzoate, Clomi otrimprazine, Pericyazine, Perphenazine, Pipotiaz pramine, Desipramine, Ephedrine, Imipramine, ine, Prochlorperazine, Promazine. Thiopropazate, Pemoline, Protryptiline, Thioproperazine. Thioridazine. Thiothixene, Trif ii. the following Psycho Analeptics (antidepressants): luoperazine, Trifluoropromazine, 1. the Tricyclic Antidepressants being: 15 a. Other Antipsychotics being: Amitryptyline, Amoxapine, Clomipramine, Clozapine, Fluspirilene, Haloperidol, Loxapine, Desipramine, Doxepin, Imipramine, Nortriptyline, Molindone, Olanzapine, Pimozide, Risperidone, Protriptyline, Trimipramine, Lithium, b. the Monamine Oxidase Inhibitors being: c. Centrally acting muscle relaxants consisting of Isocarboxazid, Phenelzine, Tranylcypromine, Baclofen, Carisoprodol, Chlorphenesin, ChlorZox c. Other Antidepressants being: aZone, Cyclobenzaprine, Dantrolene, Diazepam, Burpopion, Fluoxetine, Fluvoxamine, Maprotiline, Lorazepam, Metaxalone, Methocarbamol, Mitrazapine, Moclobemide, Nefazodone, Par Orphenadrine, and Orphenadrine citrate, Phenyloin, oxetine, Setraline, Trazodone, Venlafaxine, d. Local anaesthetics consisting of: iii. The following Respiratory Stimulants (Bronchodila 25 Articaine, Benzocaine, Bupivacaine, Chloroprocaine, tors): Cocaine, Diphenhydramine, Etidocaine, Lidocaine, Albuterol, Ephedrine, Ethylnorepinephrine, Fenot Mepivacaine, Pramoxine, Prilocalne, Procaine, Pro erol, Isoproterenol, Metaproteronol, Terbutaline, poxycaine, and Procaine, Proraracain, Ropivacaine, iv. The following Hallucinogenic medicines: Tetracaine, 1. the following Indoleamine hallucinogenics: LSD, 30 e. Medicines Affecting Autonomic Functions consisting DMT, N,N-dimethylamine, Psilocybin, of: a. the following Phenethylamines: i. The Adrenomimetics (Sympathomimetics) consisting Mescaline, Dimethoxymethylamphetamine of: (DOM), Methylenedioxyamphetamine (MDA), Phenylethylamine, Epinephrine, Norepinephrine, MDMA, 35 Dopamine, Dobutamine, Colterol, Ethylnorepi b. the Central nervous system depressants consisting of: nephrine, Isoproterenol, Isoetharine, Metaproter i. The following anaesthetics: enol, Terbutaline, Metaraminol, Clonidine, Phe Halothane, Isoflurane, Enflurane, Methoxyflurane, nylephrine, Tyramine, Hydroxyamphetamine, Sevoflurane, Desflurane, Methohexital. Thiopen Ritodrine, Prenalterol, Methoxamine, Albuterol, tal, Etomidate, Ketamine, Propofol, 40 Amphetamine, Methamphetamine, BenZphet ii. The following Sedatives and Hypnotics: amine, Ephedrine, Phenylpropanolamine, Alprazolam, Brotizolam, Chlordiazepoxide, Cloba Mephentermine, Phentermine, Fenfluramine, Pro Zam, Clonazepam, CloraZepate, Demoxepam, pylhexedrine, Diethylpropion, Phenmetrazine, Diazepam, Estazolam, Flumazenil, Flurazepam, Phendimetrazine, Halazepam, Lorazepam, Midazolam, Nitrazepam, 45 ii. The Adrenolytics (sympatholytics) consisting of: Nordazepam, Oxazepam, Prazepam, Quazepam, Phenoxybenzamine and related Haloalkylamines, Temazepam, Traizolam, Phentolamine, Prazosin, Terazosin, Doxazosin, iii. the Barbiturates being: TrimaZosin, Indoramine, Labetalol, Ketanserin, Amobarbital, Aprobarbital, Butabarbital, Butalbital, Urapidil, AlfuZosin, BunaZosin, Tamsulosin, Mephobarbital, Methohexital, Pentobarbital, Phe 50 Yohimbine, Propanolol, Metoprolol, Nadolol, nobarbital, Secobarbital. Thiopental, Atenolol, Timolol, Esmolol, Pindolol, Acebutolol, iv. the Non-barbiturates being: Labetalol, Bopindolol, Oxprenolol, Penbutolol, Buspirone, Chloral hydrate, Chlormezanone, Carvedilol, Medroxalol, Bucindolol, Levubunolol Diphenhydramine, Doxylamine, Ethchlovynol, (Betagan) glaucoma, Metipranolol, Bisoprolol. Ethinamate, Glutethemide, Hydroxyzine, Mep 55 Nebivolol, Betaxolol (Betoptic) Glaucoma, robamate, Methotrimeprazine, Methyprylon, iii. The Cholinomimetics (cholinergics) consisting of Promethazine, Propiomazine, Propofol, Zolpidem, Acetylcholine, Metacholine, Carbachol, Betanechol, Zolpiclone, Paraldehyde, Pilocarpine, Muscarine, Arecoline, Oxotremorine, V. The Anticonvulsants, (including anti-epileptics) Ambenonium, Domperidone, Edrophonium, Edro being: 60 phonium & Atropine, Metoclopramide, NeoStig Acetazolamide, Amobarbital, Carbamazepine, Clo mine, Physostigmine, Pyridostigmine, bazam, Clonazepam, CloraZepate, Corticotropin, iv. The Cholinolytics (anticholinergics) (including anti Diazepam, Divalproex, Ethosuximate, Ethotoin, Parkinsonism preparations) consisting of: Felbamate, Fosphytoin, Gabapentin, Lorazepam, Amantadine, Anisotropine, Atropine, Scopolamine Magnesium sulfate, Mephenyloin, Mephobarbital, 65 and related Belladonna alkaloids, Ipratropium bro Metharbital, Methsuximide, Nitrazepam, Paralde mide, Benztropine, piperidine, Chlorpromazine, hyde, Paramethadione, Pentobarbital, Phenace Clidinium, Dicyclomine, Diphenhydramine, Etho US 8,937,074 B2 43 44 propazine, Glycopyrollate, Homatropine, Hyos (iii) Nucleic Acid Transportation and Administration cyamine, Mepenzolate, Methantheline, Methoctra According to the present invention there is also provided a mine, Hexahydrosiladifenidol, Himbacine, method for the administration of a nucleic acid Substance to Tripitamine, Methscopolamine, Orphenadrine the cells of an animal, a plant or a micro-organism the method HCl, Pirenzepine, Procyclidine, Propantheline, being characterised in that the nucleic acid Substance is for mulated with an administration medium which comprises a Scopolamine. Thioridazine, Trihexyphenidyl, Car Solution of nitrous oxide gas in a physiologically acceptable bidopa and Levodopa, Levodopa, Pergolide, Sel carrier solvent for the gas and which administration medium egiline, includes at least one fatty acid or ester or other suitable V. Ganglion blockers consisting of derivative thereof selected from the group consisting of oleic Hexamethonium, Trimethaphan, Mecamylamine, 10 acid, linoleic acid, alpha-linolenic acid, gamma-linolenic vi. Anti-emetics and AntiVertigo preparations consisting acid, arachidonic acid, eicosapentaenoic acid C20: 5c)3. of: decosahexaenoic acid C22: 603, ricinoleic acid and deriva 5-HT Antagonists as Ondansetron, Granisetron, Tro tives thereof selected from the group consisting of the C1 to pisetron, Dolasetron, D2/5-HT Antagonist as C6 alkyl esters thereof, the glycerol-polyethylene glycol 15 esters thereof and the reaction product of hydrogenated natu Metoclopramide, Trimethobezamide, D2 Antago ral oils composed largely of ricinoleic acid based oils, such as nists as Phenotiazines namely Chlorpromazine, castor oil, with ethylene oxide. Perphenazine, Triflupromazine, D2 Antagonists as According to a further aspect of the present invention there Benzimidazole derivatives namely Domperidone, is provided a preparation adapted for use in introducing a D2 Antagonists as Butyrophenones namely Halo nucleic acid Substance into the cells of an animal, a plant or a peridol, Droperidol, Corticosteroids as Dexam micro-organism characterised in that the preparation com ethasone, Methylprednisolone, Cannabinoids as prises a formulation of the nucleic acid in an administration Dronabinol, Nabilone, H antagonists Diphenhy medium which comprises a solution of nitrous oxide gas in a dramine, Meclizine, Cyclizine, Antimuscarinic physiologically acceptable carrier Solvent for the gas and agents as Scopolamine, BenZitropiane, BenZodiaz 25 which administration medium includes at least one fatty acid epines as Lorazepam, Alprazolam, H. Antagonist or ester or other suitable derivative thereof selected from the as Dimenhydrinate, group consisting of oleic acid, linoleic acid, alpha-linolenic vii. Decongestants consisting of: acid, gamma-linolenic acid, arachidonic acid, eicosapen Oxymetazoline, Phenylephrine, Xylometazoline taenoic acid C20: 5c)3, decosahexaenoic acid C22: 6c)3. viii. -hydroxytryptamine (serotonin) and serotonin 30 ricinoleic acid and derivatives thereof selected from the group antagonists consisting of consisting of the C1 to C6 alkyl esters thereof, the glycerol 1. 5-HT Agonists being: polyethylene glycol esters thereof and the reaction product of Buspirone, ipsaperone, Sumatriptan, Cisapride, hydrogenated natural oils composed largely of ricinoleic acid 2. 5-HT Antagonists being: based oils, such as castor oil with ethylene oxide. Methysergide, Risperidone, Ketanserin, 35 The administration medium preferably includes the eicosa Ondansetron, pentaenoic acid C20: 5c)3 and/or decosahexaenoic acid 3. 5-HT transport inhibitors being: C22: 6003 as additional long chain fatty acids to at least one Fluoxetine, Sentraline, of the other components of the carrier medium defined above. f. Anti-Alzheimers agents consisting of: The reaction product of hydrogenated natural oils com Physostigmine, Tacrine and Lecithin in combination 40 posed largely of ricinoleic acid based oils with ethylene oxide with Tacrine. is preferably produced from castor oil of which the fatty acid An agent of specific relevance in this application is doxepin content is known to be predominantly composed of ricinoleic hydrochloride. Doxepin HCl is one of a class of psychothera acid. This product is known as PEG-n-Hydrogenated Castor peutic agents known as dibenZOXepin tricyclic compounds. Oil. A range of such products is marketed by BASF under the Specifically, it is an isomeric mixture of 1-Propanamine.3- 45 trade description of Cremaphor RH grades. Glycerol-poly dibenz beloxepin-1, 1(6H)ylideneN,N-dimethyl-hydrochlo ethylene glycol ester of ricinoleic acid is also marketed by the ride. In oral formulation it is useful as an antidepressant to same company but under the trade description of Cremaphor relieve symptoms of depression and anxiety Such as feelings EL. of sadness, worthlessness, or guilt; loss of interest in daily The carrier solvent for the nitrous oxide gas may be water activities; changes in appetite; tiredness; sleeping too much; 50 or any of the pharmaceutically acceptable alcohols, ethers, insomnia, and thoughts of death or Suicide. Doxepin is also oils or polymers such as a polyethyleneglycolor the like. The Sometimes used to treat certain types of pain but it is not oil may be organic or mineral oil. The organic oil may be an generally regarded as an analgesic in the ordinary sense of the essential oil based on long chain fatty acids having between word and has been described as one of a group of 'adjuvant 14 and 22 carbon atoms in the fatty acid. The oil may also be analgesics” along with other anti-depressants. In a topical 55 of either natural or synthetic origin and, if of natural origin, it formulation, doxepin is known to be used as an anti-pruritic to may be either plant oil or animal oil. As plant oils those rich relieve itching in patients with certain types of eczema. The in gamma linolenic acid GLA are preferred and as animal applicant is unaware of any prior disclosure suggesting that oil dairy cream may be used. doxepin may beneficially be used in a topical administration In the preferred form of the invention the solution is an form for alleviating pain. 60 aqueous solution saturated with nitrous oxide. Preferably the The applicant found that a potent topical formulation may water is deionised and purified to be free of microbes. be prepared according to the invention by formulating dox When the formulation containing the nucleic acid sub ipen with the medium as described. stance to be enhanced by means of the nitrous oxide is to be in It is accordingly an aspect of the present invention to pro a liquid (including an encapsulated liquid) presentation for vide a topical formulation, used either as a lotion or a cuta 65 oral administration or in a nasal or bronchial or pulmonary neous patch as described above in which doxepin is the active spray or in the form of an injectable formulation, Such for ingredient. mulation may incorporate, as part of the administration US 8,937,074 B2 45 46 medium, water or acceptable other liquid into which the The nucleic acid substance utilised in the method or for nitrous oxide is dissolved and in which the fatty acid or ester mulation according to the present invention may comprise thereof is either dissolved or suspended or emulsified along any one or more of the vast spectrum of nucleic acid Sub with the nucleic acid substance to be enhanced by being stances as herein defined. formulated therewith. In a preferred form of the invention the nucleic acid sub Likewise, where the nucleic acid Substance is to be admin stance is selected from the group comprising: DNA, RNA, istered to the patient as a topical, buccal or vaginal cream or DNA-RNA hybrids, oligonucleotides and synthetic nucleic ointment, or as a Suppository, the formulation used in making acids. up Such cream, ointment, or Suppository may incorporate, The invention has not yet been demonstrated by empirical 10 work to be applicable to all the agents or classes of agents along with the nucleic acid Substance to be enhanced, a quan referred to herein. However in respect of such nucleic acid tity of water or other liquid containing, and preferably Satu substances which have already been formulated with the rated with, nitrous oxide, the long chain fatty acid or ester aforementioned administration medium of the invention, and thereof and the nucleic acid substance formulated therewith, evaluated by different methods for the anticipated enhance and, further, such additional excipients and carriers as are 15 ment of action, no negative result has as yet been seen despite conventionally used in the pharmaceutical trade in making up the chemical and physical diversity of the nucleic acid sub Such dosage forms. stances which have been investigated. The applicant thus The carrier Solvent for the nitrous oxide gas may thus in an confidently expects on the basis of these preliminary obser alternative formulation according to the invention be essen Vations that the invention will find general application across tially non-aqueous and composed of the least one fatty acid or the entire spectrum of the nucleic acid substances embraced ester thereof selected from the group consisting of oleic acid, by the term as herein defined and of which some examples are linoleic acid, alpha-linolenic acid, gamma-linolenic acid, set out herein. arachidonic acid, eicosapentaenoic acid C20: 5c)3, decosa It is part of the applicant’s present postulations by which it hexaenoic acid C22: 603, ricinoleic acid and derivatives seeks to find an understanding of the invention and to which thereof selected from the group consisting of the C1 to C6 25 it does not wish to be bound at this stage, that while the alkyl esters thereof, the glycerol-polyethylene glycol esters administration medium of the present invention serves to thereof and the reaction product of hydrogenated natural oils transport the nucleic acid substance formulated therewith composed largely of ricinoleic acid based oils with ethylene most efficiently through the human or animal body, that oxide, required to be part of the formulation. medium also plays an important role in transferring, by an as 30 yet unexplained mechanism, the nucleic acid Substance A formulation Suited to transdermal application whether as through the membranes of and into the cells thereby to cause an ointment, cream or lotion or in the form of a skin patch an effective nucleic acid substance inter- and intracellular providing a reservoir for the formulation is also a preferred concentration of the agent rapidly to be achieved, and to be form of the formulation according to the invention. maintained. It is in this respect that the applicant believes that The essential fatty acid, or ester thereof, component of the 35 the present invention will find general application. composition preferably comprises a mixture of esters of the From amongst the nucleic acid Substances in the form of fatty acids listed above. Thus, in the most preferred form of DNA with which this invention is concerned it relates par the invention the fatty acid component of the composition is ticularly to DNA fragments making up genes or parts thereof, constituted by the complex known as Vitamin F and in this as are used in gene therapy, and in particular regard it is preferred to make use of the ester form of Vitamin 40 a) to Supplement defective genes with correct genes in F known as Vitamin F Ethyl Ester. This product is commer order to affect the outcome of diseases, as are used in cially available under the trade description of Vitamin FEthyl non-viral gene therapy; or Ester CLR 110 000 Sh.L. U./g from CLR Chemicals Labo b) to transfect cells with exogenous DNA to enable expres ratorium Dr. Kurt Richter GmbH of Berlin, Germany. The sion of Such exogenous DNA as proteins, as are used in typical fatty acid distribution of this product is as follows: 45 biotechnology; or c) to interfere in transcription of genomic DNA, as in C: 8.3% therapy of diseases where over-expression of genetic Clso: 3.5% material is a causative effect; or Cs: 21.7% d) to change the regulatory control of the expression of 50 genes, as it may relate to Some malignant and other Cs: 34.8 diseases where regulation of gene expression is caus Cs: 28.0% ative; or >Cs: 1.6% e) to transportantisense bacterial, viral or parasitic DNA or unknown: 2.1% oligonucleotides into cells, there to have an inhibitory or It is further preferred to add to the formulation the long 55 stimulatory action on the growth of Such infectious chain fatty acids known as eicosapentaenoic acid C20:5c)3 agents or a degradative action against the genome or the and decosahexaenoic acid C22:6c)3. Such a product com mRNA of the agent. bination is available from Roche Lipid Technology under the The nucleic acid substance used according to this invention trade name “Ropufa 30 n-3 oil”. may accordingly be those coding for the expression of any It has been found by microscopic studies that the formula 60 desired protein, or protein classes. The invention is thus appli tion of the nucleic acid substance with a medium as herein cable to the administration of nucleic acid sequences coding described gives rise to the formation of minute, generally for any one of the following protein classes: spherical bodies, within which, or attached to which the chemokines active ingredient is contained in a stable form and from which chemotactins it is delivered at the site of action namely on or inside the cell 65 cytokines in or from the animal body or plant or micro-organism to enzymes which it is administered. gonadotrophins US 8,937,074 B2 47 48 growth factors peptide selective for a selected receptor on a target cell. Such immunoglobulins peptide being bound to at least one of the fatty acids or interferons derivatives thereof in the composition. By the presence of interleukins Such peptide the nanolipid vesicle with its charge of a nucleic lipid-binding proteins acid substance such as DNA may be caused to be targeted to pituitary hormones a specific site in the animal or plant body or in the cells so as protease inhibitors to dock at the desired locus for the release of that charge. proteases Preliminary Hypotheses of Mechanism of Operation Somatomedins The mechanism by which the enhancement of action of More particularly the nucleic acid sequence may be 10 selected to code for any one of the following specific proteins, anti-infective drugs and CPNS drugs, and the nucleic acid namely: transportation and delivery is achieved by the present inven adenosine deamidase, tion, is currently under investigation. Some observations in bovine growth hormone (BGH) this regard have been recorded above. In addition it is brain-derived neurite factor (BDNF) 15 recorded that preliminary observations point to Some addi calcitonin tional possible explanations. The applicant again does not calcitonin wish to be bound to any of the tentative explanations it may carboxypeptidase put forward at this time. It is recorded, however, that it would catalase appear that the long chain fatty acids used in the formulation cholecystokinin (CCK) of the preparation according to the invention, or at least some chymotrypsin of these components, form, during the manufacturing process ciliary neurite transforming factor (CNTF) of the medicinal formulation, very small spherical bodies, clotting factor VIII hereinafter referred to as "nanolipid vesicles”. These nano clotting factor VIII lipid vesicles have dynamic characteristics in respect of the elastase 25 encapsulation and Subsequent delivery of compounds at pre erythropoietin (EPO) dicted areas in cells and organisms where the optimal utilisa fibroblast growth factor (acidic or arginase basic) tion of these compounds occur with resultant maximised glucagon modes of actions. glucagon-like-peptide I (GLP-1) The present model for the understanding of the invention is granulocyte colony stimulating factor gamma.-interferon 30 that the dynamic delivery characteristics of the nanolipid (G-CSF) vesicles are utilised efficiently to transport compounds to granulocyte macrophage colony Stimulating factor locations where maintenance of optimal concentrations in the human growth hormone (hGH) organisms is beneficial in combating specific infective dis IL-1 eases. The same would appear to apply or diseases responsive IL-1RA 35 to CPNS agents. The cells in this application most probably IL-2 concern neurons. The long chain fatty acids may contribute to Insulin the maintenance of the myelin sheath of the nervous system, insulin-like growth factor-1 (IGF-1) being precursors of the sphingomeilin molecules. The insulinotrophic hormone dynamic delivery characteristics of the nanolipid vesicles are insulintropin 40 apparently also utilised efficiently to transport nucleic acid interferon-alpha.2B compounds to locations where maintenance of optimal con intrinsic factor centrations in the organisms or cells is beneficial in combat lactase ing specific genetic, acquired and infective diseases. L-asparaginase Infectious diseases, especially those which are known to neurite growth factor (NGF) 45 develop resistance to compounds are known to be difficult to Ob gene product treat due to insufficient penetration of the compound into the parathyroid hormone (PTH)-like hormone causative microorganisms. These, or at least some of these pepsin appear to be particularly suited for the benefits of the present phenylalanine ammonia lyase invention. platelet derived growth factor interferon-alpha.2A 50 The composition of the invention has thus been found to (PDGF) have beneficial drug delivery effects when exposed to cells streptokinase and causative organisms harboured by Such cells. SCaS These beneficial effects are believed to be attributable to superoxide dismutase (SOD) the dynamic characteristics of the nanolipid vesicles. The thrombopoietin (TPO), 55 current hypothesis is that these characteristics include: tissue necrosis factor (TNF) 1. The Structural Characteristics of the Formulation of the tissue plasminogen activator (tPA) Preparation: transforming growth factor (TGF) Nitrous oxide and the unsaturated long chain fatty acids trypsin forming part of the administration medium are formu uricase 60 lated by being mixed with designated anti-infective urokinase agents or compounds to form the nanolipid vesicles The disease conditions which are amenable to treatment containing the compound or anti-infective agent. Two using the present invention will be readily apparent to those important observations have been made in this regard: skilled in the art and include those listed by German etal in the a) It was found that when the unsaturated long chain fatty aforementioned U.S. Pat. No. 6,258,789. 65 acids used are 20 carbons or more, the nanolipid vesicles The method and composition of the invention may further form spherical structures with Sub-compartments simi be characterised in that the carrier composition may include a lar to those seen in a sponge. US 8,937,074 B2 49 50 These structures are stable and it is our belief that antibod They can also combine to resize themselves continuously ies or other ligands would fit ideally in these sub-com without detriment to their stability. The inter-lipid rela partments so that the nanolipid vesicles bind to specific tionship is also revealed when moving through the cel epitopes or receptors at the target cell Surface. lular membrane. b) When unsaturated long chain fatty acids of 16 to 20 5 These interactive membrane characteristics make the carbons are used, the form of the nanolipid vesicles is movement of the vesicles through the cells optimal. spherical with a dynamic field of moving autofluores Although inter-relationships of the dynamic nanolipid cent particles Surrounding the vesicles. vesicles are continuously present, it has also been shown When nitrous oxide is omitted from the process the moving that the particles are stable in blood and body fluids for particles Surrounding the nanolipid vesicles move errati 10 up to 5 hours. cally and asymmetrical movements are then detected. It is believed that nitrous oxide is essential instabilising the EXAMPLES OF THE INVENTION moving autofluorescent particles Surrounding the nano lipid-vesicles, which is an essential characteristic to effi Without thereby limiting the scope of the invention some cient compound delivery. 15 examples will now be described to illustrate the invention. Preparations which do not as such form part of the claimed 2. Stability: Subject matter of this application, being preparations first The nanolipid vesicles appear to remain structurally intact disclosed in the applicants own prior patents referred to after 24 months at room temperature. Any encapsulated above, are first restated. Thereafter follows detailed disclo active compounds remain encapsulated during this time. Sures of preparations falling within the scope of the invention This stability feature is believed to be of substantial an then follows some illustrations of the utility of the inven significance and one of the contributing factors for the tion and of characteristics of the preparations according to the enhancement observed. invention. 3. Absence of Cytotoxicity: The nanolipid vesicles have no apparent cytotoxicity. 25 Preparation 1 When applied to cells in culture, at applicable concen trations they appear rather to have a beneficial effect on Preparing an Aqueous Nitrous Oxide Solution normal cell growth. 4. Mechanism of Action: A pressure vessel is charged to its operating Volume with 4.1 Loading efficiency: 30 water at 20° C. ambient temperature. The vessel is con The high loading efficiency of nanolipid vesicles has nected to a supply of nitrous oxide via a flow control valve and been demonstrated by achieving a high degree of pressure regulator. The closed vessel is supplied with nitrous encapsulation of a wide range of active drugs. oxide at a pressure of 2 bar for a period of 48 hours, it having 4.2 Transport: been determined that at the aforementioned temperature the The nanolipid vesicles behave as a transport mechanism 35 water is saturated with nitrous oxide over such period of time to carry molecules such as active compounds. under the abovementioned pressure. 4.3 Release: A resultant solution is bottled as stock solution for use in It has been shown that the nanolipid vesicles have very the formulations and applications set out below. high delivery efficiencies. The high delivery effi ciency relates to tissue penetration, cell adsorption, 40 Preparation 2 internalisation of nanolipid vesicles by cells, parasites and bacteria, intra-cellular stability, and Subsequent Preparation of Nitrous Oxide/Vitamin F Aqueous sub-cellular organelle delivery. Emulsion The result of high delivery efficiency is the release of active compounds not only at membrane sites, but 45 30g Vitamin Fethyl ester as identified and described above also at intracellular sites including the nuclei of viable was mixed with 10 g Cremophor RH40 (which is the trade cells or microorganisms. The result is an enhanced name used by BASF for a product which it describes as the efficacy of said active compound. The nanolipid reaction product of hydrogenated castor oil with ethylene vesicles and active compound appear to act synergis oxide which product is also known by the INCI name as tically in attaining enhanced efficacy. 50 PEG-n-Hydrogenated Castor Oil), 2.2g methyl paraben, 0.08 5. Elasticity: g butyl hydroxyanisole, 0.23 g butyl hydroxytoluene with Confocal laser Scanning microscopy (CLSM) shows that stirring at 80° C. the conformation of nanolipid vesicles can be changed Into 942.5 g of the stock nitrous oxide solution was dis while in movement. solved 2.5 g sodium propyl paraben and 2.5 g Germall 115 When the vesicles move through membranes, the confor 55 Imidurea with stirring at room temperature. mation changes so that the intracellular nanolipid The oily composition first described was emulsified into vesicles may have other morphological characteristics. the aqueous solution with stirring to constitute a stock nitrous While moving through membranes the nanolipid vesicles oxide/Vitamin F emulsion. It is herein referred to as “Lindil', feed the membranes with unsaturated long chain fatty “MZL or “nanolipid-vesicle' formulation. acids which in its turn will have a positive effect on 60 membrane bound processes. This process has a positive Preparation 3 effect on the metabolism of the cell and thus the survival of the cells. Preparation of a Non-Aqueous Solution of Nitrous 6. Dynamic Inter-Lipid Vesicle Relationships: Oxide in Carrier Formulation It has been shown that vesicle inter-lipid relationships do 65 exist. The lipid vesicles, can interchange the compounds The Manufacturing Process for producing a non-aqueous they respectively carry. formulation of an anti-infective agent according to the inven US 8,937,074 B2 51 52 tion will now be described with reference to the manufacture Preparation 4 of a first anti-TB medicament containing Pyrazinamide as active ingredient and a second anti-TB medicament contain Injectable Solution of Active in an Injectable ing the combination of Rifampicin, Isoniazid and Ethambutol Solution, Containing Nitrous Oxide in Carrier as active ingredients. These products are respectively desig Formulation nated as “Preparation P” and “Preparation RIE’ herein. Preparation P was Made Up According to the Following The manufacturing process for producing an injectable Protocol: formulation of an active agent according to the invention will Step 1: Weigh off the Pyrazinamide 5.00 Kg and reduce the now be described with reference to the manufacture of a 10 medicament containing Bupivacaine as active ingredient particle size to less than 40 um. Preparation X was Made Up According to the Following Step 2: Weigh off and add together the Poloxyl hydrogenated Protocol: castor oil 1.15 Kg, Vitamin F ethyl ester 2.35 Kg, d1-C.- Step 1: Weigh off the 546 g of Bupivicaine HCl and add to the Tocopherol 150.0 g and Polyethylene glycol 400 1,295 Kg pot. Mix thoroughly with NO-saturated water until the into mixing pot 2 and heat to approximately 40°C. until all 15 active compound is completely dissolved. the oil has melted. Step 2: Weigh off and add together the Poloxyl hydrogenated Step 3: Gas the oil mixture with nitrous oxide for 3 hours at 2 castor oil 1,023 Kg, Vitamin F ethyl ester 3.012 Kg, into bar in the stainless steel pressure vessel in the manner mixing pot 2 and heat to approximately 70° C. until all the described in Preparation 1 above. oil has melted. Step 4: Transfer the gassed mixture to mixing pot 1 and heat Step 3: Weigh off and add the Methyl paraben 214 g and the to approximately 70° C. Butylated hydroxytoluene 36 g to mixing pot 2, in the Step 5: Weigh off and add the Methyl paraben 50.00 g and the respective order, while continuously mixing ensuring each Butylated hydroxytoluene 5.00 g to mixing pot 1, while solid is dissolved before adding the next while still main continuously mixing ensuring each Solid is dissolved taining the temperature at 70° C. before adding the next while still maintaining the tempera 25 Step 4: Combine the contents of Step 1 and Step 3 whilst ture at 70° C. stirring during addition and continue stirring until the Step 6: Remove from the heat and allow to cool down to phases are well and truly mixed. Determine and adjust the approximately 40°C. pH to near physiological pH if necessary. Step 7: Add the Pyrazinamide 5.00 Kg stepwise while con Step 5: Sonicate and check vesicle size. 30 Step 6: Sterilise by filtration and pack into amber glass tinuously mixing. ampoules or vials. Step 8: Gas the mixture from step 7 with nitrous oxide at 20 kPa for 30 minutes with mixing until the mixture has Preparation 5 reached room temperature. Preparation RIE was Made Up According to the Following 35 Preparation of a Typical Formulation Having a Protocol: Non-Aqueous Solution of Nitrous Oxide in Carrier Step 1: Weigh off the Rifampicin 1467 Kg, Isoniazid 733.00 Formulation g and Ethambutol 2.20 Kg and reduce the particle size to less than 40 Lum. Ensure that the raw material is protected The manufacturing process for producing a non-aqueous from exposure to light at all times. 40 formulation of an CPNS agent according to the invention is Step 2: Weigh off and add together the Poloxyl hydrogenated based on the principles and ratios of ingredient described with castor oil 1.33 Kg, Vitamin F ethyl ester 3.11 Kg and reference to the manufacture of a medicament containing for Polyethylene glycol 400 1.30 Kg into mixing pot 2 and heat example Phenythoin Na as active ingredient to approximately 40°C. until all the oil has melted. Preparation Y was Made Up Based on the Following Pro 45 tocol: Step 3: Gas the oil mixture with nitrous oxide for 3 hours at 2 Step 1: Weigh of the applicable weight of Phenyloin Na for bar in the stainless steel pressure vessel in the manner as the purpose required, i.e. age of patients, severity of described above. condition. Reduce the particle size to less than 60 um. Step 4: Transfer to mixing pot 1 and continue to mix. Step 2: Weigh off and add together the Poloxyl hydroge Step 5: Weigh off and add the Methyl paraben 50.00 g, Ascor 50 nated castor oil to a final concentration of 11.5% (w/w) byl palmitate 10.00 g and the Butylated hydroxytoluene Vitamin F ethyl ester to a final concentration of 23.5% 5.00 g to mixing pot 1, while continuously mixing ensuring (w/w) and Polyethylene glycol 400 to a final concentra each solid is dissolved before adding the next. tion of 12% (W/w) into mixing pot 2 and heat to approxi Step 6: Remove from the heat and allow to cool down to mately 40° C. until all the oil has melted. dl-O-Toco approximately 40°C. 55 pherol may be added depending on the physico Step 7: Check the pH and add Potassium hydroxide while chemical characteristics of the active compound, the rate continuously mixing until the pH reads 7. of release and period of release needed, Eicosapen Step 8: Add the Rifampicin 1467 Kg, Isoniazid 733.00 g and taenoic acid and/or decosahexaenoic acid may be added Ethambutol 2.20 Kg, respectively, each stepwise allowing at this step. 60 Step 3: Gas the oil mixture with nitrous oxide for 3 hours at mixing after each addition. 2 bar in the stainless steel pressure vessel in the manner Step 9: Gas with nitrous oxide at 20 kPa for 30 minutes with described in Preparation 1 above. mixing until the mixture has reached room temperature. Step 4: Transfer the gassed mixture to mixing pot 1 and The Preparation P and Preparation RIE formulations were heat to approximately 70° C. encapsulated in Softgel capsules in the manner well known in 65 Step 5: Weigh off and add the Methyl paraben to a final the pharmaceutical trade as oral capsules for use as described volume of 0.5% and the Butylated hydroxytoluene below. 0.05% to mixing pot 1, while continuously mixing US 8,937,074 B2 53 54 ensuring each solid is dissolved before adding the next Example 1 while still maintaining the temperature at 70° C. Step 6: Remove from the heat and allow to cool down to Enhancement of Anti-Bacterial Action approximately 40°C. Step 7: Add the required amount of Phenyloin Na stepwise 5 This example pertains to the enhancement of current treat while continuously mixing. ment modalities of infectious bacterial diseases. Step 8: Gas the mixture from step 7 with nitrous oxide at 20 The increased efficacy of antibiotics carried by nanolipid kPa for 30 minutes with mixing until the mixture has vesicle in the treatment of bacterial infectious diseases was reached room temperature. demonstrated by: 10 (a) and (b) Bacterial culture studies (Bactec studies and con The Phenyloin Na Preparation prepared as set out above focal laser Scanning microscopy CLSM); was encapsulated in Soft gel capsules in the manner well (c) Infection studies involving live confocal laser Scanning known in the pharmaceutical trade as oral capsules for use as microscopy (CLSM) studies; and described below. (d) Zone of Inhibition studies according to the USP XXIII 15 Zone inhibition method. Preparation 6 The following organisms considered to be representative and hence demonstrative albeit not exhaustive of the range of Preparation of a Typical Formulation Having a organisms to which the invention relates, were used in the Non-Aqueous Solution of Nitrous Oxide in the above studies to confirm the invention: Carrier Formulation Mycobacteria Tuberculosis (ref strain H37RV) Mycobacteria Tuberculosis (MDR strains V79 & V25) The manufacturing process for producing a non-aqueous Bacillus of Calmette and Guerin (BCG) formulation of a nucleic acid Substance according to the E. Coli invention is based on the principles and ratios of the ingredi S. Aureus ents described with reference to the manufacture of a formu 25 P Aeruginosa lation containing the dedicated nucleic acids, which can be a B. Cereus gene or part thereof or a genome or part thereofas in the case A. Niger of DNA vaccines, or ODN's as in the case of the targeting of C. Albicans infectious agents as active ingredient. The anti-infective agents in the form of anti-bacterials used The non-aqueous nucleic acid preparation was made up 30 in these studies, and again considered to be representative and based on the following protocol: illustrative of the wide application of the invention, albeit not Step 1: Weigh of the applicable weight of nucleic acids as exhaustive, were Rifampicin, Ethambutol, Izoniazid, defined above for the purpose required. Pyrazinimide and Povidone-iodine, Cloxacillin, Erythromy Step 2: Weigh off and add together the Poloxyl hydroge cin E, Ciprofloxacin, Co-trimoxazole (Sulfamethoxazole and nated castor oil to a final concentration of 11.5% (w/w) 35 Trimethoprim combination) and Itraconazole. Vitamin F ethyl ester to a final concentration of 23.5% As will appear from the results discussed below the tests (w/w) and Polyethylene glycol 400 to a final concentra conducted on formulations involving the association of the tion of 12% (W/w) into mixing pot 2 and heat to approxi active ingredient with the carrier prepared as set out in Prepa mately 40° C. until all the oil has melted. dl-O-Toco ration 2 above, showed a 10 to 40-fold enhancement of the pherol may be added depending on the physico 40 efficacy of selected anti-infective agents compared to conven chemical characteristics of the active compound, the rate tional formulations thereof. of release and period of release needed. Eicosapen A. Bacterial culture studies: taenoic acid and/or decosahexaenoic acid may be added (a) The effect of the nanolipid vesicle encapsulation on the at this step. action of known anti-mycobacterials in BACTEC determi Step 3: Gas the oil mixture with nitrous oxide for 3 hours at 45 nations. 2bar in the stainless steel pressure, vessel in the manner (i) General Methodology of Collection and cultivation of described in Preparation 1 above. Samples and Evaluation Step 4: Transfer the gassed mixture to mixing pot 1 and Clinical isolates of M. tuberculosis banked at the Medical heat to approximately 70° C. School at Tygerberg Hospital were cultured on L-J slant Step 5: Weigh off and add the Methyl paraben to a final 50 cultures and used for BACTEC analysis. Drug sensitiv volume of 0.5% and the Butylated hydroxytoluene ity determinations in respect of the strains were done. M. 0.05% to mixing pot 1, while continuously mixing tuberculosis strains were selected from a bank of 1800 ensuring each solid is dissolved before adding the next clinical isolates genotyped according to their IS6110 while still maintaining the temperature at 70° C. insertion sequence profiles. The insertion sequence Step 6: Remove from the heat and allow to cool down to 55 ranges from 1 to 23 copies per Strain. These strains have approximately 40°C. been clustered into families according to their genetic Step 7: Add the required amount of stepwise while con patterns and represent recent transmission clusters tinuously mixing. because of their most frequent appearance in the com Step 8: Gas the mixture from step 7 with nitrous oxide at 20 munity. These strains may also represent more virulent kPa for 30 minutes with mixing until the mixture has 60 strains although virulence factors in M.Tb. have not yet reached room temperature. been clarified. The Nucleic acid Preparation prepared as set out above was The mycobacterial strains were carefully selected accord encapsulated in Softgel capsules in the manner well known in ing to their genetic and epidemiological type. Multidrug the pharmaceutical trade as oral capsules. This preparation resistant (MDR) strains were selected over a wide range may be further diluted with water preferably saturated with 65 from mildly resistant to highly resistant for Isoniazid, nitrous oxide to obtain the desired ratios of nanolipid vesicle Rifampicin, Ethambutoland Pyrazinamide. Clinical and and nucleic acids. laboratory strains of M. tuberculosis were cultured in a US 8,937,074 B2 55 56 medium enriched with ADC enrichment medium with Lindil alone has no bactericidal effect at the concentra continuous stirring to ensure homogenous bacterial dis tion employed in this determination. tribution and uniform aeration as described by Middle It was further found that the Rifampicin resistant strain brook, G. (1977) in Automatable radiometric detection used displayed increased Rifampicin sensitivity when in growth of mycobacterium tuberculosis in selective 5 media.” Ann. Rev. Respir. Dis. 115:1066-1069. Under Rifampicin is delivered to the bacilli via Lindil. these conditions, cultures grow reproducibly (<1.0% These results were considered to be very important. It has difference). This technique was established in the Medi now been shown that Lindil by itself has anti-mycobac cal Biochemistry laboratory at Tygerberg Hospital. terial activity. The observation that Lindil may have an At a culture density of approximately A600 nm=0.16 (1x 10 effect on M.Tb. alone could only be extrapolated from McFarland), (Siddiqui, S. H. (1995). BACTEC 460 results over the first 24 hours. It would appear that only after a 1:128 dilution of Lindil, growth of M.Tb. is the MTB system. Product and procedure manual.) M.Tb. same as for the untreated control. strain cultures were inoculated into BACTEC vials. Cul tures in BACTEC were grown until a growth index (GI) 15 It was further found that the application of Lindil/Rifampi cin combinations to the Rifampicin resistant strain of 500(+50) was reached. This culture was used as TV79 resulted in an inhibition of the same order as was starter culture for BACTEC evaluation of carrier encap observed with the Rifampicin resistant patient isolates sulated antimycobacterial drugs. BACTEC growth of which displayed inhibition by Lindil/Rifampicin at a cultures was monitored over a period of 6-10 days and concentration of 0.5ug/ml Lindil. the AGI value for every 24-hour doubling period deter mined. Every series of experiments were repeated at (iii) Evaluation in respect of Isoniazid “INH' least 4 times to allow for accurate statistical analyses. A quantity of MZL as prepared in Preparation 2 was filter Controls, with no drugs, were brought to the same con sterilized through a 0.22 uM syringe filter (Millipore). 25 MZL was diluted in the ratios of 1:2, 1:8, and 1:32. centrations. Sterility of mycobacterial cultures were These dilutions ranged from an inhibitory effect on the monitored by Ziehl-Nielsen staining. viability of M.Tb. to slight inhibition on the viability of All experiments with infectious material should be carried M.Tb. These effects were observed with serial dilutions out in a category 3 bio-safety laboratory. All experi of M.Tb. in BACTEC. ments should be carried out in Such a way as to ensure 30 INH was reconstituted with MZL dilutions in two ways: maximum safety for all other laboratory co-workers. First, dilutions of MZL were made as described before, (ii) Evaluation in respect of Rifampicin and INH was added to the dilutions to give a final con Lindil, also referred to as the nanolipid vesicle formulation centration of 0.1 ug INH/ml MZL per BACTEC vial (4 was prepared as described in Preparation 2 above and ml content). Secondly, INH was reconstituted with undi Rifampicin dissolved at a concentration of 80 micro 35 luted MZL to give a final concentration of 0.1 ug INH grams per ml, was used in an initial evaluation of the per ml MZL. The viability of drug resistant strain TV25 effect of the preparation on M. tuberculosis cultures in was evaluated in the presence of the INH/MZL combi BACTEC. This Lindil/Rif preparation was sterilized by nations in BACTEC. The Delta Growth Index (dGI) was calculated for each combination. The dGI is an indicator filtration through 0.45 micron filters so as not to give 40 background contamination by other bacteria. of Mycobacterial growth over a period of time and is M. tuberculosis Rifampicin resistant patient isolates were determined: dGI=GI (day--1)-GI (day). acquired from the South African Institute for Medical Results Research (SAIMR). Strains TV25 and TV79 were The results are set out in FIG.1. It shows that after 6 days acquired from the strain bank at Tygerberg Hospital. 45 incubation in BACTEC (dG5), the dGI for M.Tb. strain Both were determined to be resistant to Rifampicin, TV25 was still below 10 in the presence of MZL and Isoniazid an also streptomycin. The catalase activity of INH reconstituted prior to 1:2 dilution, whereas the TV79 was found to be negative and that of TV25 to be 5 sample subjected to the formulation in which the 1:2 mM. dilution of MZL was made prior to INH dilution already Rifampicin was made up in 50% ethanol at a concentration 50 showed strong positive growth with a dGI-28. of 10 g/ml. Eight ul were added to 1 ml Lindil to give a This represented an increased inhibition of 78% compared to the effect of MZL dilution prior to INHaddition. The concentration of 80 ug/ml Lindil. Of this, 0.1 ml was effect of MZL alone was 50% relative to the control for added to a BACTEC vial to give a final concentration of strain TV25. Relative to the control, the effect of MZL 2 ug/ml Rifampicin in Lindil. This is the cut-off value at 55 combined with INH followed by dilution, was 91%. This which M. tuberculosis strains are evaluated for drug gave an additional effectiveness of 41.5% of INH (INH resistance or drug sensitivity for Rifampicin. alone showed no effect) in the presence of MZL. Results: It was found that Rifampicin in Lindil kills drug sensitive (iv) Evaluation in respect of Ethambutol 60 Ethambutol was prepared in sterile deionized water and strains of M. tuberculosis to a much greater extent than dissolved in MZL at appropriate concentrations and the Rifampicin alone at concentrations below 2 g/ml. mixtures then Sonicated in a cup-horn Sonicator for one Microscopy illustrated delivery of Rifampicin by Lindil minute at frequency setting 7. It was brought into contact into the interior of the bacillus, and these results show with a culture of M. tuberculosis H37RV known to be that the Rifampicin does not stay encapsulated in the 65 resistant to Ethambutol. GI was determined every 24 bacillus, but is released from the nanolipid vesicle to the hours-tone hour. The observations are recorded in bacilli internal environment for biological activity. Table 1. US 8,937,074 B2 57 58 TABLE 1. appear to have a better effect in the untreated M.Tb. This effect could be a dose response because of the very high H37RV reference strain treated with Ethambutol. Consecutive presence of Ethambutol at the high concentrations. In treatment of H37RV with MZL carrier. the MZL treated experiments it appears as though MZL

Day 1 2 3 4 5 7 8 5 captures Ethambutolin its structure making less Etham Eth/MZL 8 g/ml 7 5 3 2 2 4 1 butol freely available for diffusion over the mycobacte Eth/MZL 4 g/ml 5 4 3 3 3 3 rial membrane. However as the concentrations become Eth/MZL 2 g/ml 6 4 3 lower it appears that MZL gives a steady delivery of Eth/MZL 1 g/ml 6 5 4 10 13 31 43 Ethambutol to the M.Tb. with the result that inhibition is Eth/MZL.5 g/ml 5 5 5 10 20 68 109 10 steadily maintained (approx 50% inhibition over the Eth/MZL.25 g/ml 5 5 4 7 16 75 136 range of 0.03125 g/ml-0.125 ug/ml). Eth/MZL.125l g/ml 5 4 4 8 19 89 142 From these results it appears that MZL maintains steady Eth;MZL.062l tg/mll 5 4 7 14 31 127 171 ; state delivery of Ethambutol to the M.Tb. over the spec Eth;MZL.O312l ?ml 5 5 7 15 33 123 182 trum of concentrations used and this effect is most Eth 8 g/ml g 16 18 17 18 15 13 15 15 prominent in the low concentration range of MZL that is Eth 4 lg/ml 21 23 26 28 27 27 25 the concentration range significant for therapeutic effi Eln 4. Eth 2 g/ml 21 24 27 32 34 39 48 cacy. an 1 sml 15, 19 23 29 31 47 55 (b) The effect of nanolipid-vesicle encapsulation of the anti Etnal biotic Pvrazinamide on the resistance of BCG (Bacillus of 5 g/ml 15 21 29 48 66 138 162 en . l 21 36 62 110 160 349 427 20 Calmette and Guerin) to Pyrazinamide: ".t 21 45 92 200 307 785 873 The encapsulation of antibiotics IZoniazid, Ethambutol

Ern .o o,ml 2O 45 97 211 338 840 999 and Rifampicin into the nanolipid vesicles formulation Eth.O312 iml 20 43 96 218 377 909 999 | of Preparation 2 described above resulted in a product is is 19 43 91 220 336 540, 534 561 suitable for use in the inhibition of Mycobacterium Control + MZL 7 5 8 21 44 186 280 : 25 Tuberculosis in bacterial isolates from patients infected OO with both drug-sensitive and multidrug resistant strains. The results were obtained with the Bactec system, with Calculations: no human cell involvement. Figures in the highlighted column (6) were used to calcu The significance of the following result stems from the fact late the effect of Ethambutol on mycobacterial growth in 30 that the BCG vaccine is extensively used for vaccination the absence of MZL. At this point the untreated TB against infection by Mycobacterium Tuberculosis control is GI-540. This is also the point where the TB (M.Tb.). BCG vaccine is classified as a non-pathogenic growth becomes stationary (decline in growth). All GI mycobacterial strain and is therefore a widely used values in this column were calculated relative to the investigative model of infection by (M.Tb.). All BCG 35 strains are resistant to Pyrazinamide (see Morbidity and control value of 415 to give '% inhibition of growth. Mortality Weekly Report: 1996, vol 45, No RR-4). The Column (9) shows the MZL treated control at approxi effect of encapsulation of this antibiotic by nanolipid mately GI=415. All GI values in this column (Ethambu vesicles according to the invention and its delivery in tol in the presence of MZL) were calculated relative to that form to BCG was investigated. the control value of 415 to give% inhibition of growth. 40 In this investigation which was performed by confocal The untreated control value was normalized to 415 to laser Scanning microscopy (CLSM), use was made of a make results comparable. The results of the calculations live/dead fluorescent stain known as Baclight. It stains are set out in Table 2. live bacteria green and dead bacteria red. General viabil ity of the BCG’s was determined by the green/red ratio TABLE 2 45 of the bacteria. The effect of equal amounts and concen Plus MZL Minus MZL trations of free and nanolipid-Vesicle encapsulated %. Inhibition %. Inhibition Pyrazinamide on BCG viability was investigated. Bacterial Viability: 8. E. East . The general viability of the BCG’s before any addition of 0.25 g/ml Ethambutol 51 51 50 antibiotics was between 85-95%. The BCG-viability 0.125 g/ml Ethambutol 52 -12 after a two-hour incubation of the applicable dosage of 0.0625 g/ml Ethambutol 49 -16 free Pyrazinamide was 68-72%. 0.0325 g/ml Ethambutol 48 -21 Bacterial Growth Characteristics: BCG generally grows in clumps. Incubation with free Results: 55 Pyrazinamide resulted in the appearance of single live The results show that the MIC for Ethambutol in M. tuber- bacteria with a few granuloma-type clumps, which culosis H37RV strain is around 1.0 g/ml. Results gradually secrete single live bacteria. The single live clearly show that from 0.125 g/ml Ethambutol, MZL bacteria were mobile. Encapsulation of Pyrazinamide in presence still maintains a strong inhibitory effect on nanolipid-vesicles led to a 65-75% decrease in BCG mycobacterial growth compared to MZL untreated bac 60 viability within a two-hour incubation. No moving BCG teria whereas there was no growth inhibition. In fact, a was observed. slight stimulation of growth (negative values) is Accordingly, BCG prelabelled with live/dead Baclight observed. This is usually observed with very low anti bacterial stain and then treated with nanolipid-vesicle biotic manipulations. At the higher Ethambutol concen encapsulated Pyrazinamide was observed by confocal trations (0.25-1.0 ug/ml) there does not appear to be 65 laser scanning microscopy. It was found that, after an much difference in growth inhibition between MZL hour, most of the bacteria were labelled red, and were treated and untreated. The very high concentrations therefore dead. No granuloma-type clumps or single US 8,937,074 B2 59 60 bacteria were observed. Such clumps and single bacte that inhibition of bacterial growth in both types of ria, all coloured green, were seen in the control in which bacteria is dramatically increased when the active is the same quantity of Pyrazinamide in water alone was, associated with the nanolipid vesicles. brought into contact with BCG also prelabelled with (ii) Enhancement of five anti-infective agents against five live/dead bacterial stain. 5 different bacterial organisms The study thus yielded the most surprising result that bac Five commercially available antibacterial compositions terial resistance to Pyrazinamide may be overcome by containing the active ingredients set out in Table 3 below were encapsulation of the antibiotic into the nanolipid compared in Zone inhibition studies with saline as control vesicles composition of Preparation 2. with compositions of the same active ingredients made up in (c) Infection studies (Live confocal laser scanning micros 10 copy studies) a carrier according to the invention. These are designated In this study the aim was to determine whether encapsula “MZL formulations in the case of aqueous made according tion of an antibiotic by nanolipid vesicles gives rise to a to preparation Zabove and “MZLA formulations in the case product by which one could overcome resistance of of non-aqueous formulations prepared according to Prepara intracellular bacteria, using infection of human mac 15 tion 3 above. This convention is also followed in other rophages by BCG in culture as a cell model. examples below as opposed to the commercial formulations The THP1 macrophage cell line (ATCC) was used for the infection study. RPMI 1640 with L. glutamine cell cul (COM) of particular active agents. The compared formula ture medium, Foetal Bovine Serum and phosphate buff tions were diluted where necessary to achieve the same con ered solution (Gibco BRL), were used for cell growth centrations. according to general cell culture methods. THP1 mac It is evident from the results set out below that the organ rophage cells were cultured and infected with pre isms were more sensitive to the active agents when encoun treated labelled BCG bacteria. Treatment consisted of tered in the carrier formulation according to the invention. TABLE 3

ZONE OF INHIBITION STUDY: FIVE COMMERCIAL ANTI-INFECTIVE FORMULATIONS AGAINST FORMULATIONS ACCORDING TO THE INVENTION FOR DIFERENT INFECTIVE AGENTS

Dose ACTIVE MZLA mg/ AGENT Com 5 ml S. Aureus P Aerugin B. Cereus E. Coii A. Niger C. Albicans Cloxacillin MZL 125 30.74 23.96 Cloxacillin COM 125 29.45 1986 Erythromycin MZL 250 26.7 29.89 Erythromycin COM 250 25.84 27.78 Ciprofloxacin MZL 250 33.05 35.78 Ciprofloxacin COM 250 30.14 33.4 Cotrimoxazole MZL 240 13.95 24.64 Cotrimoxazole COM 240 11 22.83 Itraconazole MZLA 50 16.03 14.28 Itraconazole COM 50 10.21 11.47 Control 9 9 9 9 9 9

equal concentrations namely 0.075 ug/ml of free and Example 2 nanolipid-vesicle encapsulated Pyrazinamide. CLSM 45 was used to determine infection by and survival of the Enhancement of Anti-Viral Agents bacteria. The viability of BCG’s after infection in macrophages In this study AZT at different dosages was associated with reflects that BCG’s inside macrophages treated with the nanolipid vesicles to test the efficacy of Such association nanolipid-vesicle associated Pyrazinamide is effectively 50 on the viral growth in CD4+ Helper T-cells. killed by the antibiotic Pyrazinamide, even though the The rationale for this investigation is that AZT is cytotoxic bacteria are generally recognised to be resistant to the and long-term use is associated with loss of muscle. Other antibiotic used. side effects can be nausea, anaemia, white blood cell depres (d) Zone of Inhibition Studies: Sion, mouth Sores, bone marrow damage, and headaches. It (i) Enhancement of Povidone iodide against two bacterial 55 would therefore be desirable from both a side effect and cost organisms as evidenced by Zone of inhibition studies. perspective to be able to lower the dosage of AZT adminis Inhibition of bacterial growth of two types of bacteria by tered to an HIV-infected patient, provided that such lowering the active ingredient known as Povidone iodine in of dosage does not compromise the effect of the administra formulated form with the nanolipid vesicles of Prepa tion. It was thus decided to investigate the dose/effect rela ration 2, and so used at a concentration of 6.30 g 60 tionship of AZT when formulated with the administration Povidone iodine equivalent to 0.75 g available Iodine medium of Preparation 2. in 100 g of product, was compared with the effect of Although it was the result the applicant had hoped for, it the same amount and concentration of the free active was nevertheless a surprise to find that these objects may be ingredient. The bacteria were S. Aureus and P Aerugi accomplished by increasing the effective delivery of AZT to nosa. The control used was saline. 65 HIV-infected cells, thereby maintaining a therapeutic intrac The results obtained in the study are graphically repre ellular concentration at lower dosages. From the observations sented in the graph which is FIG. 2 hereto. It is clear made it is presently postulated: US 8,937,074 B2 61 62 a) That the AZT-loaded nanolipid vesicles significantly Note that no correction has been made in the above evalu increase the kinetics of intracellular and intranuclear ation for the contribution of the cells itself to the Absor AZT delivery, with the result that AZT therapeutic dos bance/cut-off ratio. It was considered to be negligible ages can be decreased; (>0.4). b) That nanolipid vesicle-associated AZT appears to 5 Conclusions: assume the pharmacokinetics of the vesicles until Such a) The administration medium of Preparation 2 at the cor time as it is released and; rect dilution may be used to decrease the effective thera c) That the optimal loading concentration for AZT-loading peutic dosage of AZT by as much as 10 fold. It would into nanolipid vesicles can be determined with regard to considerably decrease the cytotoxicity of the AZT treat the applicable cell types. 10 The following protocols were followed in this investiga ment. At Such low dosages, it may be more attractive to tion: treat expecting mothers for HIV infection, without any a) Several cultures of CD4 helper T-cells (CEM-SS cell long-term side effects on the foetus. line: NIH AIDS Research and Reference Reagent Pro b) Optimisation of nanolipid vesicle concentration for gram) were infected with HIV subtype D viruses at 15 AZT-delivery is essential, as unloaded nanolipid applicable cell seeding densities and viral loads. Cells vesicles can favour the multiplication of HIV viruses by were maintained in tissue culture under standard cultur Supplying components for viral membranes synthesis ing conditions. and energy for viral metabolism, as the metabolism of b) The viral load in cultured cells were determined every nanolipid vesicle essential fatty acids (EFAs) may day for 7 consecutive days after infection by measuring increase the energy status of the cell, and therefore also the p24 core antigen of HIV-1 in the supernatant cultur the energy available to the HIV virus for its replication. ing fluid by ELISA. This assay is based on a colour The nanolipid vesicle concentration must therefore be change after addition of substrate that is proportionate to sufficient to serve only its transport function, without the viral replication in each culture. The colour change is Supplying either membrane components or energy for monitored by absorbance spectrophotometry at 450 nm. 25 viral multiplication. Negative control absorbance readings were used to cal The changed pharmacokinetics and intracellular biodistri culate the absorbance/cut-off for each culture. bution of AZT transported by nanolipid vesicles are probably c) Determination of the optimal concentration of nano the result of the following two mechanisms: lipid-Vesicle containing base formulation was done by a) the change in environment with regards to charge and cytotoxicity assays and cell growth curves. The cytotox 30 icity assay used was the standard MTT assay and spec hydrophobicity when nanolipid vesicles move from the trophotometry was once again used to determine cell extracellular to the intracellular environment, and viability for the CEM-SS cell line. Growth curves were b) the normal cellular pathways of the EFAS, which include its used to determine the optimal concentration of base metabolism in the mitochondria. The release of AZT is formulation for human macrophage cells (THP-1 cell 35 therefore most probably in the region of the mitochondria. line). The results obtained are graphically presented in FIG. 3 Example 3 hereto. It shows the following: a) The delivery and transport function of the nanolipid Yeasts and Fungi vesicles increase the therapeutic efficacy of AZT tenfold 40 at a dilution of 1:512 base formulation, thereby creating The Enhancement of Agents Used in the Treatment the possibility of decreasing the AZT dosage 10 fold, as of Infectious Diseases Caused by Yeasts and Fungi is shown by the relative effect of 1 nM free AZT versus 0.1 nM nanolipid-vesicle formulated AZT. The results The use of this invention for the enhancement of agents clearly show that the addition of 0.1 nM free AZT inhib 45 used in the treatment of yeasts and fungi (moulds) was inves ited viral growth and replication by 44% by day 0.7 and tigated and established by the following studies described in 8, when compared to the control (no AZT added). How greater detail below, namely: ever, the addition of 0.1 nM nanolipid vesicle-associated Comparative culture studies AZT inhibited viral growth by between 70-80% on day Infection studies (Live CLSM imaging) 8. This is comparable to the inhibition observed by 1 nM 50 Zone inhibition. free AZT (i.e. 10 times the concentration). Furthermore, (a) Comparative Culture Studies: addition of 0.1 nM nanolipid vesicle-associated AZT Fungi of the mycosis type was grown in culture medium showed a continued decrease in viral load over 8 days, (RPMI 1640 with L. glutamine cell culture medium) at 37° C. whereas the addition of even 10x that amount of free at 90% humidity, 5% CO, for a week. Equal aliquots of grown AZT resulted in a decrease in viral load up to day 6, after 55 fungi were exposed to nanolipid vesicles loaded with a com which the viral growth increased slightly. mercially available antifungal agent containing 2% Micona b) The graphs in FIG. 2 show that the association of AZT Zole nitrate. Comparison was made with free Miconazole with nanolipid vesicles changes the pharmacokinetics nitrate i.e. the commercially available form of that active and possibly the intra-cellular biodistribution of AZT. ingredient. In both instances the exposure was at a final con c) The effective delivery of the basic nanolipid vesicle 60 centration of 0.4% over similar time periods (30 minutes to 18 formulation has been established for CD4 T-cells and days), using a single initial dosage. macrophages to be a dilution of between 1:512 and Results: 1:1024 of the concentrated unfiltered formulation. The nanolipid-vesicle delivery system can be used for the Higher content of nanolipid vesicles appears to favour efficient delivery of antifungal agents to fungi. viral growth in the cells. At a dilution of base-formula 65 Miconazole nitrate association with nanolipid vesicles, but tion (1:256), viral inhibition is optimal at a higher con not free Miconazole nitrate caused a fast and unexpectedly centration of AZT (0.5-1 nM). dramatic decrease of the fungi as illustrated in Table 4. US 8,937,074 B2 63 64 TABLE 4 be capable of being treated with greatly reduced cytotoxicity and resulting lower incidence of side effects. In addition drug VLABILITY OFFUNGIAGAINST TIMEELAPSED AFTER SINGLE cost will also be lower. EXPOSURE TO MICONAZOLE NITRATE Approach and General Method Nanolipid-vesicle The specific parasite investigated was the reference drug Time encapsulated Miconazole Free Miconazole resistant strain W2 of the species Plasmodium Falciparum. 30 min 59% 80% Plasmodium Falciparum is the most commonly occurring as 4 hours O% 70% well as the most virulent malaria parasite currently known in 11 days O% 30% a. 18 days 190 15% 10 The system used in this investigation aims to mimic the live situation as closely as possible. For that reason, the parasites (b) Infection Studies: were infected into fresh primary erythrocytes isolated from Two equal aliquots of melanoma cells (UCT Mel 1 cell O+ or A+ blood donors. line) were incubated for 4 hours with fungi and 0.2% of Furthermore, human serum prepared from the same donors either free Miconazole nitrate or Miconazole nitrate for was used as an adjuvant instead of foetal calf serum. Parasite mulated with the administration medium of Preparation growth was maintained by the addition of freshly prepared 2 in order to test the effect of the formulation in terms of erythrocytes from the same donors. the invention on the efficacy of fungal treatment of The growth of the parasites was determined by visualiza human cells. tion of the parasite DNA on thin smears of the infected cul The viability of both human melanoma and human mac tures. Since mature erythrocytes contain no nucleus, and rophages was found to decline dramatically in the pres therefore no DNA, the only DNA present was of parasitic ence of free Miconazole nitrate but not when the origin. Only intracellular parasites were included in deter Miconazole is encapsulated in nanolipid vesicles. The mining the percentage parasitaemia, as extracellular parasites result is graphically presented in FIG. 4 hereto. 25 are no longer viable. FIG. 4 shows the viability of both the human melanoma Protocol: and fungi 4 hours after the addition of the therapeutic agents The protocol was typical for work of this nature and in a petri dish. included the following steps as will be readily apparent to Using a macrophage cell line, the same result was obtained. those skilled in the art. Melanoma cells were healthy after 4 hours incubation with 30 A. Culturing of malaria parasites: nanolipid-Vesicle encapsulated Miconazole nitrate and no 1. Preparation of fresh human erythrocytes fungi was present. In the presence of free Miconazole nitrate, 2. Preparation of human serum from the same donor. cells were no longer attached to the petri dish and fungi were 3. Quality control of human serum. still present in large numbers. 4. Infection of erythrocyte cultures with parasites. Initial (c) Zone of Inhibition Studies: 35 Zone of inhibition studies on yeasts and moulds namely C. infection load was 0.5% parasitaemia. Albicans, T. Mentagrophytes and E. Flocossum were . Maintenance of parasite blood cultures also carried out. Inhibition of yeast and mould growth by 6. The parasite percentage in the blood cultures was deter the active ingredient Miconazole nitrate in association mined after 36 or 48 hours, depending on the level of with nanolipid vesicles was observed. The control used 40 infection of the freshly added erythrocytes. was saline. B Basic toxicology: The result of this study is reflected in FIG. 5 hereto Determination of possible toxic effect of nanolipid formu Conclusion: lations on erythrocytes and parasite growth. It is quite clear from the results presented in FIG. 5 that the The following nanolipid formulation concentrations were active Miconazole nitrate in association with the nanolipid 45 investigated: 0.1:1500, 1:1000; 1:750; 1:500; 1:250; 1:100: vesicles inhibits the growth of both yeasts and moulds and undiluted. does so to a greatly enhanced extent. C Loading of nanolipid formulation with chloroquine: A stock solution of 10 mM chloroquine solution in a 1:250 Example 4 dilution of nanolipid formulation was made by Vortexing and 50 Sonication. All concurrent dilutions were from this stock. Parisitology D Drug delivery by MZL nanolipid formulations: The Treatment of Infectious Diseases Caused by 1. Penetrance of nanolipid formulations in red blood cells Parasites was determined by microscopic visualization, as were 55 the penetrance of chloroquine-carrying nanolipid for Summary: mulations. The effect of free and non-aqueous nanolipid formulated 2. The drug concentration series used centred around the chloroquin made according to the process described in Prepa known IC50 concentration of chloroquine in the W2 ration 3 against a resistant Falciparum strain (the reference strain. strain W2 for drug resistant malaria) was preliminarily deter 60 3. Typically 48-well plates or 96-well plates were used. 200 mined in the conventional manner. The strain is known to ul or 100ul total culture volumes were used respectively, have a 50%. Inhibition concentration value (ICs) of between of which 90% of the volume was infected erythrocyte 200-300 nmolar Chloroquine. The determined ICs value for culture. 10% Volume was used for the treatment, be it nanolipid formulated Chloroquine was about one tenth of the chloroquine in Nanolipid formulation, chloroquine in value for free chloroquine, namely 25-30 nmolar. This most 65 water, or for the controls 1:250 pure Nanolipid formu surprising result holds substantial promise for further lation, pure culturing media or water only. research as its utilisation in practice means that malaria would 4. All series were in duplicate. US 8,937,074 B2 65 66 E Visualization of intracellular parasites: released from the dosage form of the invention at a satisfac Parasitic DNA was visualised by Giemsa staining of thin torily rate and extent in comparison to the commercially Smears. Ethidium bromide oracridine orange may be used for available Comparators. fluorescent staining. The applicability of the test method for release out of the F% Parasitaemia: 5 dosage forms was confirmed by Handbook of Dissolution The % infection after applicable incubation periods was Testing: Dissolution Testing of Transdermal Delivery Sys determined as follows: tems, page 61. The Small receptor Volume to be used, in this Total parasite count per 10 microscopic fields case 12 m, is confirmed in the same reference on page 63, x 100% which refers to 5-25m. Total cell count per 10 microscopic fields 10 2. Method GQuality assurance of counting: The in vitro release from the dosage forms was determined All counts of the first series were undertaken by two sci- by a Hanson Model 57-6M, Manual Start-Up, Diffusion Cell entists. The results correlated very well. The second S1S Test System bought from Hanson Research with the follow was spot-checked by a second scientist, especially around is ing main parts: crucial concentrations. Once again no significant deviations CELL, DRIVE CONTROL were found between the two sets of results. Results: 6-CELL, DRIVE WITH CELLS The counts of the cultures are reflected in the table 5 below: VERTICAL CELLS TABLE 5

CLOL CLQ Chloroquine Cells Total % Parasit + Cells Total % concentration field cells Parasites MZL field cells Parasites Parasit

O nM 95 950 17 1.8 73 730 16 2.19 1 nM 87 870 18 2.06 127 127O 34 2.67 S M 127 1270 29 2.88. 6O 600 14 2.33 10 nM 130 1300 37 2.84 13S 13 SO 40 2.96 25 nM 120 1200 23 1.91 130 1300 37 2.84 SO M 90 900 13 1.41 111 1110 30 2.7 75 nM S4 S4O 5 O.9 129 1290 43 3.33 100 nM 125 12SO 14 1.1 64 640 18 2.81 200 nM 106 1060 4 O.3 108 108O 31 2.87 300 nM 88 880 13 1.41 1 OS 1 OSO 19 1.8 SOO nM 63 630 7 1.1 76 760 O.15 1.5 1000 nM 87 870 12 1.3 71 710 10 1.4 W2 = Chloroquine resistant strain internationally recognized and used. CLOL = Chloroquine in 1:250 dilution MZL nanolipid carrier; concentrations of chloroquine as indicated. CLC = Chloroquine in medium at the specified concentrations, Resistant strains become sensitive only at very high Chloroquine concentrations, Association of chloroquine with nanolipid formulation results in a similar susceptibility as sensitive strains, 40 Statistical analysis of the results was by Chi-square analy 3. Parts Needed sis between the two sets of data. A combination of the 3 1. Diffusion cell assembly, including donor top and recep repeats of the two sets of data gives a Chi-square value of 7.6. tor chamber (set of 6). The donor top includes a drug According to the probability tables, the difference between dosage wafer (Teflon washer), an acrylic top plate, and a pure chloroquine and MZL-associated chloroquine is highly 45 significant, with only a 0.0001 probability that the difference clamp to connect top to bottom. observed between the two treatments is due to chance. 2. Pig skin used within 24 hours from being slaughtered Conclusions: kept in Ringer Solution between 2°C.-8°C. 1. The association of chloroquine with MZL nanolipid . Davies Gold Series Dermatone, Simplex GS 102. formulation significantly decreases the ICso of chloro 50 . Application Squeegee and tweezers. quine (by 6x to 10x). . Drug dosage form. 2. Primary human cells show high tolerance for the MZL . Absorbent paper towels and tissues. nanolipid formulation, with cytotoxicity only observed . Technique at high extremely high concentrations. . Obtain skin from pig heads (jawbone skin). Use Der 3. MZL formulations may be used in the prophylaxis of ss matome according to the Operation standard operating drug resistant malaria. procedure for the Dermatone, setting it to size the skinto a thickness of 0.33 mm. The diameter of the skin should Example 5 be in excess of the drug dosage wafer. Comparative Release Properties as Determined by 2. Prepare receptor chamber of diffusion cells with slight Membrane Diffusion of Anti-Infective Agents 60 overflow of medium (pH 5 buffer with glacial acetic acid Formulated in Accordance with the Present Invention for the Test Product Acyclovir and 6.8 phosphate buffer and Commercially Available Formulations of the for the test product Miconazole) with temperature con Same Anti-Infective Agents trolled at 32° C. 3. Prepare each piece of skin with the relevant products one 1. Objective 65 at a time as follows: The scope of this study was to establish whether the Test 3.1. Lift skin with tweezers, place on tissue and blot Anti-infectives Acyclovir and Miconazole Nitrate are excess of solution, invert and blot. US 8,937,074 B2 67 68 3.2. Place skin in centered position on drug dosage TABLE 7 wafer. 3.3. Place relevant products on top of skin in dosage Table indicating release rates and percentage release per label claim for wafer cavity—0.5 ml by means of a Gilman pipette— product tested. weighed and averaged to obtain dosage applied. % Active? Release Rate % Release per 3.4. Use Squeegee to carefully smooth product over Active Agent product (ig/cm/h) label claim membrane, filling entire cavity. Acyclovir MZL O.S 69.1533 O.1214 Acyclovir O.S 54.0942 O.0952 3.5. Wipe excess dosage water with Squeegee. COM 3.6. Lift loaded dosage wafer with skin and place on top 10 Miconazole 2 389.9238 6.815S of receptor cell with skin side towards cell medium. Nitrate MZL Miconazole 2 111.2222 1946.6 Exclude bubbles during process. Place on top of Nitrate donor cell assembly, pressing down with finger, Squeezing out bubbles between top plate and dosage form. Apply clamp to lock down top donor and bot 15 7. Calculations tom receptor halves of diffusion cell. The Release Rate was calculated as follows: 5. Operation of Apparatus The apparatus must be set to 150 rpm. Samples of 150 ul are withdrawn with a micropipette at 2, 5’, 8' and 10' and 15 7.1 pig Active Released at time (min.) = minutes. The samples after being withdrawn are analysed for A San XMass Std X Vol Receptorx Acyclovir and Miconazole Nitrate respectively by means of Mass of Active Applied for Z cells X C HPLC according to the parameters set out in Table 6 below. A Std X Vol Sid XLabel Claimx Mass of Product Applied for 1 cell XZX 100 TABLE 6 25 Acyclovir Miconazole Nitrate WHERE: Injection volume 20 ul 20 ul Column Zorbax SB C18 Zorbax SB C18 A Sam Area of peak sample solution 250 mm x 4.6 mm 250 mm x 4.6 mm A std=Area of peak of standard solution Mobile Phase 0.02M GAA in H.0 70% Methanol 30 pH 3.5 30% H-0 + 1% GAA Mass std=Mass of standard taken to prepare the standard Detector HPLC at 254 mm HPLC at 224 mm solution expressed in ug Temperature Ambient (22°C.) Ambient (22°C.) Flow Rate 1.5 ml per min 1.5 ml per min Vol Std=Volume to which the standard solution is made up, Retention Time 20.8-21.9 min 9.4-9.9 min expressed in m Solvent MP adjusted to pH 5 Methanol 35 Label Claim=Amount of active present per 100 g of product Cells used 3 (1 for Comparator) 6 Mass of Product Time at which total 15 60 release determined Applied for 1 cell=Specific amount of product applied for a (Min) specific cell Mass of Active 6. Results 40 Applied for Z cells=Amount of active applied in total for all The release experiment was performed in the number of Z cells utilised per one study cells indicated above for each product and the mean release is C potency of the standard, expressed as a percentage

ug Active Released 7.2. Accumulative Dose (pig) released square cm at time (min) = (Surface Area of Exposed Skin) ug Active Released 1.767 cm2

7.3 Release Rate = Accumulative Dose (pig) released square cm Time (hours) ug Active Released X 100 7.4 Percentage of Active Released at time (min.) = ug Active Applied reported for each analysis point. The results are tabulated and 8. Conclusion graphically presented. The results as a percentage of the active released per label claim per cell at the different time 60 From the aforegoing test it was concluded that the formu intervals is also tabulated. lation according to the invention In Table 7 below is shown a summary of the release rate and (a) releases Acyclovir 1.28 faster than the Comparator percentage release per label claim for the products deter Acyclovir formulation at 15 minutes, and continues to release mined according to calculations, reporting the mean values of 6s higher quantities throughout the duration of the test; the utilised number of cells of each product after the effluxion (b) releases Miconazole Nitrate 3.51 times faster than the of the time indicated above. commercial Comparator at 60 minutes. US 8,937,074 B2 69 70 Example 6 secondly, to determine whether there are changes in the status of side effects caused by the actives; Demonstration of the Equivalent or Improved thirdly, to determine the relative safety levels of the com Bioavailability of Anti-TB Drugs in the Formulation parative products; According to the Invention Compared to a fourthly, to determine possible partitioning of the MZL Commercially Available Product nanolipid delivered drugs to cells and possible cytotoxicity as a result; and 1. Background: finally, to note the possible advantages to the Volunteers Anti-tuberculosis treatment presents with two major prob well-being (i.e. malaise, bone ache, nausea etc) when using lems—the development of drug resistance and compliance. 10 the delivery system of the invention for the administration of The nanolipid based delivery system provides a system for anti-tuberculosis drugs. single or combination tuberculosis drug treatment with a 3. Study Design significantly increased therapeutic index, using currently pre The treatment was based on the Standard Treatment Guide scribed anti-tuberculosis drugs, with a resultant decrease in lines and Essential Drugs List (1998). The study design was the development of drug resistance. The delivery system con 15 an open crossover bioavailability design of tuberculosis drugs tains the same therapeutic moieties but differs in chemical Rifampicin (R), Izoniazid (H): Ethambutol (E) and Pyrazi form, and dosage of those moieties and can therefore namide (Z) delivered by a formulated drug delivery system. regarded as a pharmaceutical alternative. Furthermore, the Bioavailability is understood to be the rate and extent to higher therapeutic index of the drug facilitates lower dosage, which the active substance or therapeutic moiety is absorbed which limits the side effects, which may in its turn be and delivered from a pharmaceutical form expected to improve compliance. Using this delivery system, a) into the general circulation and delivery of the drugs may also be expanded to tissues usually b) becomes available at the site of action. not easily reachable by current therapeutic regimes. As in other bioavailability studies, the kinetics of the thera The formulated delivery system contains components that 25 peutic moiety in the general circulation was monitored in this have been recognized as pharmaceutically safe. The public study. health authority of South Africa in concert with many other The study was conducted over two periods of 4 days each, health authorities, advise the same actives as used in this interrupted by a two week wash out period. During the last investigation as initial treatment regime for all tuberculosis day (day 4) of each period, blood samples will be taken at the patients. This protocol describes the delivery of the pre 30 times specified below to determine several pharmacokinetic scribed anti-TB drugs in 4 single daily doses by way of the parameters. Blood samples (10 ml each) were taken at the delivery system at a reduced dosage level to 1 healthy volun following intervals after administration of the drugs: 30 min teer. utes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 9 hours. In an open crossover design, pharmacokinetic parameters 3.1 Rifampicin: of the four drugs delivered by the test formulations in reduced 35 The volunteer received the currently commercially avail doses were compared to those achieved when the same drugs able prescribed Rifampicin, namely Rifampin, treatment for were administered in the reference formulations of estab 4 consecutive days, followed by a wash out period of 2 weeks. lished quality and in the standard treatment doses. The vol Rifampin contains Rifampicin as active ingredient. The Vol unteer was monitored daily during the study. The protocol unteer then received pure Rifampicin packaged into the nano below describes the dosage level, the specific drug, the time 40 lipid delivery system of the invention as described above at period of the study, the parameters investigated and the com two thirds of the prescribed dosage, again for 4 consecutive binations of drugs used. days. This nanolipid formulation of Rifampicin is herein also 2. Study Objectives referred to as Rifemzaloid. The volunteer took the medication Primary Objectives in the morning before a meal with 200 ml of tea, as food has The first primary objective of this investigation was to 45 been shown to influence absorption. Meals were standardized determine the bioavailability of generally used anti tubercu and supplied a couple of hours after first administration of the losis treatment agents, i.e. Rifampicin (R), IZoniazid (H), medication. Ethambutol (E) and Pyrazinamide (Z), each packaged into the 3.2 Combination Drugs: MZL drug delivery system in the form of the capsules pro The volunteer started with two third dosage of all four duced in the manner as described in Preparation 3 above. 50 drugs packaged into the nanolipid delivery system of the Secondly, it was to determine changes in patient global invention, followed by the same scenario with the commer assessment, i.e. significant change from baseline of the fol cially available treatment regime, namely Rifafour, after a lowing pharmacokinetic parameters: two week wash out period. The volunteer took the medication a) peak plasma concentration (Cmax), in the morning before a meal with 200 ml of tea, as food has b) the time needed to reach this concentration (Tmax), 55 been shown to influence absorption. Meals were standardized c) exposure (the area under the plasma curve (AUC 0-9 and supplied a couple of hours after first administration of the hours), and medication. d) coverage. 3.3 General Protocol Requirements: The pharmacokinetic results were compared with those of a) The subject volunteered for participation in the study. The reference formulations. The packaged drugs were adminis 60 Volunteer was a Caucasian middle-aged female and was tered at an equal or a decreased dosage of that in the commer clinically healthy i.e. blood chemistry, full blood count and cially available combination antituberculosis drugs. liver function tests of subjects fell within the normal 2.2 Secondary Objectives ranges. The Volunteer granted written informed consent The secondary objectives of the investigation were before participating in the study. firstly, to determine whether bioequivalence exists for 65 b) All drugs were orally administered in Soft Gel capsule drugs packaged into the delivery system by comparison to form at applicable doses. The volunteer did not take any reference agents; other chronic medication during the study.

US 8,937,074 B2 73 74 for PZA in the range 0.1-60 ug/ml. INH, RIF and PZA TABLE 9 and pyrazynoic acid analysis were done in triplicate. Intra- and inter-assay coefficients of variation were deter Plasma levels of Rifampicin mined. MZLA Rifampicin Rifamzaloid (ug (ig/ml Five replicate samples of four concentrations were run Time (min) Rif ml plasma) plasma) through the procedure with exactly controlled volumes, O O O as described for the extraction of the samples. To verify 30 10.31 O recovery/quality control, precision and accuracy, the 60 12.93 7.46 peak areas obtained for the extracted samples were be 10 90 11.23 10.23 120 1122 9.3 compared to those of fresh standards of the analytes in 150 10.16 8.43 mobile phase with respect to the volumes handled dur 210 8.82 9.03 ing extraction. 240 10.03 7.39 370 9.27 6.32 C18 Bondelut extraction cartridges were washed sequen 15 300 7.22 4.84 tially with 2x2 ml of methanol, 2x2 ml of water and 2 ml 330 6 4.1 of 0.05 M potassium phosphate, pH 4.5 (phosphate 360 5.77 3.45 buffer) prior to application of the sample to the columns. A 0.5 ml quantity of plasma were thawed and drawn slowly Increased Cmax onto the column and allowed to stand for 5 minutes, after The maximum plasma concentration (Cmax) of Rifampi which time unbound material were discarded. The col cin was determined to be 12.93 ug/ml and was reached umns were washed with 1 ml of phosphate buffer to be 60 minutes (Tmax) after oral administration of the active discarded, and the drugs eluted with 0.5 ml of acetoni in the nanolipid delivery system. The Cmax obtained for trile, followed by 0.5 ml of methanol with these elutes the Rifampin (10.23 ug/ml) was reached 90 minutes being pooled. 25 after administration. The delivery of Rifampicin to the plasma was therefore increased by at least 21% at Tmax 60 ul of the pooled eluates were injected immediately onto by the nanolipid carrier. Furthermore, only two thirds of the HPLC column to assay for RIF. the normal dosage was taken in the MZL formula. INH, PZA and Pyrazynoic Acid: Therefore, the increased delivery of Rifampicin to 0.5 ml of the combined eluates were dried by vacuum 30 plasma by the Nanolipid delivery system at Tmax was centrifugation and taken up in 0.5 ml of 3% acetonitrile 181% that of its comparator at equal dosages. in 0.06% TFA. Decreased Tmax 60 ul of this were injected onto the autosampler HPLC to The minimum effective concentration of Rifampicin in assay for INH and PZA, which were detected together 35 plasma is 7 g/ml. It is clear from the results that on the same column. Rifampicin reaches its effective concentration much quicker when delivered by the Nanolipid delivery sys Acceptance Criteria: tem. This is especially important in the case of unstable A validated analytical method meets the following criteria: drugs such as Rifampicin, as increased gastric exposure Precision and accuracy: The between batch CVs for low, 40 may lead to increased loss of activity. medium and high concentrations should be <15%, and Increased Coverage 20% for the LOQ QC. Coverage can be regarded as the time period during which the effective concentration is maintained. FIG. 6 illus Sensitivity: The lowest standard should be accepted as the trates that the coverage afforded by using the nanolipid LOQ if the 96 CV is <20% 45 delivery system is increased by 180% (270 minutes vs Specificity: The responses of interfering peaks at the reten 150 minutes). It is therefore possible to the increase the tion time of the analyte should be less than 20% of the time intervals between sequential dosages. response of an LOO standard. The above parameters reflect the comparative bioavailabil Stability: Stock solution stability should meet the criteria ity dynamics of the Nanolipid delivery system. The total specified in the SOP. 50 average increase in bioavailability by Nanolipid delivery system was 227%, while using only two thirds of the 5. Results dosage of the comparator. 5.1 Comparative Bioavailability of Rifampicin Differences in the above pharmacodynamic and pharma The first part of the study concerned only Rifampicin and cokinetic parameters were statistically significant (see the nanolipid-formulated Rifamzaloid. The plasma lev 55 Table 10), with a p-value of 0.0157. The statistical els determined for the indicated times are reflected in method for analysis used was Analysis of Variance with Table 9. a single factor variant. TABLE 10

SUMMARY OF STATISTICAL ANALYSIS

Groups Count Sum Average Variance

Column 1 11 103.16 9.378182 4.944216 Column 2 11 70.45 6.404545 9.408267 US 8,937,074 B2 75 76 TABLE 10-continued

SUMMARY OF STATISTICAL ANALYSIS

ANOVA

Source of Variation SS f MS F P-value F crit

Between Groups 48.63382.273 1 48.63382 6.777O6 0.017OOS894 4.3512SOO27 Within Groups 143.5248.364 2O 7.176242

FIG. 6 hereto records the observed bioavailability of (b) In similar manner a 1.7307% composition of Lidocaine Rifampicin in the MZL formulated Rifamzaloid vs its com Hydrochloride was prepared and designated LIDAREX parator Rifampin. The comparative daily Rifampicin dosages LOTION. were 400 mg/day in the case of Rifamzaloid and 600 mg/day 15 (c) A 0.3% composition of Scopolamine HBR was also in the case of Rifampin. prepared in the same manner and designated MZL Scopola 1C. 5.2 Bioavailability of Combination Drugs (d) A 0.5 nasal spray composition of Oxymetazoline HCl Similar results were obtained with Isoniazid and Pyrazina was also prepared in the same manner with some modification mide. FIG. 7 hereto illustrates the enhancement in bio and designated Oxymetazoline MZL. availability of INH, using the nanolipid delivery system, (e) A 0.5% injection formulation of Bupivicaine HC1 was and FIG. 8 that of Pyrazinamide (PZA). further prepared in the same manner but with some modifi FIG. 7 shows the enhanced bioavailability of IZoniazid when cation and designated Bupivicaine MZL: encapsulated in the MZL delivery system, even though the daily dosage of the INH in the MZL formula was only two 25 Example 8 thirds of that of the comparator, Rifafour. Study to Investigate the Release Characteristics of FIG. 8 illustrates the enhanced bioavailability of Pyrazina the Formulations Prepared in Terms of Example 1 mide in the MZL formulation. Again the daily dosage of Against Commercial Preparations the PZA in the MZL formula was only two thirds of the PZA in Rifafour, the comparator. 30 1. Objective: 5.3 Side Effects: The scope of the study was to establish whether the Test No significant side effects were found with the MZL for active agents viz. mulated liver function analysis. None of the liver Doxepin Hydrochloride, enzymes showed levels higher than the normal range. Lidocaine Hydrochloride The S-unconjugated bilirubin, which did show levels 35 Scopolamine Hydrobromide elevated above the normal range was nearly back to Oxymetazoline HCl and normal 8 hours after drug administration, and was nor Bupivicaine HC1 mal on the following day. are released from the dosage forms prepared in accordance 5.4 Volunteer's Assessment: with the invention as described in Example 1, and to The only adverse reaction to either of the MZL formulas 40 determine the extent and rate of such release. It was (Rifampicin or Combination) was nausea, whereas the further done to compare the rate of release of Doxipex to comparator Rifafour led to serious headache, jitters and that of an FDA approved 5% Doxepin Hydrochloride alSCa. topical formulation which is commercially available in 5.5 Partitioning of Nanolipid Formulated Rifampicin: Canada where it is indicated for the treatment of pruritis Between 5-10% of the Rifampicin encapsulated in the 45 associated with eczema. That product is herein referred nanolipid partitioned to blood cells rather than to to as DOXEPIN COM. The test further compares the plasma. This partitioning should increase the effective release of Lidocaine Hydrochloride from LIDAREX dosage delivered to the cells, but the partitioning is not against a commercially available Lidocaise Hydrochlo So high as to be cytotoxic. ride composition herein referred to as LIDOCAINE The study was repeated for Rifampicin and Pyrazinamide 50 COM. The test further compared the release of the with similar results. Oxymetazoline MZL to a commercially available Oxymetazoline nasal spray formulation designated Example 7 oxymetazoline Com. Finally the test compares the release of the Bupivicaine MZL formulation with a com Preparation of Topical Formulations of Doxepin 55 mercially available Bupivicaine injection formulation Hydrochloride, Lidocaine Hydrochloride, and designated Bupivicaine Com. Scopolamine Hydrochloride in the Nanolipid Vesicle The applicability of the method used to test for release out of the dosage forms is confirmed in Handbook of Dis Formulation of Preparation 2 Solution Testing; Dissolution Testing of Transdermal 60 Delivery Systems, page 61. The small receptor volume (a) A 2% composition of doxepin in the nanolipid-Vesicle to be used, in this case 12 ml, is confirmed in the same composition of Preparation 2 above was prepared by mixing reference. doxepin hydrochloride with the nanolipid vesicle formulation 2. Method as described in Preparation 2 which had been modified by the The in vitro release from the dosage forms was determined addition of Keltrol to render a thick creamy consistency, in a 65 by a Hanson Model 57-6M, Manual Start-Up, Diffusion mass/mass ratio of 2:98 with the aid of a high speed stirrer. Cell Test System available from Hanson Research with The composition is designated DOXIPEX CREAM. the following main parts: US 8,937,074 B2 77 78 CELL, DRIVE CONTROL Open the cranium with a stainless steel saw blade. 6-CELL, DRIVE WITH CELLS Divide the brain in disturbing the membrane. Loosen VERTICAL CELLS the membrane from the cranium and measure the 3. Additional Equipment Used width of the membrane with a micrometer. The thick 1. Diffusion cell assembly, including donor top and recep ness of the membrane should be between 0.19 and 0.24 mm. Cut at least 6 cells of 2.5 by 2.5 cm of tor chamber (set of 6). The donor top includes a drug membrane. dosage wafer (Teflon washer), an acrylic top plate, and a Prepare receptor chamber of diffusion cells with slight clamp to connect top to bottom. overflow of medium as specified in table below with 2. Pigskin, mucous membrane or Duramater as applicable, temperature controlled at 32°C. used within 24 hours from being slaughtered kept in 10 2. Prepare each piece of skin, mucous membrane or Ringer Solution between 2°C.-8°C. Dura mater with the relevant products one at a time as 3. Davies Gold Series Dermatome, Simplex GS 102. follows: 4. Application Squeegee and tweezers. 2.1. Lift skin, mucous membrane or Dura mater with 5. Drug dosage form. tweezers, place on tissue and blot excess of solu 6. Absorbent paper towels and tissues. 15 tion, invert and blot. 4. Technique 3.2 Place skin, mucous membrane or Dura mater in The technique used included the following steps: centered position on drug dosage wafer. 1. Obtain skin from pig heads (jawbone skin). Use Der 3.33.3 Place relevant products on top of skin, mucous matome according to the Operation standard operat membrane or Dura mater in dosage wafer cavity. ing procedure for the Dermatome, setting it to size the 2O 3.4 Use Squeegee to carefully Smooth product over skin to a thickness of 0.33 mm. The diameter of the membrane, filling entire cavity. skin should be in excess of the drug dosage wafer 3.5 Wipe excess dosage off wafer. OR 3.6 Lift loaded dosage wafer with skin, mucous mem For investigations using the active compound brane or Dura mater and place on top of receptor Oxymethalzoline MZL nasal spray, obtain the nasal 25 cell with skin, mucous membrane or Dura mater mucous membrane of the pig in the following manner: side towards cell medium. Exclude bubbles during the snout of the pig is removed just below the eyes process. Place top plate on top of donor cell assem with a stainless steel saw blade. The snout is cut inhalf bly, pressing down with finger, Squeezing out between the nostrils. The cartilage is then gently cut bubbles between top plate and dosage form. Apply and the nasal passage opened. The mucous membrane 30 clamp to lock down top donor and bottom receptor is carefully loosened and removed. Mucous mem halves of diffusion cell. brane cells of 2.5 cm by 2.5 cm are cut. The membrane 5. Operation of Apparatus: thickness is monitored with a micrometer The apparatus must be set to 150 rpm. Samples of 150 ul OR 35 are withdrawn with a micropipette at 10, 20, 30, 60, 90 For investigations using the active compound Bupivic and 120 minutes. The samples after being withdrawn are aine MZL Injectable Solution, obtain the Dura mater analysed for the active compound by means of HPLC, (the tough membrane forming the barrier between the using the parameter indicated in table below for each of brain and cranium or blood) in the following manner: the different compounds.

Oxymetazoline Doxipex Lidarex HCI Bupivicaine And And Scopol- MZL and MZL and Doxepin Lidocaine amine Oxymetazoline Bupivicaine Com Com MZL HCI Com Injection 20 ul 20 ul 20 ul 20 ul 20 ul volume Column Zorbax Zorbax Zorbax SB Zorbax Zorbax C18 C18 C18 C18 C18 15 cm 250 mm x 50 mm x 250 mm x 250 mm x 4.6 mm 4.6 mm 4.6 mm 4.6 mm Mobile Phase 40% 0.2M 50 ml GAA 70% 70% 65% NaH2PO in 930 m Phosphate Phosphate Acetonitrille: buffer with H2O at pH buffer: buffer: 35% 60% 3.4 ad 30% 30% Phosphate MeOH adj with 1N MeOH, MeOH, Buffer, pH pH to 2.5 NaOH H2.5 bH3S 7.7 with HPO Receptor PH 6.8 40% PH6.8 PH 6.8 PH 6.8 chamber phosphate MeOHA phosphate phosphate phosphate medium buffer 60% HO buffer buffer buffer Detector HPLC at HPLC at HPLC at HPLC at HPLC at 254 mm 254 nm. 240mm 280 nm. 263 nm. Temperature Ambient Ambient Ambient Ambient Ambient (22° C.) (22° C.) (22° C.) (22° C.) (22° C.) Flow Rate 1.5 ml/min 1.5 ml/min .5 ml/min ml/min 2 ml/min Retention 3-4 min 5-6 min 2.5-2.7 min 4.85-5.07 min 4.4–4.6 min Time US 8,937,074 B2 79 80 -continued Oxymetazoline Doxipex Lidarex HCI Bupivicaine And And Scopol- MZL and MZL and Doxepin Lidocaine amine Oxymetazoline Bupivicaine Com Com MZL HCI Com Solvent 80% 0.2M MeOHA KHPO. KHPO. KHPO. NaH2PO 60% H2O buffer pH buffer pH buffer pH buffer with 6.8 6.8 6.8 20% MeOH adj pH to 6.8 with 1N NaOH Cells used 6 6 6 3 Time at 60 30 15 8 which total release determined (Min)

2O 6. Results The Release Rate was Calculated as Follows: The release experiment was performed in 6 cells for each product and the mean release is reported for each analy sis point. The results were tabulated and graphically 1 Asan XMass Std X Vol Receptorx presented. The results as a percentage of the active ingre 25 Mass of Active Applied dient released per label claim per cell at the different ug Active Released for Z cells X C time intervals was also tabulated. at time (min.) T Astdx Vol Std XLabel Claimx Table 8.1 below gives a summary of the release rates and Mass of Product Applied percentage release per label claim for the products deter mined according to calculations, reporting the mean Val 30 for 1 cell XZX 100 ues at 60 minutes (1 hour) for each of these compounds. WHERE: TABLE 8.1 Asam Area of peak of sample solution Release rates and percentage release per label 35 Astd=Area of peak of standard solution claim for products tested. Mass Std=Mass of standard taken to prepare the standard % Active? Release Rate % Release per Solution expressed in Jug Active Agent product (ug/cm’/h) label claim Vol Std=Volume to which the standard solution is made up, expressed in ml Doxipex 2.OO 112.9951 O.7945 40 Doxepin Com S.OO 45.6479 O.3190 Vol Receptor Volume of diffusion cells system receptor Lidarex 1.7307 O.69S4 O.0769 Lidarex Com 1.7307 O.S18O 0.0573 chamber, expressed in ml Scopolamine MZL O.3 21.13 O.63 Label Claim=Amount of active present per 100 g of product Oxymetazoline O.S O3S O.OO3S MZL Mass of Product Oxymethazoline O.S O.33 O.OO31 45 Applied for 1 cell=Specific amount of product applied for a Com specific cell Bupivicaine HCI O.S 253.68 1.58 MZL Mass of Active Bupivicaine HCI O.S 226.75 1.35 Com Applied for Z cells=Amount of active applied in total for all 50 Z cells utilised per one study C potency of the standard, expressed as a percentage

ug Active Released 2. Accumulative Dose (pig) released square cm at time (min) = (Surface Area of Exposed Skin) ug Active Released 1.767 cm2

3. Release Rate = Accumulative Dose (pig) released square cm Time (hours) ug Active Released x 100 4. Percentage of Active Released at time (min.) = ug Active Applied US 8,937,074 B2 81 82 It was established that Previous Treatment of the Subject: A. Doxipex Cream releases Doxepin hydrochloride 2.4754 The Subject was using intramuscular and/or oral pain times faster than the commercially available Doxepin relievers and muscle relaxants on a chronic basis since her Com product; diagnosis. B. Lidarex releases Lidocaine HCl 1.3425 times faster than the commercially available Lidocaine Com product. Dosage Form and Usage of the MZL Doxipin Formula: D. C. The release of Oxymetazoline HCLI from the MZL MZL Doxipin was applied three times daily to the affected formulation compared well with that of the commercial aaS. Oxymetazoline Com formulation, but both values were 10 Progress: very low, due to the thickness of the mucous membrane After one month of usage, the Subject no longer needed the of the pig. The human mucous membrane is much thin ner and release should accordingly be much higher in Support of the armcast. Muscle degeneration due to non humans. The release of Bupivicaine from the MZL for usage was reversed to Some extent. mulation was nevertheless shown to be 10.1% faster After 4 months of usage, the Subject moved and used the than that of the commercially available Bupivicaine for 15 arm freely. The wrist remained stiff. mulation after 5 min and was about equal at all other Case Study 3: Use of Doxepin Formulation in Management times. of Severe Back Pain in a Degenerative Thoracic and Vertebral 8. Conclusion Condition It is evident from the results obtained that the percentage Subject: active ingredient released from the DOXIPEX CREAM is significantly higher than that of the comparator Doxepin Com The subject was an 80 year old Caucasian male with severe Cream. The release of Doxepin Hydrochloride from both the back pain due to degeneration of his vertebrae. He was nearly products consequently results in the effective administration completely immobilised by his condition and seldom moved of approximately the same quantity of active per dose 25 outside of his home. He was on chronic therapy of intramus although Doxipex contains 2% m/m Doxepin Hydrochloride cular pain relievers and muscle relaxants. and Doxepin Com 5% m/m Doxepin Hydrochloride in their Dosage Form and Usage of the MZL Doxepin Formula: respective formulations. In his case, synthetic polymercutaneous patches were satu A comparison of the release rates at 60 minutes indicate rated with MZL Doxepin Formulation and attached at night to that the release of Doxepin Hydrochloride from Doxipex 30 areas where he experienced extreme pain for prolonged Cream (2%) is at a rate 2.475 times greater than that from release of the active compound. Doxepin ComCream (5%). The percentage of active released Progress: from the formulation of the nanolipid vesicle formulation of Preparation 2 for this specific time is also 2.47 times greater After a year of treatment, the subject was completely 35 mobile. He stopped taking medication for pain, either of than for the comparator. commercial origin or the Doxepin Formulation. Example 9 Example 10 Clinical Studies 40 Dissolution Studies in Respect of Formulations Below is described three examples of the use of the Dox Made According to the Invention epin MZL Formulation in three patients presenting with pain due to three different clinical causes. Case Study 1. Use of Doxepin Formulation in Management 45 Formulations of the active agents referred to below were of Lower Back Pain and Depression prepared in the manner described above and were subjected to The doxepin in nanolipid formulation described above dissolution tests (2 or 3 repeats as indicated below) performed (DOXIPEX) was administered to the lower back area of a in terms of the USP24 procedure. The following results were post-menopausal Caucasian woman Suffering from manic obtained. depression associated with severe lower backache. Her 50 depression was at the time reasonably under control through the use of PROZAC. She experienced substantial relief from Product Limits Results the use of the formulation of the invention and discontinued Carbamazipine MZL NTL 75% in 30 min 123%, 112%, 114% Suspension her use of PROZAC after about 6 months on DOXIPEX. This, Carbamazipine Com NTL 75% in 30 min 111%, 112%, 1.12% is a most surprising result in view of the very low dose of 55 Suspension doxepin hydrochloride administered through the topical Zolpidem Tartrate MZL NTL 75% 92%, 98%, ND application of the formulation of the invention. SGC Zolpidem Tartrate Com NTL 75% 102%. 102% Case Study 2. Use of Doxepin Formulation in Management SGC of Pain in Reiter's Syndrome Propranolol MZL. SGC NTL 75% in 30 min 12490 126%. 130% The doxepin in nanolipid formulation described above 60 Propranolol Com SGC NTL 75% in 30 min 106%. 10.5%. 107% (DOXIPEX) was administered to the affected limbs of an Phenythion Na MZL NTL 85% in 30 min 143%. 147%. 155% SGC active post-menopausal Caucasian woman diagnosed four Phenythion Na Com NTL 85% in 30 min 53%, 46%, 46% years previously with Reiter's syndrome. SGC Clinical Symptoms in Volunteer: Incapacitated and wasted right arm of the Subject due to 65 non-movement as a result of pain in the limb. The arm was The advantages of the MZL formulations over the com carried in a light cast. mercial formulations are self-evident. US 8,937,074 B2 83 84 Example 11 Cells: The oncogenic cell line used in the study, designated UCT Study to Demonstrate the Nucleic Acid 1, was specifically cultured the University of Cape Town. Transportation and Delivery Characteristics of the Primary UCT-1 cells were cultured form a metastases of a patient's melanoma. The human melanoma cultured cells has Transport Medium of the Invention 5 all the major characteristics of fast growing cancer cells. The cells grow in adherent fashion. AIM: The dendritic cells used were either the cell line THP-1 The scope of this study is to confirm that: (ATCC) or primary macrophages, isolated and cultured from a) nucleic acids in its various forms could be stably the blood of a donor. entrapped in the nanolipid formulation of the present 10 The ODN approach was tested for the malaria parasite invention; Plasmodium Falciparum. The mixture was fed to Plasmo b) Such entrapped nucleic acids could be transported across dium infected human red blood cell cultures, cultured in the the cells membrane to cell nuclei, cell organelles or presence of human serum from a donor. plasma; Reagents: 15 Reagents used were obtained from commercial vendors, as c) the nanolipid formulation medium is not cytotoxic to specified in the procedure below. Fluorophores were from cells; Molecular Probes (Holland) and included nile red, acridine d) The nanolipid formulation medium can be used to co orange, TMRM, ethidium bromide, Alexa Green, Baclight, package various ligands with nucleic acid in the nano carboxy fluorescien and DIOC. lipid vesicles, thereby targetting the nucleic acids to 20 Analysis specific cell types by virtue of their cell Surface antigens. Confocal laser scanning microscopy (CLSM) was used to Three types of nucleic acids were selected for the investi observe entrapment of nucleic acids in the nanolipid formu gation: lation medium. The dual wavelength CLSM was done on a Linear double stranded (ds) human DNA Nikon PCM2000 microscope, with Kr/Arand He/Nelasers or Circular ds DNA: 25 a Zeiss LSM410, using the same lasers. Pinhole size gener Oligodeoxynucleotides (ODNs) ally used as 5 um and smaller. Objectives used were oil Protocol: dispersion 60x and 100x. Light and fluorescent microscopy Motivation for Investigating Linear Double Stranded (ds) on a Nikon TE300 inverted microscope was digitally cap DNA: tured, using Nikon DMX video and Nikon Coolpix900 digital 30 CaCaS. Using the vesicles of the nanolipid formulation medium as To measure the inhibition of parasite growth when using a delivery system for transgenes is considered to be body antisense ODNs, the determination of% parasitaemia was friendly and safe, without the cell damage caused by harsh by counting of the number of intracellular parasites per num physical methods or the disadvantages of viral vectors. ber of erythrocytes and by FACS analysis. Motivation for Investigating Oligodeoxynucleotids and 35 Typical Preparation of DNA for Encapsulation in Nano Ribozymes: lipid Vesicle Transport Medium: Due to the increase in drug resistance in the fight against 1. Six Lug (10 ul) of isolated circular vaccinia virus DNA infectious diseases, the use of antisense oligodeoxynucle was labelled fluorescently with 1 ul of a 0.1 ug/ml otides (ODNs) for the inhibition of intracellular organism Ethidium Bromide solution. Ethidium Bromide interch growth currently receives increasing attention. One of the 40 elates with double stranded DNA, resulting in green ways in which ODN's can be used is to ensure the presence of fluorescence at the laser excitation wavelength used. ODN's of specific design in the cytoplasm. This can result in Tenug of fragmented double stranded human placental the formation of complementary base pairing between the DNA, fragmented to sizes averaging 800 bp, was also ODNs and specific messenger RNAs, which can in turn labelled as above. inhibit the growth of the organisms. 45 One ug of FITC-labelled oligodeoxinucleotides were Motivation for Investigating Circular DNA used. In this case, the ODN's were not labelled with A number of recombinant DNA vaccines use circular ethidium bromide. DNA, containing DNA engineered for vaccination against 2. Free fluorophores were removed by washing procedures, specific infectious agents. The physical state of the DNA except where different methods are described. The typi transferred into cells by the delivery system may contribute to 50 cal washing procedure used is described below: its Survival and Subsequent expression. The coiling and com Free ethidium bromide was removed by ethanol preci paction of circular DNA differs from that of linear DNA, due pation of the DNA in the following manner: to unrelieved supercoiling of the DNA. For that reason, it was a) Three volumes of 99% ethanol and sodium acetate to considered necessary to confirm the entrapment of circular a final concentration of 0.2M were typically added to DNA into the nanolipid vesicle formulation of the invention. 55 the labelled DNA. The mix was left at overnight at Materials and Methods: -20°C. or 30 minutes at -80°C., after which the DNA Nucleic Acids: was collected by micro-centrifugation for 30 minutes The following nucleic acids were used in the study. at 15 000 rpm. a) Human placental DNA, fragmented to sizes averaging b) The collected nucleic acids were washed twice with 800 bp (commercially available form Sigma); 60 70% ethanol and air dried in laminar flow cabinet. b) recombinant circular DNA from vaccinia virus, c) The DNA was resuspended in 10 ul of sterile water obtained from University of Cape Town; and checked by CLSM to confirm that the fluorescent c) oligodeoxynucleotide designed by the Dept of Biochem labelling was successful. istry of the University of Pretoria, South Africa. The Using the Nanolipid Vesicle Formulation as a Medium to ODN’s were commercially manufactured with the 65 Encapsulate the Labelled DNA. inclusion of a FITC fluorescent label inherent in the 1. Nanolipid vesicle formulation was filter sterilized, using design. 0.45 um filters. US 8,937,074 B2 85 86 2. Typically between 100 and 500 ul of the nanolipid 7. The cells were plated in sterile polystyrene (60x15 mm) vesicle formulation was then mixed with the fluorophore tissue culture petri dishes. Nile red (Molecular Probes, Holland) to a final concen 8. Cells were cultured in Shellab CO, incubator at 37°C., tration ranging from 10 nM to 50 nM. The amount of 5% CO, and a relative humidity (RH) of +89%. nanolipid vesicle formulation and Nile red used 5 When the cells reached confluence, they were typically depended on the composition of the formulation as well plated onto 31 x0.017 mm glass cover slips with a as the proposed cell type used. In the nanolipid vesicle refractive index suitable for Confocal Laser Scanning formulation Nile red-associated fatty acids fluoresce in Microscopy (CLSM) in the following manner: the red spectrum when excited by laser energy. Nile red 9. A petri dish with a confluent layer of cells was removed not associated with or bound to fatty acids, and other 10 from the incubator and in a flow cabinet, the media was lipids do not emit red fluorescence under those condi removed under vacuum. tions. DNA itself fluoresces in the green spectrum. 10. Two ml Trypsin/EDTA (Gibco/BRL) was added to the 3. The presence of fluorescent transport vesicles in the dish and the cells returned for incubation in order to nanolipid transport medium was confirmed by CLSM. reverse adherence of the cells to the petri dish. Adher 4. Typically, the resuspended green fluorescent DNA was 15 ence of the cells to the petri dish was checked by light entrapped into nanolipid formulation medium by thor microscopy every two minutes. ough mixing with between 100-500 ul of Nile red 11. During this time, cover slips with the specifications as labelled nanolipid vesicle formulation medium, depend described above, were sterilized. Sterilizing was done by ing on the DNA load needed. Mixing was by 30 minutes dipping the cover slips in 70% ethanol and flaming the Sonication in a Sonicating bath with low frequency, or by cover slip after the alcohol evaporated. Six sterilized vigorous Vortexing, or by slow homogenization. The cover slips were placed in a polypropylene 6 well multi mixing temperatures can be varied, but was typically plate. between room temperature and 40°C. 12. When the cells have lost adherence, 5 ml media 5. Between 5 and 10 ul of the Nanolipid formulation (RPMI-10% FBS and 0.05% Gentamicin Sulphate) at medium with the entrapped DNA was spread thinly on a 25 37°C. were added to the petridish to dilute the Trypsin/ verythin glass cover slip (see below for specifications). EDTA 6. Encapsulation of the various forms of DNA referred to 13. The petri dish content was transferred to a centrifuge above into the vesicles of the nanolipid transport tube, and the cells were collected by centrifugation at medium was confirmed by CLSM. Confirmation of the 3000 rp.m. for 1 min. The supernatant was removed sizes of the entrapped DNA was done by horizontal 30 under vacuum. agarose gel electrophoresis. 14. Three ml media (RPMI-10% FBS and 0.05% Gen The nanolipid transport medium was concentrated by tamicin Sulphate) was carefully added and the cells gen 34% for use with the recombinant Vaccinia DNA and tly resuspended. 50 ul of the concentrated medium was pre-labelled 15. In each well, onto the glass coverslip, 0.5 ml of the cell with nile red. 35 Suspension was plated. This part of the study accordingly confirms that nucleic 16. Adherence of the cells was checked by microscopy and acid substances in the form of the various forms of DNA are as soon as the cells were adherent, media was added to a properly associated with the nanolipid vesicles of the formu final volume of 1 ml. lation medium. 17. The cells were incubated overnight at 37°C., 5% CO, Delivery of Exogenous DNA into the Nucleus of Cells: 40 and a RH of 89%. One of the major factors in determining the level of expres 18. If the cells cultured on the cover slip were determined sion of the genes transferred into target cells are the Survival to be typical of its parent cells by microscopy, the pro and delivery of exogenous DNA to the nucleus. The proce cedure continued as follows: dure described below for the UCT-1 melanoma cells is an 19. One of the cover slips containing the adherent cells example and can with some modifications directly be applied 45 were placed into a cellular flow cell with the side with the transfer of other types of nucleic acid to other cell types, the cells facing up. The stainless steel flow cell was Such as dendritic cells. specifically designed to fit the stage of the Nikon Preparation of Melanoma Cells for DNA Transfer: PCM2000 confocal laser scanning microscope. The UCT-1 cells, stored in liquid Nitrogen in cryovials in the cover slip with its adherent cells then forms the thin glass presence of 90% foetal bovine serum and 10% DMSO, 50 base of the flow cell. The leak-free flow cell is completed were cultured in the following manner: with the use of sterilized rubber O-rings, a polymer 1. Working as quickly as possible, the cryogenic vial is spacer and a second sterile cover slip. Working very transferred to a 37° C. water bath. quickly, 1 ml of the described media at 37°C. was very 2. Using a sterile 10 ml polypropylene pipette and gently added to the cells in the flow cell. The flow cell Pipettes(R)-akku the content of the cryovial is transferred 55 was checked for any tiny cracks in the cover slips and to centrifuge tube suitable for tissue cultures. then fixed onto the stage of the CLSM and the cells left 3. The cells were diluted with 2 ml 37° C. media compris to stabilize. ing of RPMI-10% FBS (foetal bovine serum) and 20. To determine cytotoxicity of the nanolipid formulation 0.05% Gentamicin Sulphate. medium, the same procedure was followed, but with the 4. The cells and media were gently mixed by shaking and 60 omission of any nucleic acids. A range of concentrations then centrifuged for 1 min to loosely precipitate the of the nanolipid vesicle transport medium was investi cells. gated, form undiluted to a 1000 fold dilution. The cells 5. The media was removed to eliminate any DMSO and were exposed to the transport medium for periods rang replaced with 2 ml 37° C. media comprising of RPMI ing from 2 days to 14 days, with concomitant passaging 10% FBS and 0.05% Gentamicin Sulphate. 65 of the cells when necessary. 6. Cells were gently resuspended, using a wide mouth Note: Dendritic cell, in this case THP-1 macrophages, pipette. were cultured and treated in a similar fashion, with the US 8,937,074 B2 87 88 exception that the Gentamycin was omitted or used at a much reduced concentration, and only when necessary. The percentage loading efficiency can be calculated When primary human macrophages were used, the foe Nr of vesicles containing nucleic acid tal bovine serum was replaced with 20% AB human by - Total nr of vesicles- X 100 Sel. 5 Transfer of Vaccinia Virus DNA into Melanoma Cell Nuclei by the Nanolipid Formulation Medium. Most nucleic acids are entrapped in the hydrophilic centre of the vesicles, sponges or depots. Circular DNA can be Note that this procedure can with some modifications be visualized where the DNA follows the contour of individual applied to double stranded linear DNA for delivery into the 10 vesicles, tightly associated with the interior layer of the nuclei or cell organelles or the cytoplasm of other cell types as vesicles membrane. well. Stability of Nucleic Acids Entrapped in Nanolipid Formu 1. Labelled DNA-loaded nanolipid vesicles were isolated lation Medium by differential micro-centrifugation. Care was taken that the internal water phase of the vesicles was not depleted. 15 The stability of nucleic acids in the Nanolipid formulation medium was confirmed by CLSM investigation: at room tem During the differential centrifugation steps, the status of perature, there was no obvious degradation or digestion of the the internal water phase was monitored by CLSM. entrapped nucleic acid for several weeks after preparation of 2. Typically between 5 and 100 ul of the labelled DNA the entrapped nucleic acids. The delivery of the nucleic acids loaded MZL vesicles were carefully and gradually to cells were typically done 1 week after entrapment of the added to the medium of the melanoma cells by the inlet nucleic acids in the nanolipid vesicle transport medium. in the prepared flow cell or by removing the top glass Delivery of Exogenous DNA into the Nucleus of Cells: cover slip of the flow cell. In vitro experiments with both dendritic and oncogenic cell 3. Transfer of the DNA in the nanolipid vesicle formulation lines confirmed the uptake of exogenous nucleic acids were allowed to continue for between 15 to 60 minutes, entrapped in the nanolipid vesicle transport medium. In both after which time the cell medium was removed and the oncogenic Melanoma UCT-1 cells, and in the dendritic replaced with fresh cell medium that contained no DNA THP-1 cells, delivery to the cytoplasm, the mitochondrial loaded nanolipid formulation medium. genome and the nucleus could be demonstrated. 4. Progress of DNA transport into the melanoma cell nuclei 30 In the THP-1 macrophage cell line, the uptake of the was followed and confirmed by CLSM. entrapped exogenous nucleic acid was by two mechanisms: a process of phagocytosis, or by transport across the cell mem This part of the investigation thus confirmed that the DNA brane. In the melanoma cell, transport to the nucleus and associated nanolipid vesicles duly entered the melanoma cell. mitochondria were across the cell membrane, by way of the Furthermore, the release of the labelled DNA from the 35 cytoplasm. The presence of a few intact nanolipid vesicles in nanolipid vesicle transport medium inside the cell nuclei and the cytoplasm could be demonstrated. inside the cell cytoplasm were also confirmed by CLSM. Release of the nucleic acids from the MZL vesicles was Attachment of a Peptide to the Nanolipid Formulation demonstrated in both the cytoplasm and the nucleus by opti Medium Vesicles. cal sectioning and 3D-reconstruction of the cells on the 40 1. The procedure described below can be used for a variety CLSM. The release of the nucleic acid from the vesicles in the of macromolecules, including antibodies, enzymes and nucleus is necessary before DNA can be integrated into the glycoproteins. endogenous genome for transcription. Release in the cyto plasm is especially important in the case of the ODN’s, where 2. 100 mg of the peptide was pre-labelled with Fluorescein the ODN’s would result in inhibition of protein synthesis of Diacetate (FA) to a final concentration of 10 nM. FA 45 infective agents. Research with both Susceptible and drug emits green fluorescence when exposed to the applicable resistant strain of the intracellular parasite Plasmodium Fal laser line. ciparum in the invitro human erythrocyte system showed that 3. The peptide was entrapped in the concentrated Nano antisense oligodeoxynucleotides, entrapped by the nanolipid vesicle transport medium, can be used to inhibit the growth of lipid formulation medium as described above. 50 the parasites in the red blood cell cultures. 4. The incorporation of the peptide into Nanolipid formu lation medium vesicles and Sponges were followed by Targeting of the Nanolipid Formulation Medium Vesicles: light video microscopy and CLSM as described above. The attachment of peptides into nanolipid formulation Results: vesicles was demonstrated. The current data Suggests that the 55 peptides are attached to outer membrane of the vesicles by 1. Entrapment of Nucleic Acids by the Nanolipid Formu strong electrostatic bonds. The attachment to the outer vesicle lation Medium membrane has the advantage that recognition between the The entrapment of nucleic acids in the nanolipid transport peptide and the cell recognition antigen/receptor can occur medium vesicles was illustrated by CLSM. The nucleic acids without any steric hindrance. The peptide used in the experi were entrapped in the vesicles. Nucleic acids in the process of 60 ments described above, was targeted to melanoma cells and being entrapped were visualized by its green fluorescence. fibroblasts. In additional experiment, using another ligand, Co-localization between the Nanolipid formulation medium the ligand have been shown to retain receptor recognition vesicles and green nucleic acids were similarly visualized. activity whilst packaged in nanolipid vesicle transport The loading efficiencies were high between 85% and 95% medium. These packaged vesicles canthus be targeted to cells at the concentrations of nucleic acid and Nanolipid formula 65 containing the appropriate receptor. It is our belief that the tion medium used. Nearly all vesicles showed entrapped same will hold true for other macromolecules. Such as anti DNA. bodies. US 8,937,074 B2 89 90 Toxicity and Cytotoxicity: (ii) One of the most interesting and exciting properties is Cytotoxicity: that it can be used to package ligands so that vesicles can be No cytoxicity was observed at the concentrations used targeted to specific cell Surface receptors for uptake by these under experimental conditions (1:300 to 1:1000 dilution of cells. the Nanolipid formulation medium, diluted with RPMI 5 (iii) It can be used to transfer molecules trans-dermally, medium). No adverse effects were observed at these concen orally or through the any of the mucous membranes without trations. The cells were sensitive to undiluted Transport the need for Sophisticated procedures. Medium if exposed to it for long periods (>1 day). Many variations of the invention are possible without General Safety: departing from the spirit of the invention. 10 The invention claimed is: VITAMINF: Since the constituents in vitamin F comprise 1. A method for the administration of a nucleic acid sub the n-essential fatty acids, which are fundamental to normal stance to cells of an animal, a plant or a micro-organism physiological processes, it is unlikely to be inherently toxic. It comprising administering to said animal, plant or microor could theoretically be toxic if consumed at levels far higher ganism, a composition comprising nucleic acid Substance than those normally available to the body. For example, the 15 that is formulated with an administration medium which estimated endogenous rate of formation of the intermediate, comprises an aqueous Solution of nitrous oxide gas in a physi gammalinolenic acid within the body of a normal adult was ologically acceptable carrier Solvent for the gas and which the put at 100-1000 mg. or around 2-20 mg/day. It was also administration medium includes at least one fatty acid ethyl estimated that daily intake of GLA in a human fully breastfed ester or glycerol-polyethylene glycol esters of ricinoleate, infant is of the order of 20-80 mg/kg/day. Based on these wherein said nucleic acid substance is selected from the estimates, it seems unlikely that doses of less than 100 mg/kg/ group consisting of DNA, RNA, DNA-RNA hybrids, a day will have any toxicity (Horrobin D F. Nutritional and gene or part thereof, a genome or part thereof, oligo medical importance of gamma-linolenic acid. Lipid Res, nucleotides and synthetic nucleic acids, and 1992. 31(2): 163-194). Long term animal toxicity studies wherein the nitrous oxide is dissolved to saturation. have shown that doses of evening primrose oil of up to 5 ml or 25 2. The method of claim 1, wherein the administration 10 ml/kg/day given for up to two years has no toxicity (Ever medium further comprises eicosapentaenoic acid C20: 5c)3 ett DJ, Perry CJ, Bayliss P. MedSci Res. 1988: 16:865-866). and/or decosahexaenoic acid C22: 6c)3 or ethyl esters Cremophor: From the available literature it is concluded thereof, as additional long chain fatty acids. that Cremophor administered intravenously may elicit certain 3. The method of claim 1, wherein the carrier solvent is 30 water or a pharmaceutically acceptable alcohol, ether, poly dose-dependent toxicity characteristics. There is no evidence mer or an oil. to suggest that the same is true for oral administration forms. 4. The method of claim 1, wherein the nucleic acid sub In fact, in a study using oral administration of the drug pacli stance is formulated in a liquid formulation and wherein the taxel in combination with Cremaphor EL, results showed formulation comprises, as part of the administration medium, undetectable plasma Cremaphor levels. It was concluded that 35 water or acceptable other liquid solvent into which the nitrous this finding would have a beneficial influence on the safety of oxide is dissolved and wherein the fatty acid ethyl ester is treatment with the MZL Nucleic Acid Transport Medium, dissolved or Suspended or emulsified along with the nucleic especially when it is used in its prodrug form for oral admin acid Substance. istration. In this form, any Exipient (see Textbook of Exipi 5. The method of claim 1, wherein the carrier solvent for ents) may be added, which in itself refers to the safety and 40 the nitrous oxide gas is essentially non-aqueous and com indicates the prescribed dosages. prises the least one fatty acid ethyl ester. Conclusions 6. The method of claim 5, wherein the nucleic acid sub The nanolipid vesicle administration or transport medium stance is formulated to be suitable to be applied as a topical, is a formulation capable of encapsulating macromol buccal or vaginal cream or ointment, or skin patch or as an ecules and delivering them with high efficacy to target 45 intravenous, intramuscular or subcutaneous injection, or as a sites. Furthermore, the encapsulating vesicles have been Suppository, or to be administered as an oral, nasal, or pull shown to penetrate through the cell membrane and to monary preparation. migrate to cell organelles. This study shows the efficient 7. The method of claim 1, comprising formulating the transport and delivery of DNA to such cell organelle, nucleic acid Substance to be applied as a topical, buccal or including the nuclei of the cells. 50 vaginal cream or ointment, or skin patch or as an intravenous, The major steps in the transfection process, ie the entry of intramuscular or Subcutaneous injection, or as a Suppository, DNA into the cell, the trafficking of the transgene or to be administered as an oral, nasal, or pulmonary prepa through the cytoplasm, including both endosomal and ration, wherein the formulation used in formulating said lysosomal compartments, the entry of DNA into the cream, ointment, injectable formulation, Suppository, or oral, 55 nasal, or pulmonary preparation, incorporates, comprises the nucleus or cell organelle, such as the mitochondria or the nucleic acid Substance, a quantity of water containing nitrous targeting of the nucleic acid to an intracellular infective oxide, a long chain fatty acid ethyl ester and, optionally agent, such as parasite or the targeting to the nucleic further comprises adding excipients and additional carriers as acids of Such infective agents in the cellular cytoplasm are conventionally used in pharmaceutical trade to make dos and the ability to escape intracellular mechanisms 60 age forms. designed to purge foreign nucleic acids, has in one way 8. The method of claim 1, wherein the nucleic acid sub or another been addressed by these studies, with positive stance is selected from the group consisting of DNA, RNA, results. DNA-RNA hybrids, oligonucleotides and synthetic nucleic This product has several unique advantages: acids. (i) It is a highly effective gene transfer vector and much 65 9. The method of claim 1, wherein the composition more efficient than conventional products currently on the includes a peptide, peptide antibody or peptide derivative market selective for a selected receptor orantigen or protein binding US 8,937,074 B2 91 92 element on a target cell, the peptide being bound to at least carrier Solvent for the gas and which the administration one of the fatty acid ethyl ester in the composition. medium includes at least one fatty acid ethyl ester or glycerol 10. The method of claim3, wherein said water is deionized polyethylene glycol esters of ricinoleate, Water. wherein said nucleic acid substance is selected from the 11. The method of claim 3, wherein said oil is an organic group consisting of DNA, RNA, DNA-RNA hybrids, a oil. gene or part thereof, a genome or part thereof, oligo 12. The method of claim 11, wherein said organic oil is an nucleotides and synthetic nucleic acids, and essential oil based on long chain fatty acids having between wherein the nitrous oxide is dissolved to saturation. 14 and 22 carbon atoms in the fatty acid. 18. A method for the administration of a nucleic acid sub 13. The method of claim 12, wherein said essential oil is of 10 natural origin. stance to cells of an animal, a plant or a micro-organism 14. The method of claim 13, wherein said essential oil of comprising administering to said animal, plant or microor natural oil is a plant oil rich in gammalinolenic acid IGLA. ganism, a composition comprising nucleic acid Substance 15. The method of claim 8, wherein said nucleic acid that is formulated with an administration medium which Substance is a DNA sequence coding for the expression of a 15 comprises an aqueous Solution of nitrous oxide gas in a physi chemokine, a chemotactin, a cytokine, an enzyme, a gona ologically acceptable carrier Solvent for the gas and which the dotrophin, a growth factor, an immunoglobulin, an interferon, administration medium includes Vitamin F Ethyl Ester com an interleukin, a lipid-binding protein, a pituitary hormone, a prising fatty acid distribution: protease inhibitor, a protease, or a somatomedin. 16. The method of claim 1, wherein said administration medium comprises oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapen taenoic acid C20: 5c)3, decosahexaenoic acid C22: 6c)3. C1 to C6 alkyl esters of ricinoleic acid, or a reaction product 25 of hydrogenated natural oils comprising ricinoleic acid based wherein said nucleic acid substance is selected from the oils with ethylene oxide. group consisting of DNA, RNA, DNA-RNA hybrids, 17. A composition for introducing a nucleic acid Substance a gene or part thereof, a genome or part thereof, oli into cells of an animal, a plant or a micro-organism wherein gonucleotides and synthetic nucleic acids, and the composition comprises a formulation of the nucleic acid wherein the nitrous oxide is dissolved to saturation. in an administration medium which comprises an aqueous 30 Solution of nitrous oxide gas in a physiologically acceptable k k k k k